CN111182951A

CN111182951A - Filtration and chromatography pods and methods of using the same

Info

Publication number: CN111182951A
Application number: CN201880065040.0A
Authority: CN
Inventors: W.K.麦康瑙希; S.R.汉密尔顿
Original assignee: Compass Therapeutics LLC
Current assignee: Compass Therapeutics LLC
Priority date: 2017-08-01
Filing date: 2018-08-01
Publication date: 2020-05-19
Also published as: JP2020529605A; WO2019028172A1

Abstract

Provided herein are methods of capturing and purifying recombinant proteins from liquid cell cultures comprising dual use filtration and chromatography pods, filtration and chromatography pods and uses of these pods.

Description

Filtration and chromatography pods and methods of using the same

Cross Reference to Related Applications

This application claims priority from U.S. patent application serial No.62/539,943, filed 2017, 8/1, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to the field of biotechnology, and more particularly, to the capture and purification of recombinant proteins.

Background

Current techniques and tools for producing recombinant proteins are often labor intensive, time consuming, expensive, and prone to error. Since each step requires user manipulation, recovery of the protein is affected.

Disclosure of Invention

The present disclosure is based, at least in part, on the production of novel filtration and chromatography pods for the capture and purification of recombinant proteins from liquid cell cultures. As described below and exemplified in the working examples, filtration and chromatography pods provide several benefits over previous methods of purifying recombinant proteins, including, for example, speed, ease of use, enhanced protein stability, no need for multiple culture dishes and other consumables, and rapid capture and purification of the recombinant protein of interest. Additional advantages of the invention are described herein.

Accordingly, provided herein are filtration and chromatography pods comprising a container formed by a wall and having a top end and a bottom end, wherein the wall forms a seal with the bottom end, and one or more of the wall, the top end and the bottom end comprises at least one region covered by a semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod into the interior of the pod. In some embodiments of any of the pods described herein, the bottom end includes at least one area covered by a semi-porous or porous material.

In some embodiments of any of the pods described herein, the semi-porous or porous material is characterized by one or both of: (a) does not allow cells to flow from the outside of the pod into the inside of the pod; and (b) retaining or substantially retaining the resin within the pod. In some embodiments, the semi-porous or porous material does not allow cells to flow from the exterior of the pod into the interior of the pod. In some embodiments, the semi-porous or porous material retains or substantially retains the resin inside the pod. In some embodiments of any of the pods described herein, the semi-porous or porous material is characterized by not allowing cells to flow from the exterior of the pod into the interior of the pod and retaining or substantially retaining the resin in the interior of the pod. In some embodiments of any of the pods described herein, the cell is a mammalian cell. In some embodiments of any of the pods described herein, the cell is an insect cell or a yeast cell. In some embodiments of any of the pods described herein, the cell is a bacterial cell (e.g., an escherichia coli cell). In some embodiments of any pod, method, kit, or any other aspect described herein, the cell is not a bacterial cell. In some embodiments, the semi-porous or porous material retains or substantially retains the resin inside the pod structure.

In some embodiments of any of the pods described herein, the material is a semi-permeable or porous membrane. In some embodiments of any of the pods described herein, the material is a semi-permeable or porous mesh. In some embodiments, the mesh is coated with a hydrophilic or hydrophobic material. In some embodiments of any of the pods described herein, the mesh is made of nylon or polyester. In some embodiments of any of the pods described herein, the pore size of the material is from about 50nm to about 100 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 50nm to about 50 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 100nm to about 50 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 10 μm to about 50 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 20 μm to about 50 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 100nm to about 30 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 500nm to about 20 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 1 μm to about 10 μm. In some embodiments of any of the pods described herein, the pore size of the material is from about 2 μm to about 5 μm.

In some embodiments of any of the pods described herein, the internal volume of the container is about 0.1mL to about 500 mL. In some embodiments of any of the pods described herein, the internal volume of the container is about 0.1mL to about 250 mL. In some embodiments of any of the pods described herein, the internal volume of the container is about 0.1mL to about 100 mL. In some embodiments of any of the pods described herein, the bottom end includes at least one area covered by the material. In some embodiments of any of the pods described herein, the wall includes at least one area covered by the material. In some embodiments of any of the pods described herein, the bottom end and the wall each include at least one area covered by the material. In some embodiments of any of the pods described herein, the tip includes at least one area covered by the material. In some embodiments of any of the pods described herein, each of the top end and the bottom end includes at least one area covered by a material. In some embodiments of any of the pods described herein, each of the at least one region has a surface area of about 1mm²To about 25cm². In some embodiments of any of the pods described herein, each of the at least one region has a surface area of about 1mm²To about 20cm². In some embodiments of any of the pods described herein, each of the at least one region has a surface area of about 1mm²To about 10cm². In some embodiments of any of the pods described herein, each of the at least one region has a surface area of about 1mm²To about 5cm². In some embodiments of any of the pods described herein, a surface of each of the at least one regionThe product is about 1mm²and 2.5cm². In some embodiments of any of the pods described herein, each of the at least one region has a surface area of about 1mm²and about 1cm²。

In some embodiments of any of the pods described herein, the bottom end is flat. In some embodiments, the bottom end has a hemispherical shape. In some embodiments of any of the pods described herein, the bottom end has a conical shape. In some embodiments of any of the pods described herein, the wall has a cylindrical shape. In some embodiments of any of the pods described herein, the wall has a conical shape. In some embodiments of any of the pods described herein, the walls have a spherical shape. In some embodiments of any of the pods described herein, the wall has a hemispherical shape. In some embodiments of any of the pods described herein, the wall has an hourglass shape.

In some embodiments of any of the pods described herein, the pod further includes a chromatography resin disposed within the interior of the container. In some embodiments of any of the pods described herein, the chromatography resin is a commercial chromatography resin. In some embodiments of any of the pods described herein, the chromatography resin is an affinity chromatography resin. In some embodiments of any of the pods described herein, the affinity chromatography resin is a protein a resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin. In some embodiments of any of the pods described herein, the volume of chromatography resin is about 0.1mL to about 500 mL. In some embodiments of any of the pods described herein, the volume of chromatography resin is about 0.1mL to about 250 mL. In some embodiments of any of the pods described herein, the volume of chromatography resin is about 0.1mL to about 100 mL. In some embodiments of any of the pods described herein, the volume of chromatography resin is about 0.1mL to about 50 mL. In some embodiments of any of the pods described herein, the sterility assurance level of the chromatography resin is about 1x10^-6To about 1x10^-10。

Also provided herein are kits comprising any of the pods described herein. Some embodiments of these kits may further comprise instructions for using any of the pods described herein. Some embodiments of any of the kits provided herein can further comprise a means for stably positioning the pod inside the culture dish. In some embodiments of any of the kits described herein, the culture dish is a shake flask, a shake tube, a bioreactor, or a multi-well plate. In some embodiments of any of the kits described herein, the bioreactor is a perfusion bioreactor. In some embodiments of any of the kits described herein, the bioreactor is a fed-batch bioreactor.

Also provided herein are kits comprising: (a) any pod described herein; and (b) one or more panels that may be secured to each of the one or more areas in the wall, thereby preventing fluid from leaking from the interior of the pod through the wall to the exterior of the pod.

Also provided herein are kits comprising: (a) any pod described herein; and (b) an outer sleeve securable to the pod so as to cover at least one area of the wall to prevent fluid from leaking from the interior of the pod through the at least one area of the wall to the exterior of the pod. In some embodiments, the sleeve is an empty chromatography column.

Also provided herein are kits comprising: (a) a pod of any of the pods described herein; and (b) one or both of: an elution outlet that can be stably fixed to the bottom end; a buffer reservoir that can be stably immobilized on the tip. In some embodiments of any of the kits described herein, the kit comprises a pod and an elution outlet. In some embodiments of any of the kits described herein, the kit comprises a pod and a buffer reservoir. In some embodiments of any of the kits described herein, the kit comprises a pod, an elution outlet, and a buffer reservoir. In some embodiments of any of the kits provided herein, the elution outlet is a funnel. In some embodiments of any of the kits provided herein, the elution outlet comprises a planar surface having a port. In some embodiments of any of the kits provided herein, the elution outlet is configured to allow for adjustment of a flow rate of fluid through the elution outlet. In some embodiments of any of the kits provided herein, the elution outlet is not configured to allow for adjustment of the flow rate of fluid through the elution outlet. In some embodiments of any of the kits provided herein, the elution outlet can be screwed or snapped onto the pod. In some embodiments of any of the kits provided herein, the elution outlet can be sealed to the pod using one or more (e.g., two or three) O-rings (e.g., secured to the pod or elution outlet).

Also provided herein are kits comprising: (a) any pod described herein; and (b) a movable outer sleeve connected to the wall such that rotation of the movable outer sleeve covers at least one area in the wall, thereby preventing fluid from leaking from the interior of the pod through the at least one area in the wall to the exterior of the pod. In some embodiments of any of the kits provided herein, the kit further comprises a means for stably positioning the pod inside the culture dish.

Also provided herein are kits comprising: (a) any pod described herein; and (b) means for stably positioning the long axis of the pod in a horizontal position inside the culture dish. Non-limiting examples of means for stably positioning the pods inside the culture dish are shown in figures 11-19, 36 and 37.

Also provided herein are kits comprising: (a) any pod described herein; and (b) an inlet system having a top end, an elongated cylindrical body comprising a fluid conduit, and a bottom end, wherein the top end of the inlet system comprises a fluid inlet and is configured for stable attachment to the top end of the pod and, when stably connected to the pod, allows fluid to flow into the pod through the fluid inlet and the fluid conduit and out the bottom end of the inlet system, and the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod; and (c) an outlet system comprising a fluid outlet configured to be stably attached to the bottom end of the pod and, when stably connected to the bottom end of the pod, allow fluid to flow out of the pod through the fluid outlet. In some embodiments of these kits, the top end of the inlet system and the top end of the pod comprise threads, allowing the top end of the pod to be stably attached to the top end of the inlet system. In some embodiments of these kits, the threads in the top end of the inlet system or the threads in the top end of the pod comprise an O-ring. In some embodiments of these kits, the pod comprises a chromatography resin (e.g., any of the exemplary types of chromatography resins described herein); and the inlet system and pod are configured such that the bottom end of the inlet system is proximate to the chromatography resin when the inlet system and pod are stably attached. In some embodiments of these kits, the inner surface of the pod and the bottom end of the inlet system are configured to allow for stable attachment of the pod and the bottom end of the inlet system. In some embodiments of these kits, the inner surface of the pod and the bottom end of the inlet system comprise threads, allowing the bottom ends of the pod and the inlet system to be stably attached. In some embodiments of these kits, the threads in the inner surface of the pod or the threads in the bottom end of the inlet system comprise an O-ring. In some embodiments of these kits, the outlet system and the bottom end of the pod comprise threads, allowing the outlet system to be stably attached to the bottom end of the pod. In some embodiments of these kits, the threads in the outlet system or the threads in the bottom end of the pod comprise an O-ring. In some embodiments of these kits, the fluid inlet can be attached to a conduit. In some embodiments of these kits, the fluid outlet is attachable to a conduit. Some embodiments of these kits further comprise: (d) means for stably positioning the long axis of the pod in a horizontal position inside a culture dish (e.g., any of the culture dishes described herein). Some embodiments of these kits further comprise: (d) means for stably positioning the long axis of the pod in a vertical position inside a culture dish (e.g., any of the culture dishes described herein).

Also provided herein is a method of capturing a recombinant protein produced by a liquid cell culture, the method comprising contacting any pod described herein with the liquid cell culture for a period of time, wherein the contacting step results in at least a portion of the at least one region contacting the liquid cell culture, and the contacting is performed under conditions that result in the chromatography resin capturing the recombinant protein. In some embodiments of any of the methods described herein, the liquid cell culture is placed in a culture dish.

Also provided herein is a method of purifying a recombinant protein produced by a liquid cell culture, the method comprising: (a) contacting any pod described herein with a liquid cell culture for a period of time, wherein the contacting step results in at least a portion of the at least one region being contacted with the liquid cell culture and the contacting is performed under conditions that result in the chromatography resin capturing the recombinant protein; and (b) eluting the recombinant protein from the chromatography resin after the period of time has elapsed, thereby purifying the recombinant protein.

In some embodiments of any of the methods provided herein, the method further comprises performing one or more additional unit operations on the recombinant protein after (b), (c). In some embodiments of any of the methods provided herein, the method further comprises, after (c), formulating the recombinant protein into a recombinant protein pharmaceutical product. In some embodiments of any of the methods provided herein, the method further comprises washing the chromatography resin between step (a) and step (b). In some embodiments of any of the methods described herein, the contacting is performed under sterile conditions.

In some embodiments of any of the methods described herein, the period of time is from about 5 minutes to about 2 weeks. In some embodiments of any of the methods described herein, the period of time is from about 5 minutes to about 168 hours. In some embodiments of any of the methods described herein, the period of time is from about 5 minutes to about 96 hours. In some embodiments of any of the methods described herein, the period of time is from about 5 minutes to about 48 hours. In some embodiments of any of the methods described herein, the period of time is from about 5 minutes to about 24 hours. In some embodiments of any of the methods described herein, the period of time is from about 5 minutes to about 12 hours. In some embodiments of any of the methods described herein, the period of time occurs when the liquid cell culture is in its exponential growth phase. In some embodiments of any of the methods described herein, the period of time occurs when the liquid cell culture is in its quiescent growth stage. In some embodiments, the contacting occurs while the liquid cell culture is in its death phase.

In some embodiments of any of the methods described herein, at least 30% of the total surface area of at least one zone is contacted with the liquid cell culture during the period of time in step (a). In some embodiments of any of the methods described herein, at least 50% of the total surface area of at least one zone is contacted with the liquid cell culture during the period of time in step (a). In some embodiments of any of the methods described herein, at least 75% of the total surface area of at least one zone is contacted with the liquid cell culture during the period of time in step (a). In some embodiments of any of the methods described herein, at least 90% of the total surface area of at least one zone is contacted with the liquid cell culture during the period of time in step (a).

In some embodiments of any of the methods described herein, the liquid cell culture is placed in a culture dish. In some embodiments of any of the methods described herein, the culture dish is a plate (e.g., a multi-well plate), a shake tube, a shake flask, or a bioreactor. In some embodiments of any of the methods described herein, the bioreactor is a perfusion bioreactor. In some embodiments of any of the methods described herein, the bioreactor is a fed-batch bioreactor. In some embodiments of any of the methods described herein, the liquid cell culture comprises a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. In some embodiments of any of the methods described herein, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell or an NS0 cell.

In some embodiments of any of the methods described herein, the one or more additional unit operations are selected from the group consisting of: viral inactivation, viral filtration, polishing, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and ultrafiltration/diafiltration.

In some embodiments of any of the methods described herein, the recombinant protein is a secreted protein. In some embodiments of any of the methods described herein, the recombinant protein is any protein with or without a tag (e.g., a fluorescent tag, a radioisotope, a polyhistidine tag (HIS tag), a c-myc tag, a FLAG tag, a Maltose Binding Protein (MBP) tag, an Albumin Binding Protein (ABP) tag, a bacteriophage T7 epitope (T7 tag), a Biotin Carboxyl Carrier Protein (BCCP) tag, a bluetongue virus tag (B tag), a calmodulin binding peptide tag, a cellulose binding domain tag, a chitin binding domain tag, a choline binding protein, a biotin conjugateA domain tag, a galactose-binding protein tag, a Glu-Glu epitope tag, a human influenza hemagglutinin tag, a fusion protein tag,

histidine Affinity TAG (HAT), polyarginine TAG (Asp-TAG), polyphenylalanine TAG (Phe-TAG), S-TAG, streptavidin-binding peptide, streptavidin TAG, streptomyces TAG, an enzyme (e.g., alkaline phosphatase, Chloramphenicol Acetyltransferase (CAT), glutathione S-transferase), or Fc TAG). The recombinant protein is an antibody, antigen-binding antibody fragment, enzyme or fusion protein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein. Other, suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

Fig. 1A-1E illustrate various exemplary pods and kits provided herein.

Fig. 2 illustrates an exemplary embodiment of a pod as provided herein, wherein the longitudinal axis of the pod is approximately horizontal when the pod is placed in a bioreactor.

Fig. 3 shows the exemplary pod of fig. 2 after it has been removed from the bioreactor and rotated so that the longitudinal axis is approximately vertical and its bottom end is connected to an elution outlet. Optionally, the top end of the pod may be connected to a buffer reservoir container (not shown).

Fig. 4 is an exemplary depiction of a pod as described herein disposed in a container (e.g., a bioreactor), wherein the stem transitions through apertures in the pod and in a side wall of the container.

Figure 5 is a representative image of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis comparing a traditional batch capture method (Trad.) to a capture and purification method (Cap. & Pure) under reducing and non-reducing conditions. E ═ 1 μ g of eluted sample; FT ═ the flow rate through the sample; w ═ wash sample; M.Bio-rad Precision and ladder marker (Biorad Precision Plus ladder marker); "-" ═ blank.

FIG. 6 is a comparison from day 4 after transfection

Schematic of the purification process, traditional batch capture process, and experimental schedules for capture and purification processes.

Figure 7 is a representative graph showing High Performance Liquid Chromatography (HPLC) traces under four different conditions outlined in table 2.

Figure 8 is a representative graph showing HPLC traces under four different conditions outlined in table 3.

Figure 9 is a representative graph showing HPLC traces under two different conditions outlined in table 4.

Figures 10-13 are two-dimensional views of different exemplary devices for stably positioning the pod inside the culture dish. The symmetrical two-pronged claws may hold any of the exemplary pods described herein in a culture dish.

FIGS. 14 and 15 are two-dimensional views of a portion of an exemplary apparatus for stably positioning a pod inside a culture dish; the center of the component can be attached to a symmetrical two-pronged claw that can hold any of the exemplary pods described herein in a culture dish (as shown in fig. 16-19).

Figures 16-19 are two-dimensional views of different exemplary devices for stably positioning the pod inside the culture dish. The symmetrical two-pronged claws may hold any of the exemplary pods described herein in a culture dish.

Figure 20 shows a superimposed elution profile of a 2 μ g sample injection of protein on an analytical TOSOH Supergel TSK mAb HTP column purified simultaneously using a device similar to that shown in figures 27 and 28 or using a traditional batch capture and elution method. After the device or free floating resin is removed from the clear supernatant, the clear supernatant containing the antibody is exposed to and eluted from the protein a resin prior to washing.

Figure 21 shows the elution profile of a 2 μ g sample injected on an analytical TOSOH Supergel TSK mAb HTP column using the same clear supernatant as figure 20 but using the aka ta system and proA resin contained in the packed column.

Figure 22 illustrates an exemplary pod provided herein.

Figure 23 illustrates an exemplary pod provided herein.

Fig. 24-28 illustrate an exemplary kit provided herein.

Fig. 29-31 illustrate exemplary pods provided herein.

FIGS. 32 and 33 show elution profiles from the protein A resin-containing devices shown in FIGS. 27 and 28 exposed to unfiltered supernatant and used

And (5) eluting the system.

Fig. 34 illustrates an exemplary adapter provided herein.

Fig. 35 illustrates an example adapter provided herein attached to an example pod described herein.

Figures 36 and 37 show an exemplary device for stably positioning the pod inside the culture dish.

Detailed Description

Provided herein are filtration and chromatography pods comprising a container formed by a wall and having a top end and a bottom end, wherein the wall forms a seal with the bottom end, and one or more of the wall, the top end, and the bottom end comprise at least one region covered by a semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod into the interior of the pod. Also provided herein are kits comprising any of these pods, methods of using these pods to capture recombinant proteins produced in liquid cell cultures, and methods of using these pods to purify recombinant proteins produced in liquid cell cultures.

Non-limiting aspects of these pods, kits and methods are described below and may be used in any combination without limitation. Other aspects of these pods, kits and methods are known in the art.

The pods, kits, and methods provided herein provide an unexpected reduction in the time required to capture or purify a recombinant protein (e.g., a protein expressing a recombinant protein produced by a recombinant cell, such as a stable cell) from a protein expression system. For example, a recombinant protein can be captured or purified in the following time periods: from about 5 minutes to about 20 hours, from about 5 minutes to about 18 hours, from about 5 minutes to about 16 hours, from about 5 minutes to about 14 hours, from about 5 minutes to about 12 hours, from about 5 minutes to about 10 hours, from about 5 minutes to about 8 hours, from about 5 minutes to about 6 hours, from about 5 minutes to about 4 hours, from about 5 minutes to about 3 hours, from about 5 minutes to about 2 hours, from about 5 minutes to about 1 hour, from about 5 minutes to about 30 minutes, from about 5 minutes to about 20 minutes, from about 5 minutes to about 10 minutes, from about 10 minutes to about 20 hours, from about 10 minutes to about 18 hours, from about 10 minutes to about 16 hours, from about 10 minutes to about 14 hours, from about 10 minutes to about 12 hours, from about 10 minutes to about 10 hours, from about 10 minutes to about 8 hours, from about 10 minutes to about 6 hours, from about 10 minutes to about 4 hours, from about 10 minutes to about 3 hours, from about 10 minutes to about 2 hours, about 10 minutes to about 1 hour, about 10 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 20 hours, about 20 minutes to about 18 hours, about 20 minutes to about 16 hours, about 20 minutes to about 14 hours, about 20 minutes to about 12 hours, about 20 minutes to about 10 hours, about 20 minutes to about 8 hours, about 20 minutes to about 6 hours, about 20 minutes to about 4 hours, about 20 minutes to about 3 hours, about 20 minutes to about 2 hours, about 20 minutes to about 1 hour, about 20 minutes to about 30 minutes, about 30 minutes to about 20 hours, about 30 minutes to about 18 hours, about 30 minutes to about 16 hours, about 30 minutes to about 14 hours, about 30 minutes to about 12 hours, about 30 minutes to about 10 hours, about 30 minutes to about 8 hours, about 30 minutes to about 6 hours, about 30 minutes to about 4 hours, about 30 minutes to about 3 hours, from about 30 minutes to about 2 hours, from about 30 minutes to about 1 hour, from about 1 hour to about 20 hours, from about 1 hour to about 18 hours, from about 1 hour to about 16 hours, from about 1 hour to about 14 hours, from about 1 hour to about 12 hours, from about 1 hour to about 10 hours, from about 1 hour to about 8 hours, from about 1 hour to about 6 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 20 hours, from about 2 hours to about 18 hours, from about 2 hours to about 16 hours, from about 2 hours to about 14 hours, from about 2 hours to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 2 hours to about 6 hours, from about 2 hours to about 4 hours, from about 2 hours to about 3 hours, from about 3 hours to about 20 hours, from about 3 hours to about 18 hours, from about 3 hours to about 16 hours, from about 3 hours to about 14 hours, from about 3 hours to about 12 hours, from about 3 hours to about 10 hours, from about 3 hours to about 8 hours, from about 3 hours to about 6 hours, from about 3 hours to about 4 hours, from about 4 hours to about 20 hours, from about 4 hours to about 18 hours, from about 4 hours to about 16 hours, from about 4 hours to about 14 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 4 hours to about 6 hours, from about 6 hours to about 20 hours, from about 6 hours to about 18 hours, from about 6 hours to about 16 hours, from about 6 hours to about 14 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 20 hours, from about 8 hours to about 18 hours, from about 8 hours to about 16 hours, from about 8 hours to about 14 hours, from about 8 hours to about 12 hours, about 8 hours to about 10 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 16 hours, about 10 hours to about 14 hours, about 10 hours to about 12 hours, about 12 hours to about 20 hours, about 12 hours to about 18 hours, about 12 hours to about 16 hours, about 12 hours to about 14 hours, about 14 hours to about 20 hours, about 14 hours to about 18 hours, about 14 hours to about 16 hours, about 16 hours to about 20 hours, about 16 hours to about 18 hours, or about 18 hours to about 20 hours.

Pod with removable cover

Provided herein are filtration and chromatography pods comprising a container formed by a wall and having a top end and a bottom end, wherein the wall forms a seal with the bottom end, and one or more of the wall, the top end, and the bottom end comprise at least one region covered by a semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod into the interior of the pod. The semi-porous or porous material may be attached to the walls, top end and/or bottom end of the pod by heat sealing. Other means for attaching semi-porous or porous materials to the walls, top end and/or bottom end of the pod are known in the art. Non-limiting examples of pods and kits provided herein are shown in figures 1A to 1E, figure 2, figure 3, figure 4, figures 22-31 and figure 35. In some embodiments, pod designs such as those shown in fig. 1A may optionally include one or more panels adapted to cover some or all of the surfaces of the sides of the pod formed from the semi-porous or porous material. The attachment of such a cover may further facilitate subsequent purification steps.

Semi-porous/porous materials

In some embodiments of any of the pods described herein, the semi-porous or porous material retains or substantially retains the resin within the pod structure. In some examples of any of the pods described herein, the semi-porous or porous material is characterized by one or all of: (a) not allowing cells to flow from the outside of the pod into the interior of the pod, and (b) retaining or substantially retaining the resin in the interior of the pod (c) coated with a hydrophilic or hydrophobic coating. In some embodiments, the semi-porous or porous material does not allow cells to flow from the exterior of the pod into the interior of the pod. In some embodiments, the semi-porous or porous material retains or substantially retains the resin inside the pod. In some embodiments of any of the pods described herein, the semi-porous or porous material is characterized by not allowing cells to flow from the exterior of the pod into the interior of the pod and retaining or substantially retaining the resin in the interior of the pod.

In some examples of any of the pods described herein, the semi-porous or porous material is characterized by one or both of: (a) not allowing the mammalian cells to flow from the exterior of the pod into the interior of the pod, and (b) retaining or substantially retaining the resin within the interior of the pod. In some embodiments of any of the pods described herein, the semi-porous or porous material does not allow the mammalian cells to reversibly flow from the exterior of the pod into the interior of the pod. In some examples of any of the pods described herein, the semi-porous or porous material is characterized by one or both of: (a) not allowing insect cells to flow from the outside of the pod into the inside of the pod, and (b) retaining or substantially retaining the resin within the inside of the pod. In some embodiments of any of the pods described herein, the semi-porous or porous material does not allow insect cells or yeast cells to reversibly flow from the exterior of the pod into the interior of the pod. In some embodiments of any of the pods described herein, the semi-porous or porous material does not allow the bacterial cells to reversibly flow from the exterior of the pod into the interior of the pod.

In some examples, the material is a semi-permeable or porous membrane. In some examples, the material is a semi-permeable or porous mesh (e.g., a nylon mesh).

In some examples, the material may include (at least in part) one or more of the following: polyesters, polyesters with hydrophilic coatings, cellulose acetate, cellulose nitrate, Polyethersulfone (PES), polystyrene, polypropylene, acrylic copolymers, polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (PTFE) and polycarbonates. In some embodiments, the material is a precision-knit polymer (e.g., any precision-knit polymer). In some embodiments, the material is a Sefar film (see, e.g., sefal, U.S. website www.sefar.us). In some embodiments, the material is Petex mesh sold by Sefar.

In some embodiments of any of the pods described herein, the pore size or average pore size of the material is: about 50nm to about 50 μm, about 50nm to about 45 μm, about 50nm to about 40 μm, about 50nm to about 35 μm, about 50nm to about 30 μm, about 50nm to about 25 μm, about 50nm to about 20 μm, about 50nm to about 18 μm, about 50nm to about 16 μm, about 50nm to about 14 μm, about 50nm to about 12 μm, about 50nm to about 10 μm, about 50nm to about 8 μm, about 50nm to about 6 μm, about 50nm to about 5 μm, about 50nm to about 4 μm, about 50nm to about 3 μm, about 50nm to about 2 μm, about 50nm to about 1 μm, about 50nm to about 500nm, about 50nm to about 100nm, about 100nm to about 50 μm, about 100nm to about 45 μm, about 100nm to about 40 μm, about 100nm to about 35 μm, about 30 nm to about 20 μm, about 50nm to about 100 μm, about 100nm to about 16 μm, about 100nm to about 14 μm, about 100nm to about 12 μm, about 100nm to about 10 μm, about 100nm to about 8 μm, about 100nm to about 6 μm, about 100nm to about 5 μm, about 100nm to about 4 μm, about 100nm to about 3 μm, about 100nm to about 2 μm, about 100nm to about 1 μm, about 100nm to about 500nm, about 500nm to about 50 μm, about 500nm to about 45 μm, about 500nm to about 40 μm, about 500nm to about 35 μm, about 500nm to about 30 μm, about 500nm to about 25 μm, about 500nm to about 20 μm, about 500nm to about 18 μm, about 500nm to about 16 μm, about 500nm to about 14 μm, about 500nm to about 12 μm, about 500nm to about 10 μm, about 500nm to about 8 μm, about 500nm to about 5 μm, about 500nm to about 3 μm, about 500nm to about 2 μm, about 500nm to about 1 μm, about 1 μm to about 50 μm, about 1 μm to about 45 μm, about 1 μm to about 40 μm, about 1 μm to about 35 μm, about 1 μm to about 30 μm, about 1 μm to about 25 μm, about 1 μm to about 20 μm, about 1 μm to about 18 μm, about 1 μm to about 16 μm, about 1 μm to about 14 μm, about 1 μm to about 12 μm, about 1 μm to about 10 μm, about 1 μm to about 8 μm, about 1 μm to about 6 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, about 1 μm to about 2 μm, about 2 μm to about 50 μm, about 2 μm to about 45 μm, about 2 μm to about 40 μm, about 2 μm to about 30 μm, about 1 μm to about 25 μm, about 2 μm to about 20 μm, about 2 μm to about 18 μm, about 2 μm to about 16 μm, about 2 μm to about 14 μm, about 2 μm to about 12 μm, about 2 μm to about 10 μm, about 2 μm to about 8 μm, about 2 μm to about 6 μm, about 2 μm to about 5 μm, about 2 μm to about 4 μm, about 2 μm to about 3 μm, about 3 μm to about 50 μm, about 3 μm to about 45 μm, about 3 μm to about 40 μm, about 3 μm to about 35 μm, about 3 μm to about 30 μm, about 3 μm to about 25 μm, about 3 μm to about 20 μm, about 3 μm to about 18 μm, about 3 μm to about 16 μm, about 3 μm to about 14 μm, about 3 μm to about 12 μm, about 3 μm to about 10 μm, about 3 μm to about 6 μm, about 3 μm to about 4 μm, about 4 μm to about 50 μm, about 4 μm to about 45 μm, about 4 μm to about 40 μm, about 4 μm to about 35 μm, about 4 μm to about 30 μm, about 4 μm to about 25 μm, about 4 μm to about 20 μm, about 4 μm to about 18 μm, about 4 μm to about 16 μm, about 4 μm to about 14 μm, about 4 μm to about 12 μm, about 4 μm to about 10 μm, about 4 μm to about 8 μm, about 4 μm to about 6 μm, about 4 μm to about 5 μm, about 5 μm to about 50 μm, about 5 μm to about 45 μm, about 5 μm to about 40 μm, about 5 μm to about 35 μm, about 5 μm to about 30 μm, about 5 μm to about 25 μm, about 5 μm to about 20 μm, about 14 μm to about 16 μm, about 5 μm to about 12 μm, about 5 μm to about 10 μm, about 5 μm to about 8 μm, about 5 μm to about 6 μm, about 6 μm to about 50 μm, about 6 μm to about 45 μm, about 6 μm to about 40 μm, about 6 μm to about 35 μm, about 6 μm to about 30 μm, about 6 μm to about 25 μm, about 6 μm to about 20 μm, about 6 μm to about 18 μm, about 6 μm to about 16 μm, about 6 μm to about 14 μm, about 6 μm to about 12 μm, about 6 μm to about 10 μm, about 6 μm to about 8 μm, about 8 μm to about 50 μm, about 8 μm to about 45 μm, about 8 μm to about 40 μm, about 8 μm to about 35 μm, about 8 μm to about 30 μm, about 8 μm to about 8 μm, about 8 μm to about 16 μm, about 8 μm to about 14 μm, about 8 μm to about 12 μm, about 8 μm to about 10 μm, about 10 μm to about 50 μm, about 10 μm to about 45 μm, about 10 μm to about 40 μm, about 10 μm to about 35 μm, about 10 μm to about 30 μm, about 10 μm to about 25 μm, about 10 μm to about 20 μm, about 10 μm to about 18 μm, about 10 μm to about 16 μm, about 10 μm to about 14 μm, about 10 μm to about 12 μm, about 12 μm to about 50 μm, about 12 μm to about 45 μm, about 12 μm to about 40 μm, about 12 μm to about 35 μm, about 12 μm to about 30 μm, about 12 μm to about 25 μm, about 12 μm to about 20 μm, about 12 μm to about 12 μm, about 18 μm to about 12 μm, about 14 μm to about 14 μm, about 14 μm to about 40 μm, about 14 μm to about 35 μm, about 14 μm to about 30 μm, about 14 μm to about 25 μm, about 14 μm to about 20 μm, about 14 μm to about 18 μm, about 14 μm to about 16 μm, about 16 μm to about 50 μm, about 16 μm to about 45 μm, about 16 μm to about 40 μm, about 16 μm to about 35 μm, about 16 μm to about 30 μm, about 16 μm to about 25 μm, about 16 μm to about 20 μm, about 16 μm to about 18 μm, about 18 μm to about 50 μm, about 18 μm to about 45 μm, about 18 μm to about 40 μm, about 18 μm to about 35 μm, about 18 μm to about 30 μm, about 18 μm to about 25 μm, about 18 μm to about 20 μm, about 20 μm to about 20 μm, about 20 μm to about 30 μm, about 20 μm to about 25 μm, about 25 μm to about 50 μm, about 25 μm to about 45 μm, about 25 μm to about 40 μm, about 25 μm to about 35 μm, about 25 μm to about 30 μm, about 30 μm to about 50 μm, about 30 μm to about 45 μm, about 30 μm to about 40 μm, about 30 μm to about 35 μm, about 35 μm to about 50 μm, about 35 μm to about 45 μm, about 35 μm to about 40 μm, about 40 μm to about 50 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm. In some embodiments of any of the pods described herein, the pore size or average pore size of the material is: about 30 μm to about 70 μm, about 30 μm to about 68 μm, about 30 μm to about 66 μm, about 30 μm to about 64 μm, about 30 μm to about 62 μm, about 30 μm to about 60 μm, about 30 μm to about 58 μm, about 30 μm to about 56 μm, about 30 μm to about 54 μm, about 30 μm to about 52 μm, about 30 μm to about 50 μm, about 30 μm to about 48 μm, about 30 μm to about 46 μm, about 30 μm to about 44 μm, about 30 μm to about 42 μm, about 30 μm to about 40 μm, about 30 μm to about 38 μm, about 30 μm to about 36 μm, about 30 μm to about 34 μm, about 30 μm to about 32 μm, about 32 μm to about 70 μm, about 32 μm to about 68 μm, about 66 μm to about 32 μm, about 32 μm to about 32 μm, about 32 μm to about 58 μm, about 32 μm to about 56 μm, about 32 μm to about 54 μm, about 32 μm to about 52 μm, about 32 μm to about 50 μm, about 32 μm to about 48 μm, about 32 μm to about 46 μm, about 32 μm to about 44 μm, about 32 μm to about 42 μm, about 32 μm to about 40 μm, about 32 μm to about 38 μm, about 32 μm to about 36 μm, about 32 μm to about 34 μm, about 34 μm to about 70 μm, about 34 μm to about 68 μm, about 34 μm to about 66 μm, about 34 μm to about 64 μm, about 34 μm to about 62 μm, about 34 μm to about 60 μm, about 34 μm to about 58 μm, about 34 μm to about 56 μm, about 34 μm to about 54 μm, about 34 μm to about 48 μm, about 34 μm to about 44 μm, about 34 μm to about 42 μm, about 34 μm to about 40 μm, about 34 μm to about 38 μm, about 34 μm to about 36 μm, about 36 μm to about 70 μm, about 36 μm to about 68 μm, about 36 μm to about 66 μm, about 36 μm to about 64 μm, about 36 μm to about 62 μm, about 36 μm to about 60 μm, about 36 μm to about 58 μm, about 36 μm to about 56 μm, about 36 μm to about 54 μm, about 36 μm to about 52 μm, about 36 μm to about 50 μm, about 36 μm to about 48 μm, about 36 μm to about 46 μm, about 36 μm to about 44 μm, about 36 μm to about 42 μm, about 36 μm to about 40 μm, about 36 μm to about 38 μm, about 38 μm to about 38 μm, about 38 μm to about 62 μm, about 38 μm to about 60 μm, about 38 μm to about 58 μm, about 38 μm to about 56 μm, about 38 μm to about 54 μm, about 38 μm to about 52 μm, about 38 μm to about 50 μm, about 38 μm to about 48 μm, about 38 μm to about 46 μm, about 38 μm to about 44 μm, about 38 μm to about 42 μm, about 38 μm to about 40 μm, about 40 μm to about 70 μm, about 40 μm to about 68 μm, about 40 μm to about 66 μm, about 40 μm to about 64 μm, about 40 μm to about 62 μm, about 40 μm to about 60 μm, about 40 μm to about 58 μm, about 40 μm to about 56 μm, about 40 μm to about 54 μm, about 40 μm to about 52 μm, about 40 μm to about 40 μm, about 40 μm to about 46 μm, about 40 μm to about 46 μm, about 40 μm to about 42 μm, about 42 μm to about 70 μm, about 42 μm to about 68 μm, about 42 μm to about 66 μm, about 42 μm to about 64 μm, about 42 μm to about 62 μm, about 42 μm to about 60 μm, about 42 μm to about 58 μm, about 42 μm to about 56 μm, about 42 μm to about 54 μm, about 42 μm to about 52 μm, about 42 μm to about 50 μm, about 42 μm to about 48 μm, about 42 μm to about 46 μm, about 42 μm to about 44 μm, about 44 μm to about 70 μm, about 44 μm to about 68 μm, about 44 μm to about 66 μm, about 44 μm to about 64 μm, about 44 μm to about 62 μm, about 44 μm to about 60 μm, about 44 μm to about 58 μm, about 44 μm to about 44 μm, about 44 μm to about 52 μm, about 42 μm to about 44 μm, about 44 μm to about 48 μm, about 44 μm to about 46 μm, about 46 μm to about 70 μm, about 46 μm to about 68 μm, about 46 μm to about 66 μm, about 46 μm to about 64 μm, about 46 μm to about 62 μm, about 46 μm to about 60 μm, about 46 μm to about 58 μm, about 46 μm to about 56 μm, about 46 μm to about 54 μm, about 46 μm to about 52 μm, about 46 μm to about 50 μm, about 46 μm to about 48 μm, about 48 μm to about 70 μm, about 48 μm to about 68 μm, about 48 μm to about 66 μm, about 48 μm to about 64 μm, about 48 μm to about 62 μm, about 48 μm to about 60 μm, about 48 μm to about 58 μm, about 48 μm to about 56 μm, about 48 μm to about 50 μm, about 50 μm to about 68 μm, about 50 μm to about 66 μm, about 50 μm to about 64 μm, about 50 μm to about 62 μm, about 50 μm to about 60 μm, about 50 μm to about 58 μm, about 50 μm to about 56 μm, about 50 μm to about 54 μm, about 50 μm to about 52 μm, about 52 μm to about 70 μm, about 52 μm to about 68 μm, about 52 μm to about 66 μm, about 52 μm to about 64 μm, about 52 μm to about 62 μm, about 52 μm to about 60 μm, about 52 μm to about 58 μm, about 52 μm to about 56 μm, about 52 μm to about 54 μm, about 54 μm to about 70 μm, about 54 μm to about 68 μm, about 54 μm to about 66 μm, about 54 μm to about 64 μm, about 54 μm to about 54 μm, about 54 μm to about 58 μm, about 56 μm to about 70 μm, about 56 μm to about 68 μm, about 56 μm to about 66 μm, about 56 μm to about 64 μm, about 56 μm to about 62 μm, about 56 μm to about 60 μm, about 56 μm to about 58 μm, about 58 μm to about 70 μm, about 58 μm to about 68 μm, about 58 μm to about 66 μm, about 58 μm to about 64 μm, about 58 μm to about 62 μm, about 58 μm to about 60 μm, about 60 μm to about 70 μm, about 60 μm to about 68 μm, about 60 μm to about 66 μm, about 60 μm to about 64 μm, about 60 μm to about 62 μm, about 62 μm to about 70 μm, about 62 μm to about 68 μm, about 62 μm to about 66 μm, about 62 μm to about 64 μm, about 64 μm to about 64 μm, about 62 μm to about 70 μm, about 62 μm to about 64 μm, about 64 μm to about 64 μm, about 66 μm to about 68 μm, or about 68 μm to about 70 μm.

Area covered by semi-porous/porous material

In some embodiments of the pod, one or more of the walls, top end, and bottom end comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) area covered by a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the interior of the pod. In some embodiments of these pods, the pod comprises: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas in the walls covered by a semi-porous or porous material (allowing liquid to reversibly flow from the outside of the pod into the inside of the pod), and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas in the bottom end covered by a semi-porous or porous material (allowing liquid to reversibly flow from the outside of the pod into the inside of the pod), and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas in the top end covered by a semi-porous or porous material (allowing liquid to reversibly flow from the outside of the pod into the inside of the pod).

In some examples of the pods described herein, the wall includes at least one area covered by the material. In some examples of the pods described herein, the bottom end and the wall each include at least one area covered by the material. In some examples, the tip includes at least one region covered by a material. In some examples, the top end and the bottom end each include at least one region covered by the material.

In some embodiments of the pods described herein, the total number of areas covered by the semi-porous or porous material (allowing liquid to reversibly flow from the exterior of the pod into the interior of the pod) in one or more of the walls, top end, and bottom end is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments of the pods described herein, the total number of areas covered by the semi-porous or porous material (allowing liquid to reversibly flow from the exterior of the pod to the interior of the pod) in one or more of the walls, top and bottom ends is 1 to 30, 1 to 28, 1 to 26, 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, 2 to 30, 2 to 28, 2 to 26, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 2 or 3, 3 to 30, 3 to 28, 3 to 26, 3 to 24, 3 to 22, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 30, 3 to 28, 3 to 4, or 4 to 30, 4 to 26, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 30, 6 to 28, 6 to 26, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 30, 8 to 28, 8 to 26, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 10, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 30, 14 to 14, 16 to 14, 16 to 22, 16 to 20, 16 to 18, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 22 to 22, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 30, 24 to 28, 24 to 26, 26 to 30, 26 to 28, or 28 to 30.

In some embodiments of any of the pods described herein, one or more of the at least one area covered by the semi-porous or porous material is circular, oval, square, rectangular, elongated (e.g., elongated lines or rectangles), star-shaped, saw-tooth, or serpentine.

In some embodiments, one or more (e.g., each) of the at least one region covered by the semi-porous or porous material has a surface area of: about 0.5mm²To about 60cm²About 0.5mm²To about 58cm²About 0.5mm²To about 56cm²About 0.5mm²To about 54cm²About 0.5mm²To about 52cm²About 0.5mm²To about 50cm²About 0.5mm²To about 48cm²About 0.5mm²To about 46cm²About 0.5mm²To about 44cm²About 0.5mm²To about 42cm²About 0.5mm²To about 40cm²About 0.5mm²To about 38cm²About 0.5mm²To about 36cm²About 0.5mm²To about 34cm²About 0.5mm²To about 32cm²，0.5mm²To about 30cm²About 0.5mm²To about 28cm²About 0.5mm²To about 26cm²About 0.5mm²To about 24cm²About 0.5mm²To about 22cm²About 0.5mm²To about 20cm²About 0.5mm²To about 18cm²About 0.5mm²To about 16cm²About 0.5mm²To about 14cm²About 0.5mm²To about 12cm²About 0.5mm²To about 10cm²About 0.5mm²To about 8cm²About 0.5mm²To about 6cm²About 0.5mm²To about 5cm²About 0.5mm²To about 4cm²About 0.5mm²To about 3cm²About 0.5mm²To about 2cm²About 0.5mm²To about 1cm²About 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18cm²To about 38cm²About 18cm²To about 36cm²About 18cm²To about 34cm²About 18cm²To about 32cm²About 18cm²To about 30cm²About 18cm²To about 28cm²About 18cm²To about 26cm²About 18cm²To about 24cm²About 18cm²To about 22cm²About 18cm²To about 20cm²About 20cm²To about 60cm²About 20cm²To about 58cm²About 20cm²To about 56cm²About 20cm²To about 54cm²About 20cm²To about 52cm²About 20cm²To about 50cm²About 20cm²To about 48cm²About 20cm²To about 46cm²About 20cm²To about 44cm²About 20cm²To about 42cm²About 20cm²To about 40cm²About 20cm²To about 38cm²About 20cm²To about 36cm²About 20cm²To about 34cm²About 20cm²To about 32cm²About 20cm²To about 30cm²About 20cm²To about 28cm²About 20cm²To about 26cm²About 20cm²To about 24cm²About 20cm²To about 22cm²About 22cm²To about 60cm²About 22cm²To about 58cm²About 22cm²To about 56cm²About 22cm²To about 54cm²About 22cm²To about 52cm²About 22cm²To about 50cm²About 22cm²To about 48cm²About 22cm²To about 46cm²About 22cm²To about 44cm²About 22cm²To about 42cm²About 22cm²To about 40cm²About 22cm²To about 38cm²About 22cm²To about 36cm²About 22cm²To about 34cm²About 22cm²To about 32cm²About 22cm²To about 30cm²About 22cm²To about 28cm²About 22cm²To about 26cm²About 22cm²To about 24cm²About 24cm²To about 60cm²About 24cm²To about 58cm²About 24cm²To about 56cm²About 24cm²To about 54cm²About 24cm²To about 52cm²About 24cm²To about 50cm²About 24cm²To about 48cm²About 24cm²To about 46cm²About 24cm²To about 44cm²About 24cm²To about 42cm²About 24cm²To about 40cm²About 24cm²To about 38cm²About 24cm²To about 36cm²About 24cm²To about 34cm²About 24cm²To about 32cm²About 24cm²To about 30cm²About 24cm²To about 28cm²About 24cm²To about 26cm²About 26cm²To about 60cm²About 26cm²To about 58cm²About 26cm²To about 56cm²About 26cm²To about 54cm²About 26cm²To about 52cm²About 26cm²To about 50cm²About 26cm²To about 48cm²About 26cm²To about 46cm²About 26cm²To about 44cm²About 26cm²To about 42cm²About 26cm²To about 40cm²About 26cm²To about 38cm²About 26cm²To about 36cm²About 26cm²To about 34cm²About 26cm²To about 32cm²About 26cm²To about 30cm²About 26cm²To about 28cm²About 28cm²To about 60cm²About 28cm²To about 58cm²About 28cm²To about 56cm²About 28cm²To about 54cm²About 28cm²To about 52cm²About 28cm²To about 50cm²About 28cm²To about 48cm²About 28cm²To about 46cm²About 28cm²To about 44cm²About 28cm²To about 42cm²About 28cm²To about 40cm²About 28cm²To about 38cm²About 28cm²To about 36cm²About 28cm²To about 34cm²About 28cm²To about 32cm²About 28cm²To about 30cm²About 30cm²To about 60cm²About 30cm²To about 58cm²About 30cm²To about 56cm²About 30cm²To about 54cm²About 30cm²To about 52cm²About 30cm²To about 50cm²About 30cm²To about 48cm²About 30cm²To about 46cm²About 30cm²To about 44cm²About 30cm²To about 42cm²About 30cm²To about 40cm²About 30cm²To about 38cm²About 30cm²To about 36cm²About 30cm²To about 34cm²About 30cm²To about 32cm²About 32cm²To about 60cm²About 32cm²To about 58cm²About 32cm²To about 56cm²About 32cm²To about 54cm²About 32cm²To about 52cm²About 32cm²To about 50cm²About 32cm²To about 48cm²About 32cm²To about 46cm²About 32cm²To about 44cm²About 32cm²To about 42cm²About 32cm²To about 40cm²About 32cm²To about 38cm²About 32cm²To about 36cm²About 32cm²To about 34cm²About 34cm²To about 60cm²About 34cm²To about 58cm²About 34cm²To about 56cm²About 34cm²To about 54cm²About 34cm²To about 52cm²About 34cm²To about 50cm²About 34cm²To about 48cm²About 34cm²To about 46cm²About 34cm²To about 44cm²About 34cm²To about 42cm²About 34cm²To about 40cm²About 34cm²To about 38cm²About 34cm²To about 36cm²About 36cm²To about 60cm²About 36cm²To about 58cm²About 36cm²To about 56cm²About 36cm²To about 54cm²About 36cm²To about 52cm²About 36cm²To about 50cm²About 36cm²To about 48cm²About 36cm²To about 46cm²About 36cm²To about 44cm²About 36cm²To about 42cm²About 36cm²To about 40cm²About 36cm²To about 38cm²About 38cm²To about 60cm²About 38cm²To about 58cm²About 38cm²To about 56cm²About 38cm²To about 54cm²About 38cm²To about 52cm²About 38cm²To about 50cm²About 38cm²To about 48cm²About 38cm²To about 46cm²About 38cm²To about 44cm²About 38cm²To about 42cm²About 38cm²To about 40cm²About 40cm²To about 60cm²About 40cm²To about 58cm²About 40cm²To about 56cm²About 40cm²To about 54cm²About 40cm²To about 52cm²About 40cm²To about 50cm²About 40cm²To about 48cm²About 40cm²To about 46cm²About 40cm²To about 44cm²About 40cm²To about 42cm²About 42cm²To about 60cm²About 42cm²To about 58cm²About 42cm²To about 56cm²About 42cm²To about 54cm²About 42cm²To about 52cm²About 42cm²To about 50cm²About 42cm²To about 48cm²About 42cm²To about 46cm²About 42cm²To about 44cm²About 44cm²To about 60cm²About 44cm²To about 58cm²About 44cm²To about 56cm²About 44cm²To about 54cm²About 44cm²To about 52cm²About 44cm²To about 50cm²About 44cm²To about 48cm²About 44cm²To about 46cm²About 46cm²To about 60cm²About 46cm²To about 58cm²About 46cm²To about 56cm²About 46cm²To about 54cm²About 46cm²To about 52cm²About 46cm²To about 50cm²About 46cm²To about 48cm²About 48cm²To about 60cm²About 48cm²To about 58cm²About 48cm²To about 56cm²About 48cm²To about 54cm²About 48cm²To about 52cm²About 48cm²To about 50cm²About 50cm²To about 60cm²About 50cm²To about 58cm²About 50cm²To about 56cm²About 50cm²To about 54cm²About 50cm²To about 52cm²About 52cm²To about 60cm²About 52cm²To about 58cm²About 52cm²To about 56cm²About 52cm²To about 54cm²About 54cm²To about 60cm²About 54cm²To about 58cm²About 54cm²To about 56cm²About 56cm²To about 60cm²About 56cm²To about 58cm²Or about 58cm²To about 60cm²。

In some embodiments of any of the pods described herein, the sum of the surface areas of the at least one area covered by the semi-porous or porous material is: about 0.5mm²To about 150cm²About 0.5mm²To about 140cm²About 0.5mm²To about 130cm²About 0.5mm²To about 120cm²About 0.5mm²To about 110cm²About 0.5mm²To about 100cm²About 0.5mm²To about 90cm²About 0.5mm²To about 80cm²About 0.5mm²To about 70cm²About 0.5mm²To about 60cm²About 0.5mm²To about 50cm²About 0.5mm²To about 40cm²About 0.5mm²To about 30cm²About 0.5mm²To about 20cm²About 0.5mm²To about 18cm²About 0.5mm²To about 16cm²About 0.5mm²To about 14cm²About 0.5mm²To about 12cm²About 0.5mm²To about 10cm²About 0.5mm²To about 8cm²About 0.5mm²To about 6cm²About 0.5mm²To about 5cm²About 0.5mm²To about 4cm²About 0.5mm²To about 3cm²About 0.5mm²To about 2cm²About 0.5mm²To about 1cm²About 0.5mm²To about 5mm²About 5mm²To about 150cm²About 5mm²To about 140cm²About 5mm²To about 130cm²About 5mm²To about 120cm²About 5mm²To about 110cm²About 5mm²To about 100cm²About 5mm²To about 90cm²About 5mm²To about 80cm²About 5mm²To about 70cm²About 5mm²To about 60cm²About 5mm²To about 50cm²About 5mm²To about 40cm²About 5mm²To about 30cm²About 5mm²To about 20cm²About 5mm²To about 18cm²About 5mm²To about 16cm²About 5mm²To about 14cm²About 5mm²To about 12cm²About 5mm²To about 10cm²About 5mm²To about 8cm²About 5mm²To about 6cm²About 5mm²To about 5cm²About 5mm²To about 4cm²About 5mm²To about 3cm²About 5mm²To about 2cm²About 5mm²To about 1cm²About 1cm²To about 150cm²About 1cm²To about 140cm²About 1cm²To about 130cm²About 1cm²To about 120cm²About 1cm²To about 110cm²About 1cm²To about 100cm²About 1cm²To about 90cm²About 1cm²To about 80cm²About 1cm²To about 70cm²About 1cm²To about 60cm²About 1cm²To about 50cm²About 1cm²To about 40cm²About 1cm²To about 30cm²About 1cm²To about 20cm²About 1cm²To about 18cm²About 1cm²To about 16cm²About 1cm²To about 14cm²About 1cm²To about 12cm²About 1cm²To about 10cm²About 1cm²To about 8cm²About 1cm²To about 6cm²About 1cm²To about 5cm²About 1cm²To about 4cm²About 1cm²To about 3cm²About 1cm²To about 2cm²About 2cm²To about 150cm²About 2cm²To about 140cm²About 2cm²To about 130cm²About 2cm²To about 120cm²About 2cm²To about 110cm²About 2cm²To about 100cm²About 2cm²To about 90cm²About 2cm²To about 80cm²About 2cm²To about 70cm²About 2cm²To about 60cm²About 2cm²To about 50cm²About 2cm²To about 40cm²About 2cm²To about 30cm²About 2cm²To about 20cm²About 2cm²To about 18cm²About 2cm²To about 16cm²About 2cm²To about 14cm²About 2cm²To about 12cm²About 2cm²To about 10cm²About 2cm²To about 8cm²About 2cm²To about 6cm²About 2cm²To about 5cm²About 2cm²To about 4cm²About 2cm²To about 3cm²About 3cm²To about 150cm²About 3cm²To about 140cm²About 3cm²To about 130cm²About 3cm²To about 120cm²About 3cm²To about 110cm²About 3cm²To about 100cm²About 3cm²To about 90cm²About 3cm²To about 80cm²About 3cm²To about 70cm²About 3cm²To about 60cm²About 3cm²To about 50cm²About 3cm²To about 40cm²About 3cm²To about 30cm²About 3cm²To about 20cm²About 3cm²To about 18cm²About 3cm²To about 16cm²About 3cm²To about 14cm²About 3cm²To about 12cm²About 3cm²To about 10cm²About 3cm²To about 8cm²About 3cm²To about 6cm²About 3cm²To about 5cm²About 3cm²To about 4cm²About 4cm²To about 150cm²About 4cm²To about 140cm²About 4cm²To about 130cm²About 4cm²To about 120cm²About 4cm²To about 110cm²About 4cm²To about 100cm²About 4cm²To about 90cm²About 4cm²To about 80cm²About 4cm²To about 70cm²About 4cm²To about 60cm²About 4cm²To about 50cm²About 4cm²To about 40cm²About 4cm²To about 30cm²About 4cm²To about 20cm²About 4cm²To about 18cm²About 4cm²To about 16cm²About 4cm²To about 14cm²About 4cm²To about 12cm²About 4cm²To about 10cm²About 4cm²To about 8cm²About 4cm²To about 6cm²About 4cm²To about 5cm²About 5cm²To about 150cm²About 5cm²To about 140cm²About 5cm²To about 130cm²About 5cm²To about 120cm²About 5cm²To about 110cm²About 5cm²To about 100cm²About 5cm²To about 90cm²About 5cm²To about 80cm²About 5cm²To about 70cm²About 5cm²To about 60cm²About 5cm²To about 50cm²About 5cm²To about 40cm²About 5cm²To about 30cm²About 5cm²To about 20cm²About 5cm²To about 18cm²About 5cm²To about 16cm²About 5cm²To about 14cm²About 5cm²To about 12cm²About 5cm²To about 10cm²About 5cm²To about 8cm²About 5cm²To about 6cm²About 6cm²To about 150cm²About 6cm²To about 140cm²About 6cm²To about 130cm²About 6cm²To about 120cm²About 6cm²To about 110cm²About 6cm²To about 100cm²About 6cm²To about 90cm²About 6cm²To about 80cm²About 6cm²To about 70cm²About 6cm²To about 60cm²About 6cm²To about 50cm²About 6cm²To about 40cm²About 6cm²To about 30cm²About 6cm²To about 20cm²About 6cm²To about 18cm²About 6cm²To about 16cm²About 6cm²To about 14cm²About 6cm²To about 12cm²About 6cm²To about 10cm²About 6cm²To about 8cm²About 8cm²To about 150cm²About 8cm²To about 140cm²About 8cm²To about 130cm²About 8cm²To about 120cm²About 8cm²To about 110cm²About 8cm²To about 100cm²About 8cm²To about 90cm²About 8cm²To about 80cm²About 8cm²To about 70cm²About 8cm²To about 60cm²About 8cm²To about 50cm²About 8cm²To about 40cm²About 8cm²To about 30cm²About 8cm²To about 20cm²About 8cm²To about 18cm²About 8cm²To about 16cm²About 8cm²To about 14cm²About 8cm²To about 12cm²About 8cm²To about 10cm²About 10cm²To about 150cm²About 10cm²To about 140cm²About 10cm²To about 130cm²About 10cm²To about 120cm²About 10cm²To about 110cm²About 10cm²To about 100cm²About 10cm²To about 90cm²About 10cm²To about 80cm²About 10cm²To about 70cm²About 10cm²To about 60cm²About 10cm²To about 50cm²About 10cm²To about 40cm²About 10cm²To about 30cm²About 10cm²To about 20cm²About 10cm²To about 18cm²About 10cm²To about 16cm²About 10cm²To about 14cm²About 10cm²To about 12cm²About 12cm²To about 150cm²About 12cm²To about 140cm²About 12cm²To about 130cm²About 12cm²To about 120cm²About 12cm²To about 110cm²About 12cm²To about 100cm²About 12cm²To about 90cm²About 12cm²To about 80cm²About 12cm²To about 70cm²About 12cm²To about 60cm²About 12cm²To about 50cm²About 12cm²To about 40cm²About 12cm²To about 30cm²About 12cm²To about 20cm²About 12cm²To about 18cm²About 12cm²To about 16cm²About 12cm²To about 14cm²About 14cm²To about 150cm²About 14cm²To about 140cm²About 14cm²To about 130cm²About 14cm²To about 120cm²About 14cm²To about 110cm²About 14cm²To about 100cm²About 14cm²To about 90cm²About 14cm²To about 80cm²About 14cm²To about 70cm²About 14cm²To about 60cm²About 14cm²To about 50cm²About 14cm²To about 40cm²About 14cm²To about 30cm²About 14cm²To about 20cm²About 14cm²To about 18cm²About 14cm²To about 16cm²About 16cm²To about 150cm²About 16cm²To about 140cm²About 16cm²To about 130cm²About 16cm²To about 120cm²About 16cm²To about 110cm²About 16cm²To about 100cm²About 16cm²To about 90cm²About 16cm²To about 80cm²About 16cm²To about 70cm²About 16cm²To about 60cm²About 16cm²To about 50cm²About 16cm²To about 40cm²About 16cm²To about 30cm²About 16cm²To about 20cm²About 16cm²To about 18cm²About 18cm²To about 150cm²About 18cm²To about 140cm²About 18cm²To about 130cm²About 18cm²To about 120cm²About 18cm²To about 110cm²About 18cm²To about 100cm²About 18cm²To about 90cm²About 18cm²To about 80cm²About 18cm²To about 70cm²About 18cm²To about 60cm²About 18cm²To about 50cm²About 18cm²To about 40cm²About 18cm²To about 30cm²About 18cm²To about 20cm²About 20cm²To about 150cm²About 20cm²To about 140cm²About 20cm²To about 130cm²About 20cm²To about 120cm²About 20cm²To about 110cm²About 20cm²To about 100cm²About 20cm²To about 90cm²About 20cm²To about 80cm²About 20cm²To about 70cm²About 20cm²To about 60cm²About 20cm²To about 50cm²About 20cm²To about 40cm²About 20cm²To about 30cm²About 30cm²To about 150cm²About 30cm²To about 140cm²About 30cm²To about 130cm²About 30cm²To about 120cm²About 30cm²To about 110cm²About 30cm²To about 100cm²About 30cm²To about 90cm²About 30cm²To about 80cm²About 30cm²To about 70cm²About 30cm²To about 60cm²About 30cm²To about 50cm²About 30cm²To about 40cm²About 40cm²To about 150cm²About 40cm²To about 140cm²About 40cm²To about 130cm²About 40cm²To about 120cm²About 40cm²To about 110cm²About 40cm²To about 100cm²About 40cm²To about 90cm²About 40cm²To about 80cm²About 40cm²To about 70cm²About 40cm²To about 60cm²About 40cm²To about 50cm²About 50cm²To about 150cm²About 50cm²To about 140cm²About 50cm²To about 130cm²About 50cm²To about 120cm²About 50cm²To about 110cm²About 50cm²To about 100cm²About 50cm²To about 90cm²About 50cm²To about 80cm²About 50cm²To about 70cm²About 50cm²To about 60cm²About 60cm²To about 150cm²About 60cm²To about 140cm²About 60cm²To about 130cm²About 60cm²To about 120cm²About 60cm²To about 110cm²About 60cm²To about 100cm²About 60cm²To about 90cm²About 60cm²To about 80cm²About 60cm²To about 70cm²About 70cm²To about 150cm²About 70cm²To about 140cm²About 70cm²To about 130cm²About 70cm²To about 120cm²About 70cm²To about 110cm²About 70cm²To about 100cm²About 70cm²To about 90cm²About 70cm²To about 80cm²About 80cm²To about 150cm²About 80cm²To about 140cm²About 80cm²To about 130cm²About 80cm²To about 120cm²About 80cm²To about 110cm²About 80cm²To about 100cm²About 80cm²To about 90cm²About 90cm²To about 150cm²About 90cm²To about 140cm²About 90cm²To about 130cm²About 90cm²To about 120cm²About 90cm²To about 110cm²About 90cm²To about 100cm²About 100cm²To about 150cm²About 100cm²To about 140cm²About 100cm²To about 130cm²About 100cm²To about 120cm²About 100cm²To about 110cm²About 110cm²To about 150cm²About 110cm²To about 140cm²About 110cm²To about 130cm²About 110cm²To about 120cm²About 120cm²To about 150cm²About 120cm²To about 140cm²About 120cm²To about 130cm²About 130cm²To about 150cm²About 130cm²To about 140cm²Or about 140cm²To about 150cm²。

In some embodiments of any of the pods described herein, wherein the wall includes at least one area covered by a semi-porous or porous material that allows liquid to reversibly flow from an exterior of the pod into an interior of the pod, the percentage of the total surface area of the wall represented by the at least one area covered by the semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod into the interior of the pod is: about 0.1% to about 95%, about 0.1% to about 90%, about 0.1% to about 85%, about 0.1% to about 80%, about 0.1% to about 75%, about 0.1% to about 70%, about 0.1% to about 65%, about 0.1% to about 60%, about 0.1% to about 55%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 18%, about 0.1% to about 16%, about 0.1% to about 14%, about 0.1% to about 12%, about 0.1% to about 10%, about 0.1% to about 8%, about 0.1% to about 6%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 2%, about 0.1% to about 1% to about 2%, about 0.1% to about 2%, about 1% to about 25%, about 0.1% to about 20%, about 0.2% to about 80%, about 0.2% to about 75%, about 0.2% to about 70%, about 0.2% to about 65%, about 0.2% to about 60%, about 0.2% to about 55%, about 0.2% to about 50%, about 0.2% to about 45%, about 0.2% to about 40%, about 0.2% to about 35%, about 0.2% to about 30%, about 0.2% to about 25%, about 0.2% to about 20%, about 0.2% to about 18%, about 0.2% to about 16%, about 0.2% to about 14%, about 0.2% to about 12%, about 0.2% to about 10%, about 0.2% to about 8%, about 0.2% to about 6%, about 0.2% to about 5%, about 0.2% to about 4%, about 0.2% to about 3%, about 0.2% to about 2%, about 0.2% to about 8%, about 0.2% to about 6%, about 0.2% to about 5%, about 5% to about 5%, about 5.5% to about 5%, about 0.2% to about 5%, about 0.5% to about 60%, about 0.5% to about 55%, about 0.5% to about 50%, about 0.5% to about 45%, about 0.5% to about 40%, about 0.5% to about 35%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 18%, about 0.5% to about 16%, about 0.5% to about 14%, about 0.5% to about 12%, about 0.5% to about 10%, about 0.5% to about 8%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 1%, about 1% to about 70%, about 1% to about 1%, about 1% to about 40%, about 1% to about 1%, about 1% to about 8%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 95%, about 2% to about 90%, about 2% to about 85%, about 2% to about 80%, about 2% to about 75%, about 2% to about 70%, about 2% to about 65%, about 2% to about 60%, about 2% to about 55%, about 2% to about 50%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 2% to about 18%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 95%, about 3% to about 90%, about 3% to about 85%, about 3% to about 80%, about 3% to about 75%, about 3% to about 70%, about 3% to about 65%, about 3% to about 60%, about 3% to about 55%, about 3% to about 50%, about 3% to about 45%, about 3% to about 40%, about 3% to about 35%, about 3% to about 30%, about 3% to about 25%, about 3% to about 20%, about 3% to about 18%, about 3% to about 16%, about 3% to about 14%, about 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 3% to about 4%, about 4% to about 95%, about 4% to about 90%, about 4% to about 85%, about 4% to about 80%, about 4% to about 75%, about 4% to about 70%, about 4% to about 65%, about 4% to about 60%, about 4% to about 55%, about 4% to about 50%, about 4% to about 45%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4% to about 20%, about 4% to about 18%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 18%, about 5% to about 16%, about 5% to about 14%, about 5% to about 12%, about 5% to about 10%, about 5% to about 8%, about 5% to about 6%, about 6% to about 95%, about 6% to about 90%, about 6% to about 85%, about 6% to about 80%, about 6% to about 75%, about 6% to about 70%, about 6% to about 65%, about 6% to about 60%, about 6% to about 55%, about 6% to about 50%, about 6% to about 45%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6% to about 18%, about 6% to about 14%, about 6% to about 16%, about 6% to about 12%, about 6% to about 10%, about 6% to about 8%, about 8% to about 95%, about 8% to about 90%, about 8% to about 85%, about 8% to about 80%, about 8% to about 75%, about 8% to about 70%, about 8% to about 65%, about 8% to about 60%, about 8% to about 55%, about 8% to about 50%, about 8% to about 45%, about 8% to about 40%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8% to about 14%, about 8% to about 12%, about 8% to about 10%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 95%, about 12% to about 90%, about 12% to about 85%, about 12% to about 80%, about 12% to about 75%, about 12% to about 70%, about 12% to about 65%, about 12% to about 60%, about 12% to about 55%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 12% to about 25%, about 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 95%, about 14% to about 90%, about 14% to about 85%, about 14% to about 80%, about 14% to about 75%, about 14% to about 70%, about 14% to about 65%, about 14% to about 60%, about 14% to about 55%, about 14% to about 50%, about 14% to about 45%, about 14% to about 40%, about 14% to about 35%, about 14% to about 30%, about 14% to about 25%, about 14% to about 20%, about 14% to about 18%, about 14% to about 16%, about 16% to about 95%, about 16% to about 90%, about 16% to about 85%, about 16% to about 80%, about 16% to about 75%, about 16% to about 70%, about 16% to about 65%, about 16% to about 60%, about 16% to about 55%, about 16% to about 50%, about 16% to about 45%, about 16% to about 40%, about 16% to about 35%, about 16% to about 30%, about 16% to about 25%, about 16% to about 20%, about 16% to about 18%, about 18% to about 18%, about 18% to about 90%, about 18% to about 85%, about 18% to about 80%, about 18% to about 75%, about 18% to about 70%, about 18% to about 65%, about 18% to about 60%, about 18% to about 55%, about 18% to about 50%, about 18% to about 45%, about 18% to about 40%, about 18% to about 35%, about 18% to about 30%, about 18% to about 25%, about 18% to about 20%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 95%, about 25% to about 25%, about 25% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 70% to about 75%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 85%, about 75% to about 80%, from about 85% to about 95%, from about 85% to about 90%, or from about 90% to about 95%.

In some embodiments of any of the pods described herein, wherein the bottom end includes at least one area covered by a semi-porous or porous material that allows liquid to reversibly flow from an exterior of the pod to an interior of the pod, the percentage of the total surface area of the bottom end represented by the at least one area covered by the semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod to the interior of the pod is: about 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 100%, about 2% to about 95%, about 2% to about 90%, about 2% to about 80%, about 2% to about 85%, about 2% to about 75%, about 2% to about 70%, about 2% to about 65%, about 2% to about 60%, about 2% to about 55%, about 2% to about 50%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 2% to about 18%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 100%, about 3% to about 95%, about 3% to about 90%, about 3% to about 85%, about 3% to about 80%, about 3% to about 75%, about 3% to about 70%, about 3% to about 65%, about 3% to about 60%, about 3% to about 55%, about 3% to about 50%, about 3% to about 45%, about 3% to about 40%, about 3% to about 35%, about 3% to about 30%, about 3% to about 25%, about 3% to about 20%, about 3% to about 18%, about 3% to about 16%, about 3% to about 14%, about 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 100%, about 4% to about 95%, about 4% to about 90%, about 4% to about 85%, about 4% to about 80%, about 4% to about 75%, about 4% to about 70%, about 4% to about 65%, about 4% to about 60%, about 4% to about 55%, about 4% to about 50%, about 4% to about 45%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4% to about 20%, about 18% to about 20%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 18%, about 5% to about 16%, about 5% to about 14%, about 5% to about 12%, about 5% to about 10%, about 5% to about 8%, about 5% to about 6%, about 6% to about 95%, about 100%, about 6% to about 90%, about 6% to about 85%, about 6% to about 80%, about 6% to about 75%, about 6% to about 70%, about 6% to about 65%, about 6% to about 60%, about 6% to about 55%, about 6% to about 50%, about 6% to about 45%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6% to about 18%, about 6% to about 16%, about 6% to about 14%, about 6% to about 12%, about 6% to about 10%, about 6% to about 8%, about 8% to about 100%, about 8% to about 95%, about 8% to about 90%, about 8% to about 85%, about 8% to about 80%, about 8% to about 75%, about 8% to about 70%, about 8% to about 65%, about 8% to about 60%, about 8% to about 55%, about 8% to about 45%, about 50% to about 45%, about 6% to about 10%, about 6% to about 8% to about 10%, about 8% to about 8%, about 8% to about 40%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8% to about 14%, about 8% to about 12%, about 8% to about 10%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 12%, about 100%, about 12% to about 90%, about 12% to about 85%, about 12% to about 80%, about 12% to about 75%, about 12% to about 70%, about 12% to about 65%, about 12% to about 60%, about 12% to about 55%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 12% to about 25%, about 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 100%, about 14% to about 95%, about 14% to about 90%, about 14% to about 85%, about 14% to about 80%, about 14% to about 75%, about 14% to about 70%, about 14% to about 65%, about 14% to about 60%, about 14% to about 55%, about 14% to about 50%, about 14% to about 45%, about 14% to about 40%, about 14% to about 35%, about 30% to about 30%, about 14% to about 25%, about 14% to about 20%, about 14% to about 18%, about 14% to about 16%, about 16% to about 100%, about 16% to about 95%, about 16% to about 90%, about 16% to about 85%, about 16% to about 80%, about 16% to about 75%, about 16% to about 70%, about 16% to about 65%, about 16% to about 60%, about 16% to about 55%, about 16% to about 50%, about 16% to about 45%, about 16% to about 40%, about 16% to about 35%, about 16% to about 30%, about 16% to about 25%, about 16% to about 20%, about 16% to about 18%, about 18% to about 100%, about 18% to about 95%, about 18% to about 90%, about 18% to about 85%, about 18% to about 80%, about 18% to about 75%, about 18% to about 70%, about 18% to about 65%, about 18% to about 60%, about 18% to about 55%, about 18% to about 50%, about 18% to about 45%, about 18% to about 40%, about 18% to about 35%, about 18% to about 30%, about 18% to about 25%, about 18% to about 20%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 100%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 100%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 55%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 100%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 100%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%; about 70% to about 100%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 100%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 100%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, about 90% to about 95%, or about 95% to about 100%.

In some embodiments of any of the pods described herein, wherein the top end includes at least one area covered by a semi-porous or porous material that allows liquid to reversibly flow from an exterior of the pod to an interior of the pod, the percentage of the total surface area of the top end represented by the at least one area covered by the semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod to the interior of the pod is: about 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 100%, about 2% to about 95%, about 2% to about 90%, about 2% to about 80%, about 2% to about 85%, about 2% to about 75%, about 2% to about 70%, about 2% to about 65%, about 2% to about 60%, about 2% to about 55%, about 2% to about 50%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 2% to about 18%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 100%, about 3% to about 95%, about 3% to about 90%, about 3% to about 85%, about 3% to about 80%, about 3% to about 75%, about 3% to about 70%, about 3% to about 65%, about 3% to about 60%, about 3% to about 55%, about 3% to about 50%, about 3% to about 45%, about 3% to about 40%, about 3% to about 35%, about 3% to about 30%, about 3% to about 25%, about 3% to about 20%, about 3% to about 18%, about 3% to about 16%, about 3% to about 14%, about 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 100%, about 4% to about 95%, about 4% to about 90%, about 4% to about 85%, about 4% to about 80%, about 4% to about 75%, about 4% to about 70%, about 4% to about 65%, about 4% to about 60%, about 4% to about 55%, about 4% to about 50%, about 4% to about 45%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4% to about 20%, about 18% to about 20%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 18%, about 5% to about 16%, about 5% to about 14%, about 5% to about 12%, about 5% to about 10%, about 5% to about 8%, about 5% to about 6%, about 6% to about 95%, about 100%, about 6% to about 90%, about 6% to about 85%, about 6% to about 80%, about 6% to about 75%, about 6% to about 70%, about 6% to about 65%, about 6% to about 60%, about 6% to about 55%, about 6% to about 50%, about 6% to about 45%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6% to about 18%, about 6% to about 16%, about 6% to about 14%, about 6% to about 12%, about 6% to about 10%, about 6% to about 8%, about 8% to about 100%, about 8% to about 95%, about 8% to about 90%, about 8% to about 85%, about 8% to about 80%, about 8% to about 75%, about 8% to about 70%, about 8% to about 65%, about 8% to about 60%, about 8% to about 55%, about 8% to about 45%, about 50% to about 45%, about 6% to about 10%, about 6% to about 8% to about 10%, about 8% to about 8%, about 8% to about 40%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8% to about 14%, about 8% to about 12%, about 8% to about 10%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 12%, about 100%, about 12% to about 90%, about 12% to about 85%, about 12% to about 80%, about 12% to about 75%, about 12% to about 70%, about 12% to about 65%, about 12% to about 60%, about 12% to about 55%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 12% to about 25%, about 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 100%, about 14% to about 95%, about 14% to about 90%, about 14% to about 85%, about 14% to about 80%, about 14% to about 75%, about 14% to about 70%, about 14% to about 65%, about 14% to about 60%, about 14% to about 55%, about 14% to about 50%, about 14% to about 45%, about 14% to about 40%, about 14% to about 35%, about 30% to about 30%, about 14% to about 25%, about 14% to about 20%, about 14% to about 18%, about 14% to about 16%, about 16% to about 100%, about 16% to about 95%, about 16% to about 90%, about 16% to about 85%, about 16% to about 80%, about 16% to about 75%, about 16% to about 70%, about 16% to about 65%, about 16% to about 60%, about 16% to about 55%, about 16% to about 50%, about 16% to about 45%, about 16% to about 40%, about 16% to about 35%, about 16% to about 30%, about 16% to about 25%, about 16% to about 20%, about 16% to about 18%, about 18% to about 100%, about 18% to about 95%, about 18% to about 90%, about 18% to about 85%, about 18% to about 80%, about 18% to about 75%, about 18% to about 70%, about 18% to about 65%, about 18% to about 60%, about 18% to about 55%, about 18% to about 50%, about 18% to about 45%, about 18% to about 40%, about 18% to about 35%, about 18% to about 30%, about 18% to about 25%, about 18% to about 20%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 100%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 100%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 55%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 100%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 100%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%; about 70% to about 100%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 100%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 100%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 100%, about 85% to about 95%, about 85% to about 90%, about 90% to about 100%, about 90% to about 95%, or about 95% to about 100%.

Container with a lid

In some examples, the bottom end of the container is flat, has a hemispherical shape, or has a conical shape.

In some examples of any of the pods described herein, the internal volume of the container is: about 0.05mL to about 1,000mL, about 0.05mL to about 950mL, about 0.05mL to about 900mL, about 0.05mL to about 850mL, about 0.05mL to about 800mL, about 0.05mL to about 750mL, about 0.05mL to about 700mL, about 0.05mL to about 650mL, about 0.05mL to about 600mL, about 0.05mL to about 550mL, about 0.05mL to about 500mL, about 0.05mL to about 450mL, about 0.05mL to about 400mL, about 0.05mL to about 350mL, about 0.05mL to about 300mL, about 0.05mL to about 250mL, about 0.05mL to about 200mL, about 0.05mL to about 150mL, about 0.05mL to about 100mL, about 0.05mL to about 90mL, about 0.05mL to about 80mL, about 0.05mL to about 70mL, about 0.05mL to about 0.05mL, about 0.05mL to about 20mL, about 0.05mL to about 0mL, about 0.05mL to about 20mL, about 0.05mL to about 0.05mL, about 0mL to about 20mL, about 0.05mL to about 0.05mL, about 0.05mL to about 20mL, about 0.05mL to about 6mL, about 0.05mL to about 5mL, about 0.05mL to about 4mL, about 0.05mL to about 3mL, about 0.05mL to about 2mL, about 0.05mL to about 1mL, about 0.05mL to about 0.5mL, about 0.05mL to about 0.1mL, about 0.1mL to about 1,000mL, about 0.1mL to about 950mL, about 0.1mL to about 900mL, about 0.1mL to about 850mL, about 0.1mL to about 800mL, about 0.1mL to about 750mL, about 0.1mL to about 700mL, about 0.1mL to about 650mL, about 0.1mL to about 600mL, about 0.1mL to about 550mL, about 0.1mL to about 500mL, about 0.1mL to about 450mL, about 0.1 to about 400mL, about 0.1mL to about 1mL to about 60mL, about 0.1mL to about 0mL to about 1mL, about 0.1mL to about 1mL, about 0mL to about 1mL to about 100mL, about 0.1mL to about 0.1mL, about 0 to about 1mL, about 0.1mL to about 40mL, about 0.1mL to about 35mL, about 0.1mL to about 30mL, about 0.1mL to about 25mL, about 0.1mL to about 20mL, about 0.1mL to about 15mL, about 0.1mL to about 10mL, about 0.1mL to about 8mL, about 0.1mL to about 6mL, about 0.1mL to about 5mL, about 0.1mL to about 4mL, about 0.1mL to about 3mL, about 0.1mL to about 2mL, about 0.1mL to about 1mL, about 0.1mL to about 0.5mL, about 0.5mL to about 1,000mL, about 0.5mL to about 950mL, about 0.5mL to about 900mL, about 0.5mL to about 850mL, about 0.5mL to about 800mL, about 0.5mL to about 750mL, about 0.5mL to about 5mL, about 0.5mL to about 400mL, about 5mL to about 500mL, about 0.5mL to about 400mL, about 0.5mL to about 400mL, about 0.5mL to about 100mL, about 0.5mL to about 90mL, about 0.5mL to about 80mL, about 0.5mL to about 70mL, about 0.5mL to about 60mL, about 0.5mL to about 50mL, about 0.5mL to about 45mL, about 0.5mL to about 40mL, about 0.5mL to about 35mL, about 0.5mL to about 30mL, about 0.5mL to about 25mL, about 0.5mL to about 20mL, about 0.5mL to about 15mL, about 0.5mL to about 10mL, about 0.5mL to about 8mL, about 0.5mL to about 6mL, about 0.5mL to about 5mL, about 0.5mL to about 4mL, about 0.5mL to about 3mL, about 0.5mL to about 2mL, about 0.5mL to about 1mL, about 1mL to about 500mL, about 1mL, about 500mL, about 1mL to about 500mL, about 1mL to about 400mL, about 1mL to about 350mL, about 1mL to about 300mL, about 1mL to about 250mL, about 1mL to about 200mL, about 1mL to about 150mL, about 1mL to about 100mL, about 1mL to about 90mL, about 1mL to about 80mL, about 1mL to about 70mL, about 1mL to about 60mL, about 1mL to about 50mL, about 1mL to about 45mL, about 1mL to about 40mL, about 1mL to about 35mL, about 1mL to about 30mL, about 1mL to about 25mL, about 1mL to about 20mL, about 1mL to about 15mL, about 1mL to about 10mL, about 1mL to about 8mL, about 1mL to about 6mL, about 1mL to about 5mL, about 1mL to about 4mL, about 1mL to about 3mL, about 1mL to about 2mL, about 2mL to about 2mL, about 950mL to about 2mL, about 2mL to about 900mL, about 2mL to about 800mL, about 1mL to about 800mL, about 2mL to about 650mL, about 2mL to about 600mL, about 2mL to about 550mL, about 2mL to about 500mL, about 2mL to about 450mL, about 2mL to about 400mL, about 2mL to about 350mL, about 2mL to about 300mL, about 2mL to about 250mL, about 2mL to about 200mL, about 2mL to about 150mL, about 2mL to about 100mL, about 2mL to about 90mL, about 2mL to about 80mL, about 2mL to about 70mL, about 2mL to about 60mL, about 2mL to about 50mL, about 2mL to about 45mL, about 2mL to about 40mL, about 2mL to about 35mL, about 2mL to about 30mL, about 2mL to about 25mL, about 2mL to about 20mL, about 2mL to about 15mL, about 2mL to about 10mL, about 2mL to about 8mL, about 2mL to about 6mL, about 2mL to about 5mL, about 3mL to about 3mL, about 3mL to about 900mL, about 3mL, about 3mL to about 850mL, about 3mL to about 800mL, about 3mL to about 750mL, about 3mL to about 700mL, about 3mL to about 650mL, about 3mL to about 600mL, about 3mL to about 550mL, about 3mL to about 500mL, about 3mL to about 450mL, about 3mL to about 400mL, about 3mL to about 350mL, about 3mL to about 300mL, about 3mL to about 250mL, about 3mL to about 200mL, about 3mL to about 150mL, about 3mL to about 100mL, about 3mL to about 90mL, about 3mL to about 80mL, about 3mL to about 70mL, about 3mL to about 60mL, about 3mL to about 50mL, about 3mL to about 45mL, about 3mL to about 40mL, about 3mL to about 35mL, about 3mL to about 30mL, about 3mL to about 25mL, about 3mL to about 20mL, about 3mL to about 15mL, about 3mL to about 5mL, about 3mL to about 6mL, about 3mL to about 4mL, about 4mL to about 1,000mL, about 4mL to about 950mL, about 4mL to about 900mL, about 4mL to about 850mL, about 4mL to about 800mL, about 4mL to about 750mL, about 4mL to about 700mL, about 4mL to about 650mL, about 4mL to about 600mL, about 4mL to about 550mL, about 4mL to about 500mL, about 4mL to about 450mL, about 4mL to about 400mL, about 4mL to about 350mL, about 4mL to about 300mL, about 4mL to about 250mL, about 4mL to about 200mL, about 4mL to about 150mL, about 4mL to about 100mL, about 4mL to about 90mL, about 4mL to about 80mL, about 4mL to about 70mL, about 4mL to about 60mL, about 4mL to about 50mL, about 4mL to about 45mL, about 4mL to about 40mL, about 4mL to about 35mL, about 4mL to about 30mL to about 4mL, about 4mL to about 10mL, about 4mL to about 10mL, about 4mL to about 6mL, about 4mL to about 5mL, about 5mL to about 1,000mL, about 5mL to about 950mL, about 5mL to about 900mL, about 5mL to about 850mL, about 5mL to about 800mL, about 5mL to about 750mL, about 5mL to about 700mL, about 5mL to about 650mL, about 5mL to about 600mL, about 5mL to about 550mL, about 5mL to about 500mL, about 5mL to about 450mL, about 5mL to about 400mL, about 5mL to about 350mL, about 5mL to about 300mL, about 5mL to about 250mL, about 5mL to about 200mL, about 5mL to about 150mL, about 5mL to about 100mL, about 5mL to about 90mL, about 5mL to about 80mL, about 5mL to about 70mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 45mL, about 5mL to about 5mL, about 5mL to about 10mL, about 5mL to about 8mL, about 5mL to about 6mL, about 6mL to about 1,000mL, about 6mL to about 950mL, about 6mL to about 900mL, about 6mL to about 850mL, about 6mL to about 800mL, about 6mL to about 750mL, about 6mL to about 700mL, about 6mL to about 650mL, about 6mL to about 600mL, about 6mL to about 550mL, about 6mL to about 500mL, about 6mL to about 450mL, about 6mL to about 400mL, about 6mL to about 350mL, about 6mL to about 300mL, about 6mL to about 250mL, about 6mL to about 200mL, about 6mL to about 150mL, about 6mL to about 100mL, about 6mL to about 90mL, about 6mL to about 80mL, about 6mL to about 70mL, about 6mL to about 60mL, about 6mL to about 50mL, about 6mL to about 40mL, about 6mL to about 30mL, about 6mL to about 6mL, about 6mL to about 35mL, about 6mL to about 15mL, about 6mL to about 10mL, about 6mL to about 8mL, about 8mL to about 1,000mL, about 8mL to about 950mL, about 8mL to about 900mL, about 8mL to about 850mL, about 8mL to about 800mL, about 8mL to about 750mL, about 8mL to about 700mL, about 8mL to about 650mL, about 8mL to about 600mL, about 8mL to about 550mL, about 8mL to about 500mL, about 8mL to about 450mL, about 8mL to about 400mL, about 8mL to about 350mL, about 8mL to about 300mL, about 8mL to about 250mL, about 8mL to about 200mL, about 8mL to about 150mL, about 8mL to about 100mL, about 8mL to about 90mL, about 8mL to about 80mL, about 8mL to about 70mL, about 8mL to about 60mL, about 8mL to about 50mL, about 8mL to about 40mL, about 8mL to about 8mL, about 8mL to about 20mL, about 8mL to about 8mL, about 8mL to about 15mL, about 8mL to about 10mL, about 10mL to about 1,000mL, about 10mL to about 950mL, about 10mL to about 900mL, about 10mL to about 850mL, about 10mL to about 800mL, about 10mL to about 750mL, about 10mL to about 700mL, about 10mL to about 650mL, about 10mL to about 600mL, about 10mL to about 550mL, about 10mL to about 500mL, about 10mL to about 450mL, about 10mL to about 400mL, about 10mL to about 350mL, about 10mL to about 300mL, about 10mL to about 250mL, about 10mL to about 200mL, about 10mL to about 150mL, about 10mL to about 100mL, about 10mL to about 90mL, about 10mL to about 80mL, about 10mL to about 70mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 45mL, about 10mL to about 40mL to about 10mL, about 10mL to about 20mL, about 10mL to about 10mL, about 15mL to about 1,000mL, about 15mL to about 950mL, about 15mL to about 900mL, about 15mL to about 850mL, about 15mL to about 800mL, about 15mL to about 750mL, about 15mL to about 700mL, about 15mL to about 650mL, about 15mL to about 600mL, about 15mL to about 550mL, about 15mL to about 500mL, about 15mL to about 450mL, about 15mL to about 400mL, about 15mL to about 350mL, about 15mL to about 300mL, about 15mL to about 250mL, about 15mL to about 200mL, about 15mL to about 150mL, about 15mL to about 100mL, about 15mL to about 90mL, about 15mL to about 80mL, about 15mL to about 70mL, about 15mL to about 60mL, about 15mL to about 50mL, about 15mL to about 45mL, about 15mL to about 40mL, about 15mL to about 35mL, about 15mL to about 30mL to about 20mL, about 20mL to about 900mL, about 20mL to about 20mL, about 20mL to about 850mL, about 20mL to about 800mL, about 20mL to about 750mL, about 20mL to about 700mL, about 20mL to about 650mL, about 20mL to about 600mL, about 20mL to about 550mL, about 20mL to about 500mL, about 20mL to about 450mL, about 20mL to about 400mL, about 20mL to about 350mL, about 20mL to about 300mL, about 20mL to about 250mL, about 20mL to about 200mL, about 20mL to about 150mL, about 20mL to about 100mL, about 20mL to about 90mL, about 20mL to about 80mL, about 20mL to about 70mL, about 20mL to about 60mL, about 20mL to about 50mL, about 20mL to about 45mL, about 20mL to about 40mL, about 20mL to about 35mL, about 20mL to about 30mL, about 20mL to about 25mL, about 25mL to about 1mL, about 25mL to about 25mL, about 950mL to about 25mL, about 25mL to about 900mL, about 800mL to about 800mL, about 25mL to about 650mL, about 25mL to about 600mL, about 25mL to about 550mL, about 25mL to about 500mL, about 25mL to about 450mL, about 25mL to about 400mL, about 25mL to about 350mL, about 25mL to about 300mL, about 25mL to about 250mL, about 25mL to about 200mL, about 25mL to about 150mL, about 25mL to about 100mL, about 25mL to about 90mL, about 25mL to about 80mL, about 25mL to about 70mL, about 25mL to about 60mL, about 25mL to about 50mL, about 25mL to about 45mL, about 25mL to about 40mL, about 25mL to about 35mL, about 25mL to about 30mL, about 30mL to about 1,000mL, about 30mL to about 950mL, about 30mL to about 900mL, about 30mL to about 850mL, about 30mL to about 800mL, about 30mL to about 30mL, about 30mL to about 600mL, about 30mL to about 500mL, about 500mL to about 500mL, about 30mL to about 400mL, about 30mL to about 350mL, about 30mL to about 300mL, about 30mL to about 250mL, about 30mL to about 200mL, about 30mL to about 150mL, about 30mL to about 100mL, about 30mL to about 90mL, about 30mL to about 80mL, about 30mL to about 70mL, about 30mL to about 60mL, about 30mL to about 50mL, about 30mL to about 45mL, about 30mL to about 40mL, about 30mL to about 35mL, about 35mL to about 1,000mL, about 35mL to about 950mL, about 35mL to about 900mL, about 35mL to about 850mL, about 35mL to about 800mL, about 35mL to about 750mL, about 35mL to about 700mL, about 35mL to about 650mL, about 35mL to about 600mL, about 35mL to about 550mL, about 35mL to about 500mL, about 35mL to about 450mL to about 35mL, about 35mL to about 400mL, about 35mL to about 300mL, about 35mL to about 35mL, about 35mL to about 100mL, about 35mL to about 90mL, about 35mL to about 80mL, about 35mL to about 70mL, about 35mL to about 60mL, about 35mL to about 50mL, about 35mL to about 45mL, about 35mL to about 40mL, about 40mL to about 1,000mL, about 40mL to about 950mL, about 40mL to about 900mL, about 40mL to about 850mL, about 40mL to about 800mL, about 40mL to about 750mL, about 40mL to about 700mL, about 40mL to about 650mL, about 40mL to about 600mL, about 40mL to about 550mL, about 40mL to about 500mL, about 40mL to about 450mL, about 40mL to about 400mL, about 40mL to about 350mL, about 40mL to about 300mL, about 40mL to about 250mL, about 40mL to about 200mL, about 40mL to about 150mL, about 40mL to about 100mL, about 40mL to about 90mL to about 40mL, about 40mL to about 70mL, about 40mL to about 40mL, about 45mL to about 1,000mL, about 45mL to about 950mL, about 45mL to about 900mL, about 45mL to about 850mL, about 45mL to about 800mL, about 45mL to about 750mL, about 45mL to about 700mL, about 45mL to about 650mL, about 45mL to about 600mL, about 45mL to about 550mL, about 45mL to about 500mL, about 45mL to about 450mL, about 45mL to about 400mL, about 45mL to about 350mL, about 45mL to about 300mL, about 45mL to about 250mL, about 45mL to about 200mL, about 45mL to about 150mL, about 45mL to about 100mL, about 45mL to about 90mL, about 45mL to about 80mL, about 45mL to about 70mL, about 45mL to about 60mL, about 45mL to about 50mL, about 50mL to about 1,000mL, about 50mL to about 900mL, about 50mL to about 50mL, about 50mL to about 650mL, about 50mL to about 600mL, about 650mL, about 50mL to about 700mL, about 50mL to about 550mL, about 50mL to about 500mL, about 50mL to about 450mL, about 50mL to about 400mL, about 50mL to about 350mL, about 50mL to about 300mL, about 50mL to about 250mL, about 50mL to about 200mL, about 50mL to about 150mL, about 50mL to about 100mL, about 50mL to about 90mL, about 50mL to about 80mL, about 50mL to about 70mL, about 50mL to about 60mL, about 60mL to about 1,000mL, about 60mL to about 950mL, about 60mL to about 900mL, about 60mL to about 850mL, about 60mL to about 800mL, about 60mL to about 750mL, about 60mL to about 700mL, about 60mL to about 650mL, about 60mL to about 600mL, about 60mL to about 550mL, about 60mL to about 500mL, about 60mL to about 450mL, about 60mL to about 400mL, about 60mL to about 350mL to about 300mL, about 60mL to about 250mL, about 60mL, about 60mL to about 90mL, about 60mL to about 80mL, about 60mL to about 70mL, about 70mL to about 1,000mL, about 70mL to about 950mL, about 70mL to about 900mL, about 70mL to about 850mL, about 70mL to about 800mL, about 70mL to about 750mL, about 70mL to about 700mL, about 70mL to about 650mL, about 70mL to about 600mL, about 70mL to about 550mL, about 70mL to about 500mL, about 70mL to about 450mL, about 70mL to about 400mL, about 70mL to about 350mL, about 70mL to about 300mL, about 70mL to about 250mL, about 70mL to about 200mL, about 70mL to about 150mL, about 70mL to about 100mL, about 70mL to about 90mL, about 70mL to about 80mL, about 80mL to about 1,000mL, about 80mL to about 900mL, about 80mL to about 80mL, about 850mL to about 80mL, about 800mL, about 80mL to about 550mL, about 80mL to about 500mL, about 80mL to about 450mL, about 80mL to about 400mL, about 80mL to about 350mL, about 80mL to about 300mL, about 80mL to about 250mL, about 80mL to about 200mL, about 80mL to about 150mL, about 80mL to about 100mL, about 80mL to about 90mL, about 90mL to about 1,000mL, about 90mL to about 950mL, about 90mL to about 900mL, about 90mL to about 850mL, about 90mL to about 800mL, about 90mL to about 750mL, about 90mL to about 700mL, about 90mL to about 650mL, about 90mL to about 600mL, about 90mL to about 550mL, about 90mL to about 500mL, about 90mL to about 450mL, about 90mL to about 400mL, about 90mL to about 350mL, about 90mL to about 300mL, about 90mL to about 200mL, about 90mL to about 100mL, about 100mL to about 100mL, about 100mL to about 850mL, about 100mL to about 800mL, about 100mL to about 750mL, about 100mL to about 700mL, about 100mL to about 650mL, about 100mL to about 600mL, about 100mL to about 550mL, about 100mL to about 500mL, about 100mL to about 450mL, about 100mL to about 400mL, about 100mL to about 350mL, about 100mL to about 300mL, about 100mL to about 250mL, about 100mL to about 200mL, about 100mL to about 150mL, about 150mL to about 1,000mL, about 150mL to about 950mL, about 150mL to about 900mL, about 150mL to about 850mL, about 150mL to about 800mL, about 150mL to about 750mL, about 150mL to about 700mL, about 150mL to about 650mL, about 150mL to about 600mL, about 150mL to about 550mL, about 150mL to about 500mL, about 150mL to about 450mL to about 150mL, about 150mL to about 200mL, about 150mL to about 200mL, about 200mL to about 950mL, about 200mL to about 900mL, about 200mL to about 850mL, about 200mL to about 800mL, about 200mL to about 750mL, about 200mL to about 700mL, about 200mL to about 650mL, about 200mL to about 600mL, about 200mL to about 550mL, about 200mL to about 500mL, about 200mL to about 450mL, about 200mL to about 400mL, about 200mL to about 350mL, about 200mL to about 300mL, about 200mL to about 250mL, about 250mL to about 1,000mL, about 250mL to about 950mL, about 250mL to about 900mL, about 250mL to about 850mL, about 250mL to about 800mL, about 250mL to about 750mL, about 250mL to about 700mL, about 250mL to about 650mL, about 250mL to about 600mL, about 250mL to about 550mL, about 250mL to about 500mL, about 250mL to about 450mL to about 250mL, about 250mL to about 300mL, about 250mL to about 300mL, about 300mL to about 850mL, about 300mL to about 800mL, about 300mL to about 750mL, about 300mL to about 700mL, about 300mL to about 650mL, about 300mL to about 600mL, about 300mL to about 550mL, about 300mL to about 500mL, about 300mL to about 450mL, about 300mL to about 400mL, about 300mL to about 350mL, about 350mL to about 1,000mL, about 350mL to about 950mL, about 350mL to about 900mL, about 350mL to about 850mL, about 350mL to about 800mL, about 350mL to about 750mL, about 350mL to about 700mL, about 350mL to about 650mL, about 350mL to about 600mL, about 350mL to about 550mL, about 350mL to about 500mL, about 350mL to about 450mL, about 350mL to about 400mL, about 400mL to about 1,000mL, about 400mL to about 900mL, about 400mL to about 400mL, about 400mL to about 400mL, about 400mL to about 550mL, about 400mL to about 500mL, about 400mL to about 450mL, about 450mL to about 1,000mL, about 450mL to about 950mL, about 450mL to about 900mL, about 450mL to about 850mL, about 450mL to about 800mL, about 450mL to about 750mL, about 450mL to about 700mL, about 450mL to about 650mL, about 450mL to about 600mL, about 450mL to about 550mL, about 450mL to about 500mL, about 500mL to about 1,000mL, about 500mL to about 950mL, about 500mL to about 900mL, about 500mL to about 850mL, about 500mL to about 800mL, about 500mL to about 750mL, about 500mL to about 700mL, about 500mL to about 650mL, about 500mL to about 600mL, about 500mL to about 550mL, about 550mL to about 1,000mL, about 550mL to about 550mL, about 600mL to about 1,000mL, about 600mL to about 950mL, about 600mL to about 900mL, about 600mL to about 850mL, about 600mL to about 800mL, about 600mL to about 750mL, about 600mL to about 700mL, about 600mL to about 650mL, about 650mL to about 1,000mL, about 650mL to about 950mL, about 650mL to about 900mL, about 650mL to about 850mL, about 650mL to about 800mL, about 650mL to about 750mL, about 650mL to about 700mL, about 700mL to about 1,000mL, about 700mL to about 950mL, about 700mL to about 900mL, about 700mL to about 850mL, about 700mL to about 800mL, about 700mL to about 750mL, about 750mL to about 1,000mL, about 750mL to about 950mL, about 750mL to about 900mL, about 750mL to about 850mL, about 750mL to about 800mL, about 750mL to about 850mL, about 800mL, about 850mL to about 850mL, about 850mL to about 800mL, about 850mL to about 850mL, about 800mL, about 750mL, about 850mL to about 850mL, about 800mL, about 850mL, about 800mL, about 900mL to about 1,000mL, about 900mL to about 950mL, or about 950mL to about 1,000 mL.

Wall(s)

The wall may be composed of, or in part of: polyethylene (PE), polypropylene (PP), Polystyrene (PS), polyvinyl chloride (PVC), acrylic polymers, polyesters, silicone rubber, silicon, polyurethane, halogenated plastics (e.g., chlorinated polyethylene, chlorinated polyvinyl chloride, chlorosulfonated polyethylene, polychloroprene, fluorinated ethylene propylene), biaxially oriented polypropylene (BOPP), bioplastic, cellophane, ethane-derived plastics, nylon, polylactic acid (PLA), polyethylene terephthalate (PE or PETE plastic code 1), polypropylene, polystyrene, polycarbonate, polydiene, or poly (methyl methacrylate). Other exemplary compositions that may comprise the wall include: bakelite (phenolic), Kevlar or Twaron (para-aramid), Kynar (polyvinylidene fluoride), Mylar (polyethylene terephthalate film), neoprene (polychloroprene), nylon (polyamide 6,6), or lon (polyacrylonitrile), Rilsan (polyamides 11 and 12), Techn or a (copolyamide), Teflon (polytetrafluoroethylene), Ultem (polyimide), Vectran (aramid), Viton (polytetrafluoroethylene), or Zylon (poly-p-phenylene-2, 6-benzobisoxazole (PBO). in some embodiments of the pod, the walls may be composed of or partially composed of nylon, polypropylene, or polyvinyl alcohol (PVA).

In some embodiments of the pod, the wall has a cylindrical shape, a conical shape, a rectangle, a square, a sphere, a hemisphere, an hourglass, or an ellipse.

Chromatography resin

In some embodiments of any of the methods described herein, the pod further comprises a chromatography resin disposed inside the container. In some examples, the chromatography resin is a commercial chromatography resin. In other examples, the chromatography resin is not a commercial chromatography resin.

The chromatography resin may be any chromatography resin known in the art. For example, the chromatography resin may be an affinity chromatography resin (e.g., a protein A resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin), a hydrophobic interaction chromatography resin, a mixed mode chromatography resin, or a molecular sieve chromatography resin. In some embodiments, the resin is a specific affinity resin, e.g., a resin conjugated to an agent (e.g., an antibody, antibody fragment, peptide, nucleic acid, etc.) specific for the recombinant protein of interest. In some embodiments, the chromatography resin is an alkali-resistant resin.

The volume of chromatography resin placed inside the container may be: about 0.1mL to about 500mL, about 0.1mL to about 490mL, about 0.1mL to about 480mL, about 0.1mL to about 460mL, about 0.1mL to about 440mL, about 0.1mL to about 420mL, about 0.1mL to about 400mL, about 0.1mL to about 380mL, about 0.1mL to about 360mL, about 0.1mL to about 340mL, about 0.1mL to about 320mL, about 0.1mL to about 300mL, about 0.1mL to about 280mL, about 0.1mL to about 260mL, about 0.1mL to about 250mL, about 0.1mL to about 240mL, about 0.1mL to about 220mL, about 0.1mL to about 210mL, about 0.1mL to about 200mL, about 0.1mL to about 190mL, about 0.1mL to about 180mL, about 0.1mL to about 170mL, about 0.1mL to about 60mL, about 0mL to about 1mL to about 100mL, about 0.1mL to about 100mL, about 1mL, about 0.1mL to about 100mL, about 0.1mL to about 100mL, about 0.1mL to about 50mL, about 0.1mL to about 40mL, about 0.1mL to about 30mL, about 0.1mL to about 25mL, about 0.1mL to about 20mL, about 0.1mL to about 15mL, about 0.1mL to about 10mL, about 0.1mL to about 5mL, about 0.1mL to about 2mL, about 0.1mL to about 1mL, about 0.2mL to about 500mL, about 0.2mL to about 490mL, about 0.2mL to about 480mL, about 0.2mL to about 460mL, about 0.2mL to about 440mL, about 0.2mL to about 420mL, about 0.2mL to about 400mL, about 0.2mL to about 380mL, about 0.2mL to about 360mL, about 0.2mL to about 340mL, about 0.2mL to about 320mL, about 0.2mL to about 300mL, about 0.2mL to about 200mL, about 0.2mL to about 0.180 mL, about 0mL to about 0.2mL to about 200mL, about 0.2mL to about 180mL, about 0.2mL to about 150mL, about 0.2mL to about 140mL, about 0.2mL to about 130mL, about 0.2mL to about 120mL, about 0.2mL to about 110mL, about 0.2mL to about 100mL, about 0.2mL to about 90mL, about 0.2mL to about 80mL, about 0.2mL to about 70mL, about 0.2mL to about 60mL, about 0.2mL to about 50mL, about 0.2mL to about 40mL, about 0.2mL to about 30mL, about 0.2mL to about 25mL, about 0.2mL to about 20mL, about 0.2mL to about 15mL, about 0.2mL to about 10mL, about 0.2mL to about 5mL, about 0.2mL to about 2mL, about 0.2mL to about 1mL, about 0.4mL to about 500mL, about 0.4mL to about 10mL, about 0.2mL to about 5mL, about 0.2mL to about 2mL, about 0.2mL to about 4mL, about 0.2mL to about 1mL, about 0.4mL to about 400mL, about 0.4mL to about 4mL, about 4mL to about 400mL, about 0.400 mL to about 4mL, about 4mL to about 400mL, about 4mL to about 4mL, about 0.4mL to about 260mL, about 0.4mL to about 250mL, about 0.4mL to about 240mL, about 0.4mL to about 220mL, about 0.4mL to about 210mL, about 0.4mL to about 200mL, about 0.4mL to about 190mL, about 0.4mL to about 180mL, about 0.4mL to about 170mL, about 0.4mL to about 160mL, about 0.4mL to about 150mL, about 0.4mL to about 140mL, about 0.4mL to about 130mL, about 0.4mL to about 120mL, about 0.4mL to about 110mL, about 0.4mL to about 100mL, about 0.4mL to about 90mL, about 0.4mL to about 80mL, about 0.4mL to about 70mL, about 0.4mL to about 60mL, about 0.4mL to about 50mL, about 0.4mL to about 40mL, about 0.4mL to about 5mL, about 0.5mL to about 5mL, about 0.4mL to about 5mL, about 0.5mL to about 5mL, about 0.5mL to about 460mL, about 0.5mL to about 440mL, about 0.5mL to about 420mL, about 0.5mL to about 400mL, about 0.5mL to about 380mL, about 0.5mL to about 360mL, about 0.5mL to about 340mL, about 0.5mL to about 320mL, about 0.5mL to about 300mL, about 0.5mL to about 280mL, about 0.5mL to about 260mL, about 0.5mL to about 250mL, about 0.5mL to about 240mL, about 0.5mL to about 220mL, about 0.5mL to about 210mL, about 0.5mL to about 200mL, about 0.5mL to about 190mL, about 0.5mL to about 180mL, about 0.5mL to about 170mL, about 0.5mL to about 160mL, about 0.5mL to about 150mL, about 0.5mL to about 190mL, about 0.5mL to about 120mL to about 5mL, about 0.5mL to about 100mL, about 0mL to about 5mL, about 100mL, about 0.5mL to about 100mL, about 0.5mL to about 25mL, about 0.5mL to about 20mL, about 0.5mL to about 15mL, about 0.5mL to about 10mL, about 0.5mL to about 5mL, about 0.5mL to about 2mL, about 0.5mL to about 1mL, about 0.6mL to about 500mL, about 0.6mL to about 490mL, about 0.6mL to about 480mL, about 0.6mL to about 460mL, about 0.6mL to about 440mL, about 0.6mL to about 420mL, about 0.6mL to about 400mL, about 0.6mL to about 380mL, about 0.6mL to about 360mL, about 0.6mL to about 340mL, about 0.6mL to about 320mL, about 0.6mL to about 300mL, about 0.6mL to about 280mL, about 0.6mL to about 260mL, about 0.6mL to about 250mL, about 0.6mL to about 100mL, about 6mL to about 6mL, about 0.6mL to about 100mL, about 6mL to about 100mL, about 0.6mL to about 180mL, about 6mL to about 180mL, about 0.6mL to about 120mL, about 0.6mL to about 110mL, about 0.6mL to about 100mL, about 0.6mL to about 90mL, about 0.6mL to about 80mL, about 0.6mL to about 70mL, about 0.6mL to about 60mL, about 0.6mL to about 50mL, about 0.6mL to about 40mL, about 0.6mL to about 30mL, about 0.6mL to about 25mL, about 0.6mL to about 20mL, about 0.6mL to about 15mL, about 0.6mL to about 10mL, about 0.6mL to about 5mL, about 0.6mL to about 2mL, about 0.6mL to about 1mL, about 0.8mL to about 500mL, about 0.8mL to about 490mL, about 0.8mL to about 480mL, about 0.8mL to about 460mL, about 0.8mL to about 440mL, about 0.8mL to about 0mL to about 8mL, about 0.8mL to about 8mL, about 8mL to about 8mL, about 0.8mL to about 8mL, about 8mL to about 8mL, about 0.8mL to about 8mL, about 8mL to about 0.8mL to about 300mL, about 0.8mL to about 220mL, about 0.8mL to about 210mL, about 0.8mL to about 200mL, about 0.8mL to about 190mL, about 0.8mL to about 180mL, about 0.8mL to about 170mL, about 0.8mL to about 160mL, about 0.8mL to about 150mL, about 0.8mL to about 140mL, about 0.8mL to about 130mL, about 0.8mL to about 120mL, about 0.8mL to about 110mL, about 0.8mL to about 100mL, about 0.8mL to about 90mL, about 0.8mL to about 80mL, about 0.8mL to about 70mL, about 0.8mL to about 60mL, about 0.8mL to about 50mL, about 0.8mL to about 40mL, about 0.8mL to about 30mL, about 0.8mL to about 25mL, about 0.8mL to about 20mL, about 0.8mL to about 1mL, about 1mL to about 8mL, about 1mL to about 1mL, about 1mL to about 400mL, about 1mL to about 380mL, about 1mL to about 360mL, about 1mL to about 340mL, about 1mL to about 320mL, about 1mL to about 300mL, about 1mL to about 280mL, about 1mL to about 260mL, about 1mL to about 250mL, about 1mL to about 240mL, about 1mL to about 220mL, about 1mL to about 210mL, about 1mL to about 200mL, about 1mL to about 190mL, about 1mL to about 180mL, about 1mL to about 170mL, about 1mL to about 160mL, about 1mL to about 150mL, about 1mL to about 140mL, about 1mL to about 130mL, about 1mL to about 120mL, about 1mL to about 110mL, about 1mL to about 100mL, about 1mL to about 90mL, about 1mL to about 80mL, about 1mL to about 70mL, about 1 to about 60mL, about 1mL to about 50mL, about 1mL to about 20mL, about 1mL to about 1mL, about 1mL to about 20mL, about 1mL to about 10mL, about 1mL to about 5mL, about 1mL to about 2mL, about 2mL to about 500mL, about 2mL to about 490mL, about 2mL to about 480mL, about 2mL to about 460mL, about 2mL to about 440mL, about 2mL to about 420mL, about 2mL to about 400mL, about 2mL to about 380mL, about 2mL to about 360mL, about 2mL to about 340mL, about 2mL to about 320mL, about 2mL to about 300mL, about 2mL to about 280mL, about 2mL to about 260mL, about 2mL to about 250mL, about 2mL to about 240mL, about 2mL to about 220mL, about 2mL to about 210mL, about 2mL to about 200mL, about 2mL to about 190mL, about 2mL to about 180mL, about 2mL to about 170mL, about 2mL to about 160mL, about 2mL to about 150mL, about 2mL to about 120mL, about 2mL to about 100mL, about 2mL to about 80mL, about 2mL to about 70mL, about 2mL to about 60mL, about 2mL to about 50mL, about 2mL to about 40mL, about 2mL to about 30mL, about 2mL to about 25mL, about 2mL to about 20mL, about 2mL to about 15mL, about 2mL to about 10mL, about 2mL to about 5mL, about 5mL to about 500mL, about 5mL to about 490mL, about 5mL to about 480mL, about 5mL to about 460mL, about 5mL to about 440mL, about 5mL to about 420mL, about 5mL to about 400mL, about 5mL to about 380mL, about 5mL to about 360mL, about 5mL to about 340mL, about 5mL to about 320mL, about 5mL to about 300mL, about 5mL to about 280mL, about 5mL to about 260mL, about 5mL to about 250mL, about 5mL to about 240mL, about 5mL to about 220mL, about 5mL to about 5mL, about 5mL to about 180mL, about 5mL to about 180mL, about 5mL to about 160mL, about 5mL to about 150mL, about 5mL to about 140mL, about 5mL to about 130mL, about 5mL to about 120mL, about 5mL to about 110mL, about 5mL to about 100mL, about 5mL to about 90mL, about 5mL to about 80mL, about 5mL to about 70mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 25mL, about 5mL to about 20mL, about 5mL to about 15mL, about 5mL to about 10mL, about 10mL to about 500mL, about 10mL to about 490mL, about 10mL to about 480mL, about 10mL to about 460mL, about 10mL to about 440mL, about 10mL to about 420mL, about 10mL to about 400mL, about 10mL to about 380mL, about 10 to about 10mL, about 10mL to about 360mL, about 250mL to about 250mL, about 10mL to about 240mL, about 10mL to about 220mL, about 10mL to about 210mL, about 10mL to about 200mL, about 10mL to about 190mL, about 10mL to about 180mL, about 10mL to about 170mL, about 10mL to about 160mL, about 10mL to about 150mL, about 10mL to about 140mL, about 10mL to about 130mL, about 10mL to about 120mL, about 10mL to about 110mL, about 10mL to about 100mL, about 10mL to about 90mL, about 10mL to about 80mL, about 10mL to about 70mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 40mL, about 10mL to about 30mL, about 10mL to about 25mL, about 10mL to about 20mL, about 10mL to about 15mL, about 15mL to about 500mL, about 15mL to about 490mL, about 15mL to about 480mL, about 15mL to about 460mL to about 440mL, about 15mL to about 400mL, about 400mL to about 380mL, about 380mL to about 380mL, about 15mL to about 340mL, about 15mL to about 320mL, about 15mL to about 300mL, about 15mL to about 280mL, about 15mL to about 260mL, about 15mL to about 250mL, about 15mL to about 240mL, about 15mL to about 220mL, about 15mL to about 210mL, about 15mL to about 200mL, about 15mL to about 190mL, about 15mL to about 180mL, about 15mL to about 170mL, about 15mL to about 160mL, about 15mL to about 150mL, about 15mL to about 140mL, about 15mL to about 130mL, about 15mL to about 120mL, about 15mL to about 110mL, about 15mL to about 100mL, about 15mL to about 90mL, about 15mL to about 80mL, about 15mL to about 70mL, about 15mL to about 60mL, about 15mL to about 50mL, about 15mL to about 40mL, about 15mL to about 30mL, about 15mL to about 25mL to about 20mL, about 20mL to about 20mL, about 500mL to about 20mL, about 20mL to about 440mL, about 20mL to about 420mL, about 20mL to about 400mL, about 20mL to about 380mL, about 20mL to about 360mL, about 20mL to about 340mL, about 20mL to about 320mL, about 20mL to about 300mL, about 20mL to about 280mL, about 20mL to about 260mL, about 20mL to about 250mL, about 20mL to about 240mL, about 20mL to about 220mL, about 20mL to about 210mL, about 20mL to about 200mL, about 20mL to about 190mL, about 20mL to about 180mL, about 20mL to about 170mL, about 20mL to about 160mL, about 20mL to about 150mL, about 20mL to about 140mL, about 20mL to about 130mL, about 20mL to about 120mL, about 20mL to about 110mL, about 20mL to about 100mL, about 20mL to about 90mL, about 20 to about 80mL, about 20mL to about 70mL, about 20mL to about 20mL, about 25mL to about 500mL, about 25mL to about 490mL, about 25mL to about 480mL, about 25mL to about 460mL, about 25mL to about 440mL, about 25mL to about 420mL, about 25mL to about 400mL, about 25mL to about 380mL, about 25mL to about 360mL, about 25mL to about 340mL, about 25mL to about 320mL, about 25mL to about 300mL, about 25mL to about 280mL, about 25mL to about 260mL, about 25mL to about 250mL, about 25mL to about 240mL, about 25mL to about 220mL, about 25mL to about 210mL, about 25mL to about 200mL, about 25mL to about 190mL, about 25mL to about 180mL, about 25mL to about 170mL, about 25mL to about 160mL, about 25mL to about 150mL, about 25mL to about 140mL, about 25mL to about 130mL, about 25mL to about 120mL, about 25mL to about 110mL to about 25mL, about 25mL to about 80mL, about 25mL to about 60mL, about 25mL to about 50mL, about 25mL to about 40mL, about 25mL to about 30mL, about 30mL to about 500mL, about 30mL to about 490mL, about 30mL to about 480mL, about 30mL to about 460mL, about 30mL to about 440mL, about 30mL to about 420mL, about 30mL to about 400mL, about 30mL to about 380mL, about 30mL to about 360mL, about 30mL to about 340mL, about 30mL to about 320mL, about 30mL to about 300mL, about 30mL to about 280mL, about 30mL to about 260mL, about 30mL to about 250mL, about 30mL to about 240mL, about 30mL to about 220mL, about 30mL to about 210mL, about 30mL to about 200mL, about 30mL to about 190mL, about 30mL to about 180mL, about 30mL to about 170mL, about 30mL to about 160mL, about 30 to about 150mL, about 30mL to about 120mL, about 30mL to about 100mL, about 30mL to about 80mL, about 30mL to about 70mL, about 30mL to about 60mL, about 30mL to about 50mL, about 30mL to about 40mL, about 40mL to about 500mL, about 40mL to about 490mL, about 40mL to about 480mL, about 40mL to about 460mL, about 40mL to about 440mL, about 40mL to about 420mL, about 40mL to about 400mL, about 40mL to about 380mL, about 40mL to about 360mL, about 40mL to about 340mL, about 40mL to about 320mL, about 40mL to about 300mL, about 40mL to about 280mL, about 40mL to about 260mL, about 40mL to about 250mL, about 40mL to about 240mL, about 40mL to about 220mL, about 40mL to about 210mL, about 40mL to about 200mL, about 40mL to about 190mL, about 40mL to about 180mL, about 40mL to about 170mL, about 40mL to about 160mL to about 40mL, about 40mL to about 130mL, about 40mL to about 140mL, about 40mL to about 130mL, about 40mL to about 100mL, about 40mL to about 90mL, about 40mL to about 80mL, about 40mL to about 70mL, about 40mL to about 60mL, about 40mL to about 50mL, about 50mL to about 500mL, about 50mL to about 490mL, about 50mL to about 480mL, about 50mL to about 460mL, about 50mL to about 440mL, about 50mL to about 420mL, about 50mL to about 400mL, about 50mL to about 380mL, about 50mL to about 360mL, about 50mL to about 340mL, about 50mL to about 320mL, about 50mL to about 300mL, about 50mL to about 280mL, about 50mL to about 260mL, about 50mL to about 250mL, about 50mL to about 240mL, about 50mL to about 220mL, about 50mL to about 210mL, about 50mL to about 200mL, about 50mL to about 190mL, about 50 to about 180mL, about 50mL to about 160mL to about 170mL, about 50mL to about 140mL, about 50mL to about 50mL, about 50mL to about 140mL, about 50mL to about 110mL, about 50mL to about 100mL, about 50mL to about 90mL, about 50mL to about 80mL, about 50mL to about 70mL, about 50mL to about 60mL, about 60mL to about 500mL, about 60mL to about 490mL, about 60mL to about 480mL, about 60mL to about 460mL, about 60mL to about 440mL, about 60mL to about 420mL, about 60mL to about 400mL, about 60mL to about 380mL, about 60mL to about 360mL, about 60mL to about 340mL, about 60mL to about 320mL, about 60mL to about 300mL, about 60mL to about 280mL, about 60mL to about 260mL, about 60mL to about 250mL, about 60mL to about 240mL, about 60mL to about 220mL, about 60mL to about 210mL, about 60mL to about 200mL, about 60mL to about 190mL, about 60 to about 180mL, about 60mL to about 170mL, about 60mL to about 140mL, about 60mL to about 60mL, about 60mL to about 140mL, about 60mL to about 110mL, about 60mL to about 100mL, about 60mL to about 90mL, about 60mL to about 80mL, about 60mL to about 70mL, about 70mL to about 500mL, about 70mL to about 490mL, about 70mL to about 480mL, about 70mL to about 460mL, about 70mL to about 440mL, about 70mL to about 420mL, about 70mL to about 400mL, about 70mL to about 380mL, about 70mL to about 360mL, about 70mL to about 340mL, about 70mL to about 320mL, about 70mL to about 300mL, about 70mL to about 280mL, about 70mL to about 260mL, about 70mL to about 250mL, about 70mL to about 240mL, about 70mL to about 220mL, about 70mL to about 210mL, about 70mL to about 200mL, about 70mL to about 190mL, about 70mL to about 180mL, about 70mL to about 170mL, about 160mL to about 70mL, about 70mL to about 70mL, about 70mL to about 100mL, about 70mL to about 90mL, about 70mL to about 80mL, about 80mL to about 500mL, about 80mL to about 490mL, about 80mL to about 480mL, about 80mL to about 460mL, about 80mL to about 440mL, about 80mL to about 420mL, about 80mL to about 400mL, about 80mL to about 380mL, about 80mL to about 360mL, about 80mL to about 340mL, about 80mL to about 320mL, about 80mL to about 300mL, about 80mL to about 280mL, about 80mL to about 260mL, about 80mL to about 250mL, about 80mL to about 240mL, about 80mL to about 220mL, about 80mL to about 210mL, about 80mL to about 200mL, about 80mL to about 190mL, about 80mL to about 180mL, about 80mL to about 170mL, about 80mL to about 160mL, about 80 to about 150mL, about 80mL to about 120mL, about 80mL to about 100mL, about 80mL to about 80mL, about 90mL to about 500mL, about 90mL to about 490mL, about 90mL to about 480mL, about 90mL to about 460mL, about 90mL to about 440mL, about 90mL to about 420mL, about 90mL to about 400mL, about 90mL to about 380mL, about 90mL to about 360mL, about 90mL to about 340mL, about 90mL to about 320mL, about 90mL to about 300mL, about 90mL to about 280mL, about 90mL to about 260mL, about 90mL to about 250mL, about 90mL to about 240mL, about 90mL to about 220mL, about 90mL to about 210mL, about 90mL to about 200mL, about 90mL to about 190mL, about 90mL to about 180mL, about 90mL to about 170mL, about 90mL to about 160mL, about 90mL to about 150mL, about 90mL to about 140mL, about 90mL to about 130mL, about 90mL to about 120mL, about 90mL to about 110mL to about 100mL, about 100mL to about 100mL, about 100mL to about 440mL, about 100mL to about 420mL, about 100mL to about 400mL, about 100mL to about 380mL, about 100mL to about 360mL, about 100mL to about 340mL, about 100mL to about 320mL, about 100mL to about 300mL, about 100mL to about 280mL, about 100mL to about 260mL, about 100mL to about 250mL, about 100mL to about 240mL, about 100mL to about 220mL, about 100mL to about 210mL, about 100mL to about 200mL, about 100mL to about 190mL, about 100mL to about 180mL, about 100mL to about 170mL, about 100mL to about 160mL, about 100mL to about 150mL, about 100mL to about 140mL, about 100mL to about 130mL, about 100mL to about 120mL, about 100mL to about 110mL, about 110mL to about 500mL, about 110mL to about 490mL, about 110mL to about 480mL, about 110mL to about 110mL, about 110mL to about 400mL, about 400mL to about 380mL, about 110mL to about 340mL, about 110mL to about 320mL, about 110mL to about 300mL, about 110mL to about 280mL, about 110mL to about 260mL, about 110mL to about 250mL, about 110mL to about 240mL, about 110mL to about 220mL, about 110mL to about 210mL, about 110mL to about 200mL, about 110mL to about 190mL, about 110mL to about 180mL, about 110mL to about 170mL, about 110mL to about 160mL, about 110mL to about 150mL, about 110mL to about 140mL, about 110mL to about 130mL, about 110mL to about 120mL, about 120mL to about 500mL, about 120mL to about 490mL, about 120mL to about 480mL, about 120mL to about 460mL, about 120mL to about 440mL, about 120mL to about 420mL, about 120mL to about 400mL, about 120mL to about 380mL, about 120mL to about 360mL, about 120mL to about 340mL, about 250mL to about 250mL, about 120mL to about 240mL, about 120mL to about 220mL, about 120mL to about 210mL, about 120mL to about 200mL, about 120mL to about 190mL, about 120mL to about 180mL, about 120mL to about 170mL, about 120mL to about 160mL, about 120mL to about 150mL, about 120mL to about 140mL, about 120mL to about 130mL, about 130mL to about 500mL, about 130mL to about 490mL, about 130mL to about 480mL, about 130mL to about 460mL, about 130mL to about 440mL, about 130mL to about 420mL, about 130mL to about 400mL, about 130mL to about 380mL, about 130mL to about 360mL, about 130mL to about 340mL, about 130mL to about 320mL, about 130mL to about 300mL, about 130mL to about 280mL, about 130mL to about 260mL, about 130mL to about 250mL, about 130mL to about 220mL, about 130mL to about 180mL, about 130mL to about 130mL, about 130mL to about 180mL, about 130mL to about 180mL, about 130mL to about 160mL, about 130mL to about 150mL, about 130mL to about 140mL, about 140mL to about 500mL, about 140mL to about 490mL, about 140mL to about 480mL, about 140mL to about 460mL, about 140mL to about 440mL, about 140mL to about 420mL, about 140mL to about 400mL, about 140mL to about 380mL, about 140mL to about 360mL, about 140mL to about 340mL, about 140mL to about 320mL, about 140mL to about 300mL, about 140mL to about 280mL, about 140mL to about 260mL, about 140mL to about 250mL, about 140mL to about 240mL, about 140mL to about 220mL, about 140mL to about 210mL, about 140mL to about 200mL, about 140mL to about 190mL, about 140mL to about 180mL, about 140mL to about 170mL, about 140mL to about 160mL, about 140mL to about 150mL, about 150mL to about 150mL, about 440mL to about 150mL, about 150mL to about 440mL, about 150mL to about 400mL, about 150mL to about 380mL, about 150mL to about 360mL, about 150mL to about 340mL, about 150mL to about 320mL, about 150mL to about 300mL, about 150mL to about 280mL, about 150mL to about 260mL, about 150mL to about 250mL, about 150mL to about 240mL, about 150mL to about 220mL, about 150mL to about 210mL, about 150mL to about 200mL, about 150mL to about 190mL, about 150mL to about 180mL, about 150mL to about 170mL, about 150mL to about 160mL, about 160mL to about 500mL, about 160mL to about 490mL, about 160mL to about 480mL, about 160mL to about 460mL, about 160mL to about 440mL, about 160mL to about 420mL, about 160mL to about 400mL, about 160mL to about 380mL, about 160mL to about 360mL, about 160mL to about 340mL, about 160mL to about 260mL, about 160mL to about 300mL, about 160mL to about 250mL, about 160mL to about 220mL, about 160mL to about 210mL, about 160mL to about 200mL, about 160mL to about 190mL, about 160mL to about 180mL, about 160mL to about 170mL, about 170mL to about 500mL, about 170mL to about 490mL, about 170mL to about 480mL, about 170mL to about 460mL, about 170mL to about 440mL, about 170mL to about 420mL, about 170mL to about 400mL, about 170mL to about 380mL, about 170mL to about 360mL, about 170mL to about 340mL, about 170mL to about 320mL, about 170mL to about 300mL, about 170mL to about 280mL, about 170mL to about 260mL, about 170mL to about 250mL, about 170mL to about 240mL, about 170mL to about 220mL, about 170mL to about 210mL, about 170mL to about 200mL, about 170mL to about 190mL, about 170mL to about 180mL, about 180mL to about 180mL, about 180mL to about 400mL, about 180mL to about 380mL, about 180mL to about 360mL, about 180mL to about 340mL, about 180mL to about 320mL, about 180mL to about 300mL, about 180mL to about 280mL, about 180mL to about 260mL, about 180mL to about 250mL, about 180mL to about 240mL, about 180mL to about 220mL, about 180mL to about 210mL, about 180mL to about 200mL, about 180mL to about 190mL, about 190mL to about 500mL, about 190mL to about 490mL, about 190mL to about 480mL, about 190mL to about 460mL, about 190mL to about 440mL, about 190mL to about 420mL, about 190mL to about 400mL, about 190mL to about 380mL, about 190mL to about 360mL, about 190mL to about 340mL, about 190mL to about 320mL, about 190mL to about 300mL, about 190 to about 280mL, about 190mL to about 260mL, about 190mL to about 220mL, about 190mL to about 190mL, about 200mL to about 500mL, about 200mL to about 490mL, about 200mL to about 480mL, about 200mL to about 460mL, about 200mL to about 440mL, about 200mL to about 420mL, about 200mL to about 400mL, about 200mL to about 380mL, about 200mL to about 360mL, about 200mL to about 340mL, about 200mL to about 320mL, about 200mL to about 300mL, about 200mL to about 280mL, about 200mL to about 260mL, about 200mL to about 250mL, about 200mL to about 240mL, about 200mL to about 220mL, about 200mL to about 210mL, about 210mL to about 500mL, about 210mL to about 490mL, about 210mL to about 480mL, about 210mL to about 460mL, about 210mL to about 440mL, about 210mL to about 420mL, about 210mL to about 400mL, about 210mL to about 380mL, about 210mL to about 210mL, about 360mL to about 340mL, about 250mL to about 250mL, about 210mL to about 250mL, about 210mL to about 240mL, about 210mL to about 220mL, about 220mL to about 500mL, about 220mL to about 490mL, about 220mL to about 480mL, about 220mL to about 460mL, about 220mL to about 440mL, about 220mL to about 420mL, about 220mL to about 400mL, about 220mL to about 380mL, about 220mL to about 360mL, about 220mL to about 340mL, about 220mL to about 320mL, about 220mL to about 300mL, about 220mL to about 280mL, about 220mL to about 260mL, about 220mL to about 250mL, about 220mL to about 240mL, about 240mL to about 500mL, about 240mL to about 490mL, about 240mL to about 480mL, about 240mL to about 460mL, about 240mL to about 440mL, about 240mL to about 420mL, about 240mL to about 400mL, about 240mL to about 380mL, about 240mL to about 360mL, about 240mL to about 240mL, about 250mL to about 500mL, about 250mL to about 490mL, about 250mL to about 480mL, about 250mL to about 460mL, about 250mL to about 440mL, about 250mL to about 420mL, about 250mL to about 400mL, about 250mL to about 380mL, about 250mL to about 360mL, about 250mL to about 340mL, about 250mL to about 320mL, about 250mL to about 300mL, about 250mL to about 280mL, about 250mL to about 260mL, about 260mL to about 500mL, about 260mL to about 490mL, about 260mL to about 480mL, about 260mL to about 460mL, about 260mL to about 440mL, about 260mL to about 420mL, about 260mL to about 400mL, about 260mL to about 380mL, about 260mL to about 360mL, about 260mL to about 340mL, about 260mL to about 320mL, about 260mL to about 300mL, about 260 to about 280mL, about 280mL to about 400mL, about 280mL to about 380mL, about 280mL to about 360mL, about 280mL to about 340mL, about 280mL to about 320mL, about 280mL to about 300mL, about 300mL to about 500mL, about 300mL to about 490mL, about 300mL to about 480mL, about 300mL to about 460mL, about 300mL to about 440mL, about 300mL to about 420mL, about 300mL to about 400mL, about 300mL to about 380mL, about 300mL to about 360mL, about 300mL to about 340mL, about 300mL to about 320mL, about 320mL to about 500mL, about 320mL to about 490mL, about 320mL to about 480mL, about 320mL to about 460mL, about 320mL to about 440mL, about 320mL to about 420mL, about 320mL to about 400mL, about 320mL to about 380mL, about 320mL to about 360mL, about 320mL to about 340mL, about 340mL to about 340mL, about 340mL to about 400mL, about 340mL to about 380mL, about 340mL to about 360mL, about 360mL to about 500mL, about 360mL to about 490mL, about 360mL to about 480mL, about 360mL to about 460mL, about 360mL to about 440mL, about 360mL to about 420mL, about 360mL to about 400mL, about 360mL to about 380mL, about 380mL to about 500mL, about 380mL to about 490mL, about 380mL to about 480mL, about 380mL to about 460mL, about 380mL to about 440mL, about 380mL to about 420mL, about 380mL to about 400mL, about 400mL to about 500mL, about 400mL to about 490mL, about 400mL to about 480mL, about 400mL to about 460mL, about 400mL to about 440mL, about 400mL to about 420mL, about 420mL to about 500mL, about 420mL to about 490mL, about 420 to about 480mL, about 420mL to about 440mL, about 440mL to about 440mL, about 400mL to about 440mL, about 440mL to about 440mL, about 420mL to about 440mL, about 440mL to about 440mL, about 460mL to about 500mL, about 460mL to about 490mL, about 460mL to about 480mL, about 480mL to about 500mL, about 480mL to about 490mL, or about 490mL to about 500 mL.

In some embodiments, the chromatography resin disposed in the pod has a sterility assurance level of: about 1x10^-6To about 1x10^-10About 1x10^-6To about 0.5x10^-10About 1x10^-6To about 1x10^-9About 1x10^-6To about 0.5x10^-9About 1x10^-6To about 1x10^-8About 1x10^-6To about 0.5x10^-8About 1x10^-6To about 1x10^-7About 1x10^-6To about 0.5x10^-7About 0.5x10^-7To about 1x10^-10About 0.5x10^-7To about 0.5x10^-10About 0.5x10^-7To about 1x10^-9About 0.5x10^-7To about 0.5x10^-9About 0.5x10^-7To about 1x10^-8About 0.5x10^-7To about 0.5x10^-8About 0.5x10^-7To about 1x10^-7About 1x10^-7To about 1x10^-10About 1x10^-7To about 0.5x10^-10About 0.5x10^-7To about 1x10^-9About 1x10^-7To about 0.5x10^-9About 1x10^-7To about 1x10^-8About 1x10^-7To about 0.5x10^-8About 0.5x10^-8To about 1x10^-10About 0.5x10^-8To about 0.5x10^-10About 0.5x10^-8To about 1x10^-9About 0.5x10^-8To about 0.5x10^-9About 0.5x10^-8To about 1x10^-8About 1x10^-8To about 1x10^-10About 1x10^-8To about 0.5x10^-10About 1x10^-8To about 1x10^-9About 1x10^-8To about 0.5x10^-9About 0.5x10^-9To about 1x10^-10About 0.5x10^-9To about 0.5x10^-10About 0.5x10^-9To about 1x10^-9About 1x10^-9To about 1x10^-10About 1x10^-9To about 0.5x10^-10Or about 0.5x10^-10To about 1x10^-10。

External member

Also provided herein are kits comprising any of the pods described herein. Also provided herein are kits comprising (a) any of the pods described herein (wherein, in a wall of the container, the pod comprises one or more areas covered by a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the inside of the pod), and (b) one or more panels that can be secured over each of the one or more areas on the wall to prevent leakage of fluid from inside the pod through the wall to outside the pod. Also provided herein are kits comprising (a) any of the pods described herein (wherein, in a wall of the container, the pod comprises one or more areas covered by a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the inside of the pod), and (b) an outer sleeve that can be secured over the pod to cover the one or more areas in the wall to prevent leakage of fluid from the inside of the pod to the outside of the pod through at least one area in the wall.

Also provided are kits that include (a) any of the pods described herein, and (b) one or both of an elution outlet that can be stably immobilized on the bottom end and a buffer reservoir that can be stably immobilized on the top end. Also provided herein are kits comprising (a) any of the pods described herein (wherein in a wall of the container the pod comprises one or more regions covered by a semi-porous or porous material that allows liquid to reversibly flow from outside the pod into the inside of the pod), and (b) a movable outer sleeve connected to the wall such that rotation of the movable outer sleeve can cover the one or more regions in the wall to prevent leakage of fluid from the inside of the pod to the outside of the pod through the at least one region in the wall. Also provided herein is a kit comprising (a) any of the pods described herein, and (b) means for stably positioning the long axis of the pod in a horizontal position inside a culture dish.

Any of the kits described herein can include a means for stably positioning the pod inside the culture dish. An exemplary means for stably positioning the pod inside a culture dish (e.g., any of the culture dishes described herein) may include a bracket, a stand, and an adapter that fits over an opening in the culture dish and allows the pod to be inserted into the culture dish (without dropping the pod into the culture dish). In some embodiments, the means for stably positioning the pod inside the culture dish (e.g., any of the culture dishes described herein) is flexible and allows the pod to move with the movement of the liquid culture without falling into the culture dish. In some embodiments, the means for stably positioning the pods inside a culture dish (e.g., any of the culture dishes described herein) is rigid and allows the pods to withstand the movement of the liquid culture without falling into the culture dish. Non-limiting examples of devices for stably positioning a pod inside a culture dish (e.g., any of the culture dishes described herein) are shown in fig. 10-19, 36, and 37.

In some embodiments, the pod is stably secured by gluing the pod inside a petri dish. In some embodiments, the pod is stably secured by snapping the pod inside the culture dish.

In some embodiments, the kit includes an adapter manifold to allow assembly of multiple pods into a plate array, e.g., to further facilitate automated high throughput purification methods.

Non-limiting examples of culture dishes include multi-well plates (e.g., 6-well plates, 12-well plates, 18-well plates, 24-well plates, 48-well plates, 96-well plates, or 384-well plates), shake flasks (e.g., erlenmeyer flasks or T-flasks), shake tubes (e.g., Eppend or f-tubes), wave bags, bioreactors (e.g., bench-top bioreactors, perfusion bioreactors, fed-batch bioreactors), or rolling tube bioreactors. Other examples of bioreactors are known in the art.

Some embodiments of any of the kits described herein can further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) panels that can be secured over one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) areas in the wall to prevent fluid from leaking from the interior of the pod through the wall to the exterior of the pod. The one or more panels may be made from one or more of any of the exemplary materials described herein that may be used to make the walls. One or more panels are designed to cover any of the areas described herein. One or more panels may be designed to snap, snap or screw into the wall to cover one or more areas in the wall as described herein.

Some embodiments of the kits described herein may further comprise an outer sleeve that may be secured to the pod so as to cover at least one area in the wall to prevent fluid from leaking from the interior of the pod through the at least one area in the wall to the exterior of the pod. As can be appreciated in the art, the sleeve may be made at least in part of a flexible rubber or silicon such that it can be slid over the container to cover at least one region in the wall. In some embodiments, the cartridge may be an empty chromatography column fitted on the container.

Some embodiments of the kits described herein may further include a movable outer sleeve connected to the wall such that rotation of the movable outer sleeve may cover at least one area in the wall to prevent fluid from leaking from the interior of the pod to the exterior of the pod through the at least one area in the wall. In some embodiments, the necessary rotation of the movable outer sleeve for covering at least one region in the wall is clockwise. In some embodiments, the necessary rotation of the movable outer sleeve for covering at least one region in the wall is counterclockwise. In an alternative embodiment, the movable outer sleeve is connected to the wall such that sliding the movable outer sleeve upward or downward may cover at least one area in the wall to prevent fluid from leaking from the interior of the pod through the at least one area in the wall to the exterior of the pod.

Some embodiments of the kit further comprise an elution outlet that can be stably secured to the bottom end. The elution outlet may be a funnel with ports or a flat surface. In some embodiments, the elution outlet may comprise means for controlling the flow of fluid from the interior of the container. Non-limiting examples of means for controlling the flow of fluid from the interior of the container include a screw valve or a plug valve. Additional exemplary means for controlling the flow of fluid from the interior of the container are known in the art. In other examples, the elution outlet does not include a means for controlling the flow of fluid from the interior of the container. The elution outlet may be designed to snap, click or screw onto the bottom end of the container. In some embodiments of any of the kits provided herein, the buffer reservoir can be sealed to the pod using one or more (e.g., two or three) O-rings (e.g., affixed to the pod or buffer reservoir).

Some embodiments of any of the kits described herein further comprise a buffer reservoir that can be stably affixed to the tip. The buffer reservoir may snap, snap or screw onto the top end of the container. The buffer reservoir may have any shape such that it can hold a certain amount of fluid that can flow into the container. For example, the buffer reservoir may be cylindrical, rectangular, hemispherical, spherical or elliptical in shape. In some embodiments, the buffer reservoir may include means for controlling the flow of fluid from the buffer reservoir into the container interior. Non-limiting examples of means for controlling the flow of fluid from the buffer reservoir into the container include a screw valve or a stopcock valve. Other exemplary means for controlling the flow of fluid from the buffer reservoir into the container are known in the art. In other examples, the buffer reservoir does not include a means for controlling the flow of fluid out of the buffer reservoir into the interior of the container. The buffer reservoir may be designed to hold a quantity of fluid: 0.1mL to about 500mL, about 0.1mL to about 450mL, about 0.1mL to about 400mL, about 0.1mL to about 350mL, about 0.1mL to about 300mL, about 0.1mL to about 250mL, about 0.1mL to about 200mL, about 0.1mL to about 150mL, about 0.1mL to about 100mL, about 0.1mL to about 80mL, about 0.1mL to about 60mL, about 0.1mL to about 50mL, about 0.1mL to about 40mL, about 0.1mL to about 30mL, about 0.1mL to about 20mL, about 0.1mL to about 10mL, about 0.1mL to about 5mL, about 0.1mL to about 2mL, about 0.1mL to about 1mL, about 0.2mL to about 500mL, about 0.2mL to about 450mL, about 0.2mL to about 400mL, about 0.1mL to about 2mL, about 0.2mL to about 0mL to about 2mL, about 0.1mL to about 0mL, about 0.2mL to about 2mL, about 0.1mL to about 0.2mL to about 0.1mL, about 0 to about 2mL, about 0.2mL to about 20mL, about 0.2mL to about 10mL, about 0.2mL to about 5mL, about 0.2mL to about 2mL, about 0.2mL to about 1mL, 0.5mL to about 500mL, about 0.5mL to about 450mL, about 0.5mL to about 400mL, about 0.5mL to about 350mL, about 0.5mL to about 300mL, about 0.5mL to about 250mL, about 0.5mL to about 200mL, about 0.5mL to about 150mL, about 0.5mL to about 100mL, about 0.5mL to about 80mL, about 0.5mL to about 60mL, about 0.5mL to about 50mL, about 0.5mL to about 40mL, about 0.5mL to about 30mL, about 0.5mL to about 20mL, about 0.5mL to about 10mL, about 0.5mL to about 5mL, about 5mL to about 5mL, about 2mL to about 500mL, about 2mL to about 400mL, about 0.5mL to about 200mL, about 400mL to about 400mL, about 2mL to about 60mL, about 2mL to about 50mL, about 2mL to about 40mL, about 2mL to about 30mL, about 2mL to about 20mL, about 2mL to about 10mL, about 2mL to about 5mL, about 5mL to about 500mL, about 5mL to about 450mL, about 5mL to about 400mL, about 5mL to about 350mL, about 5mL to about 300mL, about 5mL to about 250mL, about 5mL to about 200mL, about 5mL to about 150mL, about 5mL to about 100mL, about 5mL to about 80mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 20mL, about 5mL to about 10mL, about 10mL to about 500mL, about 10mL to about 450mL, about 10mL to about 400mL, about 10mL to about 350mL, about 10mL to about 300mL to about 10mL, about 10mL to about 100mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 40mL, about 10mL to about 30mL, about 10mL to about 20mL, about 20mL to about 500mL, about 20mL to about 450mL, about 20mL to about 400mL, about 20mL to about 350mL, about 20mL to about 300mL, about 20mL to about 250mL, about 20mL to about 200mL, about 20mL to about 150mL, about 20mL to about 100mL, about 20mL to about 80mL, about 20mL to about 60mL, about 20mL to about 50mL, about 20mL to about 40mL, about 20mL to about 30mL, about 30mL to about 500mL, about 30mL to about 450mL, about 30mL to about 400mL, about 30mL to about 350mL, about 30mL to about 300mL, about 30mL to about 250mL, about 30mL to about 200mL, about 30 to about 150mL, about 30mL to about 80mL, about 30mL to about 30mL, about 30mL to about 50mL, about 30mL to about 30mL, about 50mL to about 450mL, about 50mL to about 400mL, about 50mL to about 350mL, about 50mL to about 300mL, about 50mL to about 250mL, about 50mL to about 200mL, about 50mL to about 150mL, about 50mL to about 100mL, about 50mL to about 80mL, or about 50mL to about 60mL.

Also provided herein are kits comprising: (a) any pod described herein (e.g., a pod having any combination of any feature or aspect described herein); (b) an inlet system having a top end, an elongated cylindrical body comprising a fluid conduit, and a bottom end, wherein the top end of the inlet system comprises a fluid inlet and is configured for stable attachment to the top end of the pod and, when stably connected to the pod, allows fluid to flow into the pod through the fluid inlet and the fluid conduit and out the bottom end of the inlet system, and the elongated cylindrical body of the inlet system is configured to fit within the wall of the pod; and (c) an outlet system comprising a fluid outlet configured for stable attachment to the bottom end of the pod and allowing fluid to flow out of the pod through the fluid outlet when stably connected to the bottom end of the pod. In some embodiments of these kits, the top end of the inlet system and the top end of the pod comprise threads that allow the top end of the pod to be stably attached to the top end of the inlet system. In some embodiments of these kits, the top end of the inlet system and the top end of the pod form a seal (e.g., a fluid seal) when the inlet system and pod are stably attached. In some embodiments of these kits, the threads in the top end of the inlet system or the threads in the top end of the pod comprise an O-ring. Other means of providing a stable connection between the top end of the inlet system and the top end of the pod are known in the art. In some embodiments of these kits, the pod comprises a chromatography resin; and the inlet system and pod are configured such that the bottom end of the inlet system is proximate to the chromatography resin when the inlet system and pod are stably attached. In some embodiments of these kits, the inner surface of the pod and the bottom end of the inlet system are configured to allow for stable attachment of the pod and the bottom end of the inlet system. In some embodiments of these kits, the inner surface of the pod and the bottom end of the inlet system form a seal (e.g., a fluid seal) when the inlet system and pod are stably attached. In some embodiments of these kits, the inner surface of the pod and the bottom end of the inlet system comprise threads that allow the bottom ends of the pod and the inlet system to be stably attached. In some embodiments of these kits, the threads in the inner surface of the pod or the threads in the bottom end of the inlet system comprise an O-ring. Other means of providing a stable connection between the inner surface of the pod and the bottom end of the inlet system are known in the art. In some embodiments of these kits, the outlet system and the bottom end of the pod comprise threads that allow the outlet system to be stably attached to the bottom end of the pod. In some embodiments of these kits, the threads in the outlet system or the threads in the bottom end of the pod comprise an O-ring. In some embodiments of these kits, the fluid inlet can be attached to a tubing (e.g., a tubing connected to a chromatography system). In some embodiments of these kits, the fluid outlet can be attached to a tubing (e.g., a tubing connected to a chromatography system). Some embodiments of these kits further comprise: (d) means for stably positioning the long axis of the pod in a horizontal position inside the culture dish. Some embodiments of these kits further comprise: (d) means for stably positioning the long axis of the pod in a vertical position inside the culture dish. In some embodiments of these kits, the elongated cylindrical body of the inlet system has a length of: from about 0.1cm to about 50cm, from about 0.1cm to about 45cm, from about 0.1cm to about 40cm, from about 0.1cm to about 35cm, from about 0.1cm to about 30cm, from about 0.1cm to about 25cm, from about 0.1cm to about 20cm, from about 0.1cm to about 18cm, from about 0.1cm to about 16cm, from about 0.1cm to about 14cm, from about 0.1cm to about 12cm, from about 0.1cm to about 10cm, from about 0.1cm to about 8cm, from about 0.1cm to about 6cm, from about 0.1cm to about 5cm, from about 0.1cm to about 4.5cm, from about 0.1cm to about 4.0cm, from about 0.1cm to about 3.5cm, from about 0.1cm to about 3.0cm, from about 0.1cm to about 2.5cm, from about 0.1cm to about 2.0cm, from about 0cm to about 0cm, from about 0.1cm to about 0cm, from about 0cm to about 0.1cm, from about 0cm to about 5cm, from about 0cm, from about 0.1cm to about 5cm, from about 0cm to about 0cm, from about 0.1cm to about 5cm, from about 0cm to about 0cm, from about 0cm to about 5cm, from about 0cm to about 0cm, from about 0.1cm to about 5cm to about 0cm, from about 0cm to about 5cm, from about 0cm to about 0, from about 0.5cm to about 14cm, from about 0.5cm to about 12cm, from about 0.5cm to about 10cm, from about 0.5cm to about 8cm, from about 0.5cm to about 6cm, from about 0.5cm to about 5cm, from about 0.5cm to about 4.5cm, from about 0.5cm to about 4.0cm, from about 0.5cm to about 3.5cm, from about 0.5cm to about 3.0cm, from about 0.5cm to about 2.5cm, from about 0.5cm to about 2.0cm, from about 0.5cm to about 1.5cm, from about 0.5cm to about 1.0cm, from about 1.0cm to about 50cm, from about 1.0cm to about 45cm, from about 1.0cm to about 40cm, from about 1.0cm to about 35cm, from about 1.0cm to about 30cm, from about 1.0cm to about 25cm, from about 1.0cm to about 0cm, from about 1.0cm to about 1cm, from about 1.0cm to about 1cm to about 1.0cm, from about 1cm to about 1cm, from about 1.1 cm to about 1cm, from about 1cm to about 1.0cm to about 1cm, from about 1cm to about 1, about 1.0cm to about 2.5cm, about 1.0cm to about 2.0cm, about 1.0cm to about 1.5cm, about 1.5cm to about 50cm, about 1.5cm to about 45cm, about 1.5cm to about 40cm, about 1.5cm to about 35cm, about 1.5cm to about 30cm, about 1.5cm to about 25cm, about 1.5cm to about 20cm, about 1.5cm to about 18cm, about 1.5cm to about 16cm, about 1.5cm to about 14cm, about 1.5cm to about 12cm, about 1.5cm to about 10cm, about 1.5cm to about 8cm, about 1.5cm to about 6cm, about 1.5cm to about 5cm, about 1.5cm to about 4.5cm, about 1.5cm to about 4.0cm, about 1.5cm to about 3cm to about 6cm, about 1.5cm to about 1.0cm, about 2cm to about 2.0cm, about 1.5cm to about 2cm, about 2cm to about 2cm, about 1.5cm to about 2cm, from about 2.0cm to about 14cm, from about 2.0cm to about 12cm, from about 2.0cm to about 10cm, from about 2.0cm to about 8cm, from about 2.0cm to about 6cm, from about 2.0cm to about 5cm, from about 2.0cm to about 4.5cm, from about 2.0cm to about 4.0cm, from about 2.0cm to about 3.5cm, from about 2.0cm to about 3.0cm, from about 2.0cm to about 2.5cm, from about 2.5cm to about 50cm, from about 2.5cm to about 45cm, from about 2.5cm to about 40cm, from about 2.5cm to about 35cm, from about 2.5cm to about 30cm, from about 2.5cm to about 25cm, from about 2.5cm to about 20cm, from about 2.5cm to about 18cm, from about 2.5cm to about 16cm, from about 2.5cm to about 30cm, from about 2.5cm to about 2cm, from about 2.5cm to about 5cm, from about 2cm to about 5cm, from about 3.0cm to about 5cm, from about 3cm to about 5cm, from about 2.5cm to about 3cm, from about 2.5cm to about 5cm, from about 5cm to about 5cm, from about 3.0cm to about 35cm, from about 3.0cm to about 30cm, from about 3.0cm to about 25cm, from about 3.0cm to about 20cm, from about 3.0cm to about 18cm, from about 3.0cm to about 16cm, from about 3.0cm to about 14cm, from about 3.0cm to about 12cm, from about 3.0cm to about 10cm, from about 3.0cm to about 8cm, from about 3.0cm to about 6cm, from about 3.0cm to about 5cm, from about 3.0cm to about 4.5cm, from about 3.0cm to about 4.0cm, from about 3.0cm to about 3.5cm, from about 3.5cm to about 50cm, from about 3.5cm to about 45cm, from about 3.5cm to about 40cm, from about 3.5cm to about 35cm, from about 3.5cm to about 30cm, from about 3.5cm to about 25cm, from about 3.0cm to about 5cm, from about 3.5cm to about 3cm, from about 3.5cm to about 5cm, from about 3.5cm to about 3cm to about 5cm, from about 3cm to about 5cm, from about 5cm to about 3.5cm, from about 4.0cm to about 45cm, from about 4.0cm to about 40cm, from about 4.0cm to about 35cm, from about 4.0cm to about 30cm, from about 4.0cm to about 25cm, from about 4.0cm to about 20cm, from about 4.0cm to about 18cm, from about 4.0cm to about 16cm, from about 4.0cm to about 14cm, from about 4.0cm to about 12cm, from about 4.0cm to about 10cm, from about 4.0cm to about 8cm, from about 4.0cm to about 6cm, from about 4.0cm to about 5cm, from about 4.0cm to about 4.5cm, from about 4.5cm to about 50cm, from about 4.5cm to about 45cm, from about 4.5cm to about 40cm, from about 4.5cm to about 35cm, from about 4.5cm to about 30cm, from about 4.5cm to about 25cm, from about 4.5cm to about 45cm, from about 4.5cm to about 4cm to about 5cm, from about 5cm to about 5cm, from about 4.5cm to about 5cm, from about 5cm to about 5cm, from about 4.5cm to about 5cm, from about 5cm to about 5cm, from about 5cm to about 5, from about 5cm to about 35cm, from about 5cm to about 30cm, from about 5cm to about 25cm, from about 5cm to about 20cm, from about 5cm to about 18cm, from about 5cm to about 16cm, from about 5cm to about 14cm, from about 5cm to about 12cm, from about 5cm to about 10cm, from about 5cm to about 8cm, from about 5cm to about 6cm, from about 6cm to about 50cm, from about 6cm to about 45cm, from about 6cm to about 40cm, from about 6cm to about 35cm, from about 6cm to about 30cm, from about 6cm to about 25cm, from about 6cm to about 20cm, from about 6cm to about 18cm, from about 6cm to about 16cm, from about 6cm to about 14cm, from about 6cm to about 12cm, from about 6cm to about 10cm, from about 6cm to about 8cm, from about 8cm to about 50cm, from about 8cm to about 45cm, from about 8cm to about 40cm, from about 8cm to about 35cm, from about 8cm to about 8cm, from about 8cm to about 8cm, from about 8cm, from about 8cm to about 14cm, from about 8cm to about 12cm, from about 8cm to about 10cm, from about 10cm to about 50cm, from about 10cm to about 45cm, from about 10cm to about 40cm, from about 10cm to about 35cm, from about 10cm to about 30cm, from about 10cm to about 25cm, from about 10cm to about 20cm, from about 10cm to about 18cm, from about 10cm to about 16cm, from about 10cm to about 14cm, from about 10cm to about 12cm, from about 12cm to about 50cm, from about 12cm to about 45cm, from about 12cm to about 40cm, from about 12cm to about 35cm, from about 12cm to about 30cm, from about 12cm to about 25cm, from about 12cm to about 20cm, from about 12cm to about 18cm, from about 12cm to about 16cm, from about 12cm to about 14cm, from about 14cm to about 50cm, from about 14cm to about 45cm, from about 14cm to about 40cm, from about 14cm to about 35cm, from about 14cm to about 30cm, from about 14cm to about 14cm, from about 14cm to about 14cm, from about 16cm to about 50cm, from about 16cm to about 45cm, from about 16cm to about 40cm, from about 16cm to about 35cm, from about 16cm to about 30cm, from about 16cm to about 25cm, from about 16cm to about 20cm, from about 16cm to about 18cm, from about 18cm to about 50cm, from about 18cm to about 45cm, from about 18cm to about 40cm, from about 18cm to about 35cm, from about 18cm to about 30cm, from about 18cm to about 25cm, from about 18cm to about 20cm, from about 20cm to about 50cm, from about 20cm to about 45cm, from about 20cm to about 40cm, from about 20cm to about 35cm, from about 20cm to about 30cm, from about 20cm to about 25cm, from about 25cm to about 50cm, from about 25cm to about 45cm, from about 25cm to about 40cm, from about 25cm to about 35cm, from about 25cm to about 30cm, from about 30cm to about 50cm, from about 30cm to about 35cm, from about 35cm to about 35cm, from about 35cm to about 35cm, from about 40cm to about 50cm, from about 40cm to about 45cm, or from about 45cm to about 50 cm; and/or a diameter of about 0.1cm to about 30cm, about 0.1cm to about 28cm, about 0.1cm to about 26cm, about 0.1cm to about 24cm, about 0.1cm to about 22cm, about 0.1cm to about 20cm, about 0.1cm to about 18cm, about 0.1cm to about 16cm, about 0.1cm to about 14cm, about 0.1cm to about 12cm, about 0.1cm to about 10cm, about 0.1cm to about 9cm, about 0.1cm to about 8cm, about 0.1cm to about 7cm, about 0.1cm to about 6cm, about 0.1cm to about 5cm, about 0.1cm to about 4cm, about 0.1cm to about 3cm, about 0.1cm to about 2cm, about 0.1cm to about 1cm, about 0.1cm to about 5cm, about 5cm to about 5cm, about 0.1cm to about 5cm, about 5cm to about 5cm, about 20cm, about 0.1cm to about 5cm, from about 0.5cm to about 10cm, from about 0.5cm to about 9cm, from about 0.5cm to about 8cm, from about 0.5cm to about 7cm, from about 0.5cm to about 6cm, from about 0.5cm to about 5cm, from about 0.5cm to about 4cm, from about 0.5cm to about 3cm, from about 0.5cm to about 2cm, from about 0.5cm to about 1cm, from about 1cm to about 30cm, from about 1cm to about 28cm, from about 1cm to about 26cm, from about 1cm to about 24cm, from about 1cm to about 22cm, from about 1cm to about 20cm, from about 1cm to about 18cm, from about 1cm to about 16cm, from about 1cm to about 14cm, from about 1cm to about 12cm, from about 1cm to about 10cm, from about 1cm to about 9cm, from about 1cm to about 8cm, from about 1cm to about 7cm, from about 1cm to about 6cm, from about 1cm to about 2cm, from about 1cm to about 2cm, from about 2cm to about 22cm, from about 2cm to about 20cm, from about 2cm to about 18cm, from about 2cm to about 16cm, from about 2cm to about 14cm, from about 2cm to about 12cm, from about 2cm to about 10cm, from about 2cm to about 9cm, from about 2cm to about 8cm, from about 2cm to about 7cm, from about 2cm to about 6cm, from about 2cm to about 5cm, from about 2cm to about 4cm, from about 2cm to about 3cm, from about 3cm to about 30cm, from about 3cm to about 28cm, from about 3cm to about 26cm, from about 3cm to about 24cm, from about 3cm to about 22cm, from about 3cm to about 20cm, from about 3cm to about 18cm, from about 3cm to about 16cm, from about 3cm to about 14cm, from about 3cm to about 12cm, from about 3cm to about 10cm, from about 3cm to about 9cm, from about 3cm to about 8cm, from about 3cm to about 7cm to about 4cm, from about 3cm to about 4cm, from about 6cm, from about 3cm to about 4cm, from about 4cm to about 26cm, from about 4cm to about 24cm, from about 4cm to about 22cm, from about 4cm to about 20cm, from about 4cm to about 18cm, from about 4cm to about 16cm, from about 4cm to about 14cm, from about 4cm to about 12cm, from about 4cm to about 10cm, from about 4cm to about 9cm, from about 4cm to about 8cm, from about 4cm to about 7cm, from about 4cm to about 6cm, from about 4cm to about 5cm, from about 5cm to about 30cm, from about 5cm to about 28cm, from about 5cm to about 26cm, from about 5cm to about 24cm, from about 5cm to about 22cm, from about 5cm to about 20cm, from about 5cm to about 18cm, from about 5cm to about 16cm, from about 5cm to about 14cm, from about 5cm to about 12cm, from about 5cm to about 10cm, from about 5cm to about 9cm, from about 5cm to about 8cm, from about 5cm to about 7cm to about 6cm, from about 6cm, from about 6cm to about 22cm, from about 6cm to about 20cm, from about 6cm to about 18cm, from about 6cm to about 16cm, from about 6cm to about 14cm, from about 6cm to about 12cm, from about 6cm to about 10cm, from about 6cm to about 9cm, from about 6cm to about 8cm, from about 6cm to about 7cm, from about 7cm to about 30cm, from about 7cm to about 28cm, from about 7cm to about 26cm, from about 7cm to about 24cm, from about 7cm to about 22cm, from about 7cm to about 20cm, from about 7cm to about 18cm, from about 7cm to about 16cm, from about 7cm to about 14cm, from about 7cm to about 12cm, from about 7cm to about 10cm, from about 7cm to about 9cm, from about 7cm to about 8cm, from about 8cm to about 30cm, from about 8cm to about 28cm, from about 8cm to about 26cm, from about 8cm to about 24cm, from about 8cm to about 8cm, from about 8cm to about 12cm, from about 8cm to about 10cm, from about 8cm to about 9cm, from about 9cm to about 30cm, from about 9cm to about 28cm, from about 9cm to about 26cm, from about 9cm to about 24cm, from about 9cm to about 22cm, from about 9cm to about 20cm, from about 9cm to about 18cm, from about 9cm to about 16cm, from about 9cm to about 14cm, from about 9cm to about 12cm, from about 9cm to about 10cm, from about 10cm to about 30cm, from about 10cm to about 28cm, from about 10cm to about 26cm, from about 10cm to about 24cm, from about 10cm to about 22cm, from about 10cm to about 20cm, from about 10cm to about 18cm, from about 10cm to about 16cm, from about 10cm to about 14cm, from about 10cm to about 12cm, from about 12cm to about 30cm, from about 12cm to about 28cm, from about 12cm to about 26cm, from about 12cm to about 24cm, from about 12cm to about 12cm, from about 12cm to about 12cm, from about 14cm to about 28cm, from about 14cm to about 26cm, from about 14cm to about 24cm, from about 14cm to about 22cm, from about 14cm to about 20cm, from about 14cm to about 18cm, from about 14cm to about 16cm, from about 16cm to about 30cm, from about 16cm to about 28cm, from about 16cm to about 26cm, from about 16cm to about 24cm, from about 16cm to about 22cm, from about 16cm to about 20cm, from about 16cm to about 18cm, from about 18cm to about 30cm, from about 18cm to about 28cm, from about 18cm to about 26cm, from about 18cm to about 24cm, from about 18cm to about 22cm, from about 18cm to about 20cm, from about 20cm to about 30cm, from about 20cm to about 28cm, from about 20cm to about 26cm, from about 20cm to about 24cm, from about 20cm to about 22cm, from about 22cm to about 30cm, from about 22cm to about 28cm, from about 24cm to about 24cm, from about 26cm to about 28cm, or from about 28cm to about 30 cm.

In some embodiments, the inner diameter of the fluid conduit in the inlet system is: about 0.1mm to about 10mm, about 0.1mm to about 9.5mm, about 0.1mm to about 9.0mm, about 0.1mm to about 8.5mm, about 0.1mm to about 8.0mm, about 0.1mm to about 7.5mm, about 0.1mm to about 7.0mm, about 0.1mm to about 6.5mm, about 0.1mm to about 6.0mm, about 0.1mm to about 5.5mm, about 0.1mm to about 5.0mm, about 0.1mm to about 4.5mm, about 0.1mm to about 4.0mm, about 0.1mm to about 3.5mm, about 0.1mm to about 3.0mm, about 0.1mm to about 2.5mm, about 0.1mm to about 2.0mm, about 0.1mm to about 0.0 mm, about 0.1mm to about 5mm, about 0.5mm, about 0mm to about 5mm, about 0.5mm, about 0.0 mm to about 5mm, about 0mm, about 0.5mm to about 5mm, about 0.0 mm, about 0mm to about 5mm, about 0.5mm, about 0mm to about 0.5mm, about 0.0 mm to about 0mm, about 0mm to about 5mm, about 0mm, about 5mm to about 0.5mm, about 0mm to about 0mm, about 5mm to about 0.0.0 mm, about 5mm, about 0mm to about 5mm, about 0mm, about 0.0 mm to about 5mm, about 0.0.0 mm to about 5mm, about 0mm, about 0.0 mm, about 0mm, about 0.5mm to about 4.5mm, about 0.5mm to about 4.0mm, about 0.5mm to about 3.5mm, about 0.5mm to about 3.0mm, about 0.5mm to about 2.5mm, about 0.5mm to about 2.0mm, about 0.5mm to about 1.5mm, about 0.5mm to about 1.0mm, about 1.0mm to about 10mm, about 1.0mm to about 9.5mm, about 1.0mm to about 9.0mm, about 1.0mm to about 8.5mm, about 1.0mm to about 8.0mm, about 1.0mm to about 7.5mm, about 1.0mm to about 7.0mm, about 1.0mm to about 6.5mm, about 1.0mm to about 6.0mm, about 1.0mm to about 5.0mm, about 1.0mm to about 5mm, about 1.0mm to about 1.5mm, about 1.0mm to about 2 mm, about 1.0mm to about 1.0mm, about 3 mm, about 1.0mm to about 1.0mm, about 1mm, about 1.0mm, about 3 mm, about 1.0mm, about 1.

In some embodiments of these kits, the pod contains a chromatography resin (e.g., any type of chromatography resin described herein), and the inlet system and pod are configured such that the bottom end of the inlet system is approximately about 1mm to about 30mm (e.g., about 1mm to about 25mm, about 1mm to about 20mm, about 1mm to about 15mm, about 1mm to about 10mm, about 1mm to about 5mm) from the chromatography resin when the inlet system and pod are stably attached. In some embodiments of these kits, the pod may contain any volume of chromatography resin described herein.

In some embodiments of these kits, the inlet system has an approximately conical shape. In some embodiments of these kits, the outlet system has an approximately conical shape. Non-limiting examples of kits comprising pods, inlet systems and outlet systems are shown in fig. 27 (unassembled) and fig. 28 (assembled). Non-limiting examples of other kits described herein are shown in fig. 1A-1E, fig. 3, fig. 23-26, and fig. 35.

Some embodiments of these kits may further comprise: (d) an adapter having a top end and a bottom end, wherein the top end of the adapter is configured for stable attachment to the bottom end of the pod, and the bottom end of the adapter has one or more blades, wherein when the top end of the adapter is stably attached to the bottom end of the pod, the positioning of the one or more blades facilitates fluid flow into the pod (e.g., by allowing liquid from outside the pod to reversibly flow into the material inside the pod). A non-limiting example of such an adapter is shown in fig. 34. A diagram illustrating an exemplary adapter attached to an exemplary pod is shown in figure 35.

In some embodiments of these kits, the inner surface of the pod and the top end of the adapter are configured to allow for stable attachment of the pod and the top end of the adapter. In some embodiments of these kits, the inner surface of the pod and the top end of the adapter form a seal (e.g., a fluid seal) when the adapter and the pod are stably attached. In some embodiments of these kits, the inner surface of the pod and the top end of the adapter comprise threads that allow for stable attachment of the pod and the top end of the adapter. In some embodiments of these kits, the threads in the inner surface of the pod or the threads in the top end of the adapter comprise an O-ring. Other means of providing a stable connection between the inner surface of the pod and the top end of the adapter are known in the art.

Some embodiments of any kit described herein may further comprise instructions for performing any of the methods described herein.

Method of producing a composite material

Provided herein are methods of capturing a recombinant protein produced by a liquid cell culture, the method comprising: contacting any pod provided herein with a liquid cell culture for a period of time, wherein the contacting step results in at least a portion of at least one region being contacted with the liquid cell culture and the contacting is performed under conditions that result in the chromatography resin capturing the recombinant protein.

Also provided herein is a method of purifying a recombinant protein produced by a liquid cell culture, the method comprising: (a) contacting any pod provided herein with a liquid cell culture for a period of time, wherein the contacting step results in a portion of at least one region being contacted with the liquid cell culture and the contacting is performed under conditions that result in the chromatography resin capturing the recombinant protein; and (b) eluting the recombinant protein from the chromatography resin after a period of time, thereby purifying the recombinant protein. In some embodiments, a chromatography system (e.g.,

system) is eluted (and optionally a washing step).

In some embodiments of any of the methods described herein, the pod is any pod described herein, wherein the pod comprises a chromatography resin (e.g., any chromatography resin described herein or known in the art) disposed inside the container. In some embodiments, the pods are configured such that the longitudinal axis of the pod is positioned about horizontally (e.g., horizontally) in the liquid cell culture. In some embodiments, the pods are arranged such that the longitudinal axes of the pods are disposed about vertically (e.g., vertically) in the liquid cell culture. In some embodiments, the pod comprises a chromatography resin, which is a commercial chromatography resin. In some embodiments, the chromatography resin is an affinity chromatography resin (e.g., a protein a resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin). In some embodiments, after the contacting step, the pod or chromatography resin may be frozen or stored for a period of time before the recombinant protein is eluted from the chromatography resin in the pod. In some embodiments where the pod or chromatography resin is frozen or stored for a period of time, the pod or chromatography resin may be frozen or stored for a period of time after the contacting step and after the pod or chromatography resin is contacted with the wash buffer.

In some embodiments, the contacting results in at least a portion of the at least one region (e.g., at least 10% of the at least one region, at least 15% of the at least one region, at least 20% of the at least one region, at least 25% of the at least one region, at least 30% of the at least one region, at least 35% of the at least one region, at least 40% of the at least one region, at least 45% of the at least one region, at least 50% of the at least one region, at least 55% of the at least one region, at least 60% of the at least one region, at least 65% of the at least one region, at least 70% of the at least one region, at least 75% of the at least one region, at least 80% of the at least one region, at least 85% of the at least one region, at least 90% of the at least, at least 99% of at least one region) is contacted with the liquid cell culture. In some embodiments, at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) of the total surface area of at least one region is contacted with a liquid culture.

In some embodiments, the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin. In some embodiments, the capture can result in a level of binding of the recombinant protein to the chromatography resin of up to 60% to 100% (e.g., up to 60% to 70%, up to 60% to 75%, up to 60% to 80%, up to 60% to 85%, up to 60% to 90%, up to 60% to 95%, up to 65% to 70%, up to 65% to 75%, up to 65% to 80%, up to 65% to 85%, up to 65% to 90%, up to 65% to 95%, up to 65% to 100%, up to 70% to 75%, up to 70% to 80%, up to 70% to 85%, up to 70% to 90%, up to 70% to 95%, up to 70% to 100%, up to 75% to 80%, up to 75% to 85%, up to 75% to 90%, up to 75% to 95%, up to 75% to 100%, up to 80% to 85%, up to 80% to 90%, up to 80% to 95%, up to 80% to 100%, up to 85% to 90%, up to 85% to 95%, up to 85% to 100%, up to 90% to 95%, up to 95% to 100%, up to 50% to 55%, up to 50% to 60%, up to 50% to 65%, up to 50% to 70%, up to 50% to 75%, up to 50% to 80%, up to 50% to 85%, up to 50% to 90%, up to 50% to 95%, up to 50% to 100%, up to 55% to 60%, up to 55% to 65%, up to 55% to 70%, up to 55% to 75%, up to 55% to 80%, up to 55% to 85%, up to 55% to 90%, up to 55% to 95%, up to 55% to 100%, up to 50%, up to 51%, up to 52%, up to 53%, up to 54%, up to 55%, up to 56%, up to 57%, up to 58%, up to 59%, up to 61%, up to 62%, up to 63%, up to 64%, up to 65%, up to 66%, up to 67%, up to 68%, up to 69%, up to 70%, up to 71%, up to 72%, up to 73%, up to 74%, up to 75%, up to 76%, up to 77%, up to 78%, up to 79%, up to 80%, up to 81%, up to 82%, up to 83%, up to 84%, up to 85%, up to 86%, up to 87%, up to 88%, up to 89%, up to 90%, up to 91%, up to 92%, up to 93%, up to 94%, up to 95%, up to 96%, up to 97%, up to 98%, up to 99%, or 100%.

In some embodiments, the capturing may result in capturing from about 0.05mg to about 80mg of the recombinant protein (e.g., from about 0.05mg to about 0.1mg, from about 0.05mg to about 0.2mg, from about 0.05mg to about 0.5mg, from about 0.05mg to about 0.6mg, from about 0.05mg to about 0.8mg, from about 0.05mg to about 1mg, from about 0.05mg to about 2mg, from about 0.05mg to about 5mg, from about 0.05mg to about 10mg, from about 0.05mg to about 15mg, from about 0.05mg to about 20mg, from about 0.05mg to about 25mg, from about 0.05mg to about 30mg, from about 0.05mg to about 35mg, from about 0.05mg to about 40mg, from about 0.05mg to about 45mg, from about 0.05mg to about 50mg, from about 0.05mg to about 55mg, from about 0.05mg to about 35mg, from about 0.05mg to about 0.05mg, from about 0.05mg to about 1mg, from about 0.05mg to about 1mg, about 0.1mg to about 2mg, about 0.1mg to about 5mg, about 0.1mg to about 10mg, about 0.1mg to about 15mg, about 0.1mg to about 20mg, about 0.1mg to about 25mg, about 0.1mg to about 30mg, about 0.1mg to about 35mg, about 0.1mg to about 40mg, about 0.1mg to about 45mg, about 0.1mg to about 50mg, about 0.1mg to about 55mg, about 0.1mg to about 60mg, about 0.1mg to about 65mg, about 0.1mg to about 70mg, about 0.1mg to about 75mg, about 0.1mg to about 80mg, about 0.2mg to about 0.5mg, about 0.2mg to about 0.6mg, about 0.2mg to about 0.8mg, about 0.2mg to about 1mg, about 2mg to about 2mg, about 0.2mg to about 0.5mg, about 0.2mg to about 0.2mg, about 0.2mg to about 0.5mg, about 0.2mg to about 2mg, about 0.2mg to about 0.2mg, about 0.2mg to about 0.5mg, about 0.2mg to about 0.2mg, about 2mg to about 2mg, about 0.2mg to about 0.2mg, about 0., about 0.2mg to about 60mg, about 0.2mg to about 65mg, about 0.2mg to about 70mg, about 0.2mg to about 75mg, about 0.2mg to about 80mg, about 0.5mg to about 0.6mg, about 0.5mg to about 0.8mg, about 0.5mg to about 1mg, about 0.5mg to about 2mg, about 0.5mg to about 5mg, about 0.5mg to about 10mg, about 0.5mg to about 15mg, about 0.5mg to about 20mg, about 0.5mg to about 25mg, about 0.5mg to about 30mg, about 0.5mg to about 35mg, about 0.5mg to about 40mg, about 0.5mg to about 45mg, about 0.5mg to about 50mg, about 0.5mg to about 55mg, about 0.5mg to about 60mg, about 0.5mg to about 65mg, about 0.5mg to about 0.6mg, about 0.5mg to about 0.5mg, about 0.6mg to about 0.5mg, about 0.5mg to about 6mg, about 0.5mg to about 0.5mg, about 6mg to about 0.6mg, about 0.6mg to about 30mg, about 0.6mg to about 35mg, about 0.6mg to about 40mg, about 0.6mg to about 45mg, about 0.6mg to about 50mg, about 0.6mg to about 55mg, about 0.6mg to about 60mg, about 0.6mg to about 65mg, about 0.6mg to about 70mg, about 0.6mg to about 75mg, about 0.6mg to about 80mg, about 0.8mg to about 1mg, about 0.8mg to about 2mg, about 0.8mg to about 5mg, about 0.8mg to about 10mg, about 0.8mg to about 15mg, about 0.8mg to about 20mg, about 0.8mg to about 25mg, about 0.8mg to about 30mg, about 0.8mg to about 35mg, about 0.8mg to about 40mg, about 0.8mg to about 45mg, about 0.8mg to about 0.8mg, about 0.8mg to about 1mg, about 0.8mg to about 8mg, about 0.8mg, about 1mg to about 1mg, about 1mg to about 20mg, about 1mg to about 25mg, about 1mg to about 30mg, about 1mg to about 35mg, about 1mg to about 40mg, about 1mg to about 45mg, about 1mg to about 50mg, about 1mg to about 55mg, about 1mg to about 60mg, about 1mg to about 65mg, about 1mg to about 70mg, about 1mg to about 75mg, about 1mg to about 80mg, about 2mg to about 5mg, about 2mg to about 10mg, about 2mg to about 15mg, about 2mg to about 20mg, about 2mg to about 25mg, about 2mg to about 30mg, about 2mg to about 35mg, about 2mg to about 40mg, about 2mg to about 45mg, about 2mg to about 50mg, about 2mg to about 55mg, about 2mg to about 60mg, about 2mg to about 65mg, about 2mg to about 70mg, about 2mg to about 5mg, about 5mg to about 30mg, about 5mg to about 35mg, about 5mg to about 40mg, about 5mg to about 45mg, about 5mg to about 50mg, about 5mg to about 55mg, about 5mg to about 60mg, about 5mg to about 65mg, about 5mg to about 70mg, about 5mg to about 75mg, about 5mg to about 80mg, about 10mg to about 15mg, about 10mg to about 20mg, about 10mg to about 25mg, about 10mg to about 30mg, about 10mg to about 35mg, about 10mg to about 40mg, about 10mg to about 45mg, about 10mg to about 50mg, about 10mg to about 55mg, about 10mg to about 60mg, about 10mg to about 65mg, about 10mg to about 70mg, about 10mg to about 75mg, about 10mg to about 80mg, about 15mg to about 20mg, about 15mg to about 15mg, about 15mg to about 55mg, about 15mg to about 60mg, about 15mg to about 65mg, about 15mg to about 70mg, about 15mg to about 75mg, about 15mg to about 80mg, about 20mg to about 25mg, about 20mg to about 30mg, about 20mg to about 35mg, about 20mg to about 40mg, about 20mg to about 45mg, about 20mg to about 50mg, about 20mg to about 55mg, about 20mg to about 60mg, about 20mg to about 65mg, about 20mg to about 70mg, about 20mg to about 75mg, about 20mg to about 80mg, about 25mg to about 30mg, about 25mg to about 35mg, about 25mg to about 40mg, about 25mg to about 45mg, about 25mg to about 50mg, about 25mg to about 55mg, about 25mg to about 60mg, about 25mg to about 65mg, about 25mg to about 70mg, about 25mg to about 30mg, about 30mg to about 35mg, about 25mg to about 30mg, about 30mg to about 55mg, about 30mg to about 60mg, about 30mg to about 65mg, about 30mg to about 70mg, about 30mg to about 75mg, about 30mg to about 80mg, about 35mg to about 40mg, about 35mg to about 45mg, about 35mg to about 50mg, about 35mg to about 55mg, about 35mg to about 60mg, about 35mg to about 65mg, about 35mg to about 70mg, about 35mg to about 75mg, about 35mg to about 80mg, about 40mg to about 45mg, about 40mg to about 50mg, about 45mg to about 55mg, about 45mg to about 60mg, about 45mg to about 65mg, about 45mg to about 70mg, about 45mg to about 75mg, about 45mg to about 80mg, about 50mg to about 55mg, about 50mg to about 60mg, about 50mg to about 65mg, about 50mg to about 70mg, about 50mg to about 80mg, about 55mg to about 55mg, about 50mg to about 65mg, about 50mg to about 70mg, about 50mg to about 55mg, about 55mg to about 80mg, about 60mg to about 65mg, about 60mg to about 70mg, about 60mg to about 75mg, about 60mg to about 80mg, about 65mg to about 70mg, about 65mg to about 75mg, about 65mg to about 80mg, about 70mg to about 75mg, about 70mg to about 80mg, about 75mg to about 80 mg).

In some embodiments, the purity of the capture is at least 45% (e.g., at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.2%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%).

For a period of time

In some embodiments of any of the methods provided herein, contacting of any pod provided herein with a liquid cell culture is for a period of time (e.g., about 30 seconds to about 2 weeks). In some embodiments of any of the methods provided herein, the recombinant protein is eluted from the chromatography resin after a period of time (e.g., about 30 seconds to about 2 weeks).

In some embodiments, the period of time for contacting and/or eluting the recombinant protein from the chromatography resin is: from about 30 seconds to about 1 minute, from about 30 seconds to 45 seconds, from about 30 seconds to about 90 seconds, from about 30 seconds to about 2 minutes, from about 30 seconds to about 150 seconds, from about 30 seconds to about 3 minutes, from about 30 seconds to about 210 seconds, from about 30 seconds to about 4 minutes, from about 30 seconds to about 270 seconds, from about 30 seconds to about 5 minutes, from about 1 minute to about 2 minutes, from about 1 minute to about 3 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 5 minutes, from about 2 minutes to about 3 minutes, from about 2 minutes to about 4 minutes, from about 2 to about 5 minutes, from about 3 minutes to about 4 minutes, from about 3 minutes to about 5 minutes, from about 4 minutes to about 5 minutes, from about 5 minutes to about 2 weeks, from about 5 minutes to about 168 hours, from about 5 minutes to about 162 hours, from about 5 minutes to about 156 hours, from about 5 minutes to about 150 hours, from about 5 minutes to about 144 hours, from about 5 minutes to about 138 hours, from about 5 minutes to about 132 hours, from about 5 minutes to about 126 hours, from about 5 minutes to about 120 hours, from about 5 minutes to about 114 hours, from about 5 minutes to about 108 hours, from about 5 minutes to about 102 hours, from about 5 minutes to about 96 hours, from about 5 minutes to about 90 hours, from about 5 minutes to about 84 hours, from about 5 minutes to about 78 hours, from about 5 minutes to about 72 hours, from about 5 minutes to about 66 hours, from about 5 minutes to about 60 hours, from about 5 minutes to about 54 hours, from about 5 minutes to about 48hours, from about 5 minutes to about 42 hours, from about 5 minutes to about 36 hours, from about 5 minutes to about 30 hours, from about 5 minutes to about 24 hours, from about 5 minutes to about 18 hours, from about 5 minutes to about 12 hours, from about 5 minutes to about 6 hours, from about 5 minutes to about 5 hours, from about 5 minutes to about 240 minutes, from about 5 minutes to about 210 minutes, from about 5 minutes to about 180 minutes, from about 5 minutes to about 150 minutes, from about 5 minutes to about 120 minutes, from about 5 minutes to about 90 minutes, from about 5 minutes to about 60 minutes, from about 5 minutes to about 30 minutes, from about 5 minutes to about 20 minutes, from about 5 minutes to about 15 minutes, from about 5 minutes to about 10 minutes; from about 10 minutes to about 2 weeks, from about 10 minutes to about 168 hours, from about 10 minutes to about 162 hours, from about 10 minutes to about 156 hours, from about 10 minutes to about 150 hours, from about 10 minutes to about 144 hours, from about 10 minutes to about 138 hours, from about 10 minutes to about 132 hours, from about 10 minutes to about 126 hours, from about 10 minutes to about 120 hours, from about 10 minutes to about 114 hours, from about 10 minutes to about 108 hours, from about 10 minutes to about 102 hours, from about 10 minutes to about 96 hours, from about 10 minutes to about 90 hours, from about 10 minutes to about 84 hours, from about 10 minutes to about 78 hours, from about 10 minutes to about 72 hours, from about 10 minutes to about 66 hours, from about 10 minutes to about 60 hours, from about 10 minutes to about 54 hours, from about 10 minutes to about 48hours, from about 10 minutes to about 42 hours, from about 10 minutes to about 36 hours, from about 10 minutes to about 30 hours, from about 10 minutes to about 24 hours, from about 10 minutes to about 18 hours, from about 10 minutes to about 12 hours, from about 10 minutes to about 6 hours, from about 10 minutes to about 5 hours, from about 10 minutes to about 240 minutes, from about 10 minutes to about 210 minutes, from about 10 minutes to about 180 minutes, from about 10 minutes to about 150 minutes, from about 10 minutes to about 120 minutes, from about 10 minutes to about 90 minutes, from about 10 minutes to about 60 minutes, from about 10 minutes to about 30 minutes, from about 10 minutes to about 15 minutes, from about 10 minutes to about 20 minutes, from about 10 minutes to about 25 minutes; about 20 minutes to about 2 weeks, about 20 minutes to about 168 hours, about 20 minutes to about 162 hours, about 20 minutes to about 156 hours, about 20 minutes to about 150 hours, about 20 minutes to about 144 hours, about 20 minutes to about 138 hours, about 20 minutes to about 132 hours, about 20 minutes to about 126 hours, about 20 minutes to about 120 hours, about 20 minutes to about 114 hours, about 20 minutes to about 108 hours, about 20 minutes to about 102 hours, about 20 minutes to about 96 hours, about 20 minutes to about 90 hours, about 20 minutes to about 84 hours, about 20 minutes to about 78 hours, about 20 minutes to about 72 hours, about 20 minutes to about 66 hours, about 20 minutes to about 60 hours, about 20 minutes to about 54 hours, about 20 minutes to about 48hours, about 20 minutes to about 42 hours, about 20 minutes to about 36 hours, about 20 minutes to about 30 hours, about 20 minutes to about 24 hours, from about 20 minutes to about 18 hours, from about 20 minutes to about 12 hours, from about 20 minutes to about 6 hours, from about 20 minutes to about 5 hours, from about 20 minutes to about 240 minutes, from about 20 minutes to about 210 minutes, from about 20 minutes to about 180 minutes, from about 20 minutes to about 150 minutes, from about 20 minutes to about 120 minutes, from about 20 minutes to about 90 minutes, from about 20 minutes to about 60 minutes, from about 20 minutes to about 30 minutes, from about 20 minutes to about 25 minutes; about 30 minutes to about 2 weeks, about 30 minutes to about 168 hours, about 30 minutes to about 162 hours, about 30 minutes to about 156 hours, about 30 minutes to about 150 hours, about 30 minutes to about 144 hours, about 30 minutes to about 138 hours, about 30 minutes to about 132 hours, about 30 minutes to about 126 hours, about 30 minutes to about 120 hours, about 30 minutes to about 114 hours, about 30 minutes to about 108 hours, about 30 minutes to about 102 hours, about 30 minutes to about 96 hours, about 30 minutes to about 90 hours, about 30 minutes to about 84 hours, about 30 minutes to about 78 hours, about 30 minutes to about 72 hours, about 30 minutes to about 66 hours, about 30 minutes to about 60 hours, about 30 minutes to about 54 hours, about 30 minutes to about 48hours, about 30 minutes to about 42 hours, about 30 minutes to about 36 hours, about 30 minutes to about 30 hours, about 30 minutes to about 24 hours, from about 30 minutes to about 18 hours, from about 30 minutes to about 12 hours, from about 30 minutes to about 6 hours, from about 30 minutes to about 5 hours, from about 30 minutes to about 240 minutes, from about 30 minutes to about 210 minutes, from about 30 minutes to about 180 minutes, from about 30 minutes to about 150 minutes, from about 30 minutes to about 120 minutes, from about 30 minutes to about 90 minutes, from about 30 minutes to about 60 minutes; about 40 minutes to about 2 weeks, about 40 minutes to about 168 hours, about 40 minutes to about 162 hours, about 40 minutes to about 156 hours, about 40 minutes to about 150 hours, about 40 minutes to about 144 hours, about 40 minutes to about 138 hours, about 40 minutes to about 132 hours, about 40 minutes to about 126 hours, about 40 minutes to about 120 hours, about 40 minutes to about 114 hours, about 40 minutes to about 108 hours, about 40 minutes to about 102 hours, about 40 minutes to about 96 hours, about 40 minutes to about 90 hours, about 40 minutes to about 84 hours, about 40 minutes to about 78 hours, about 40 minutes to about 72 hours, about 40 minutes to about 66 hours, about 40 minutes to about 60 hours, about 40 minutes to about 54 hours, about 40 minutes to about 48hours, about 40 minutes to about 42 hours, about 40 minutes to about 36 hours, about 40 minutes to about 30 hours, about 40 minutes to about 24 hours, from about 40 minutes to about 18 hours, from about 40 minutes to about 12 hours, from about 40 minutes to about 6 hours, from about 40 minutes to about 5 hours, from about 40 minutes to about 240 minutes, from about 40 minutes to about 210 minutes, from about 40 minutes to about 180 minutes, from about 40 minutes to about 150 minutes, from about 40 minutes to about 120 minutes, from about 40 minutes to about 90 minutes, from about 40 minutes to about 60 minutes, from about 40 minutes to about 50 minutes, from about 40 minutes to about 45 minutes; about 45 minutes to about 2 weeks, about 45 minutes to about 168 hours, about 45 minutes to about 162 hours, about 45 minutes to about 156 hours, about 45 minutes to about 150 hours, about 45 minutes to about 144 hours, about 45 minutes to about 138 hours, about 45 minutes to about 132 hours, about 45 minutes to about 126 hours, about 45 minutes to about 120 hours, about 45 minutes to about 114 hours, about 45 minutes to about 108 hours, about 45 minutes to about 102 hours, about 45 minutes to about 96 hours, about 45 minutes to about 90 hours, about 45 minutes to about 84 hours, about 45 minutes to about 78 hours, about 45 minutes to about 72 hours, about 45 minutes to about 66 hours, about 45 minutes to about 60 hours, about 45 minutes to about 54 hours, about 45 minutes to about 48hours, about 45 minutes to about 42 hours, about 45 minutes to about 36 hours, about 45 minutes to about 30 hours, about 45 minutes to about 24 hours, from about 45 minutes to about 18 hours, from about 45 minutes to about 12 hours, from about 45 minutes to about 6 hours, from about 45 minutes to about 5 hours, from about 45 minutes to about 240 minutes, from about 45 minutes to about 210 minutes, from about 45 minutes to about 180 minutes, from about 45 minutes to about 150 minutes, from about 45 minutes to about 120 minutes, from about 45 minutes to about 90 minutes, from about 45 minutes to about 60 minutes, from about 45 minutes to about 50 minutes, from about 45 minutes to about 55 minutes; about 60 minutes to about 2 weeks, about 60 minutes to about 168 hours, about 60 minutes to about 162 hours, about 60 minutes to about 156 hours, about 60 minutes to about 150 hours, about 60 minutes to about 144 hours, about 60 minutes to about 138 hours, about 60 minutes to about 132 hours, about 60 minutes to about 126 hours, about 60 minutes to about 120 hours, about 60 minutes to about 114 hours, about 60 minutes to about 108 hours, about 60 minutes to about 102 hours, about 60 minutes to about 96 hours, about 60 minutes to about 90 hours, about 60 minutes to about 84 hours, about 60 minutes to about 78 hours, about 60 minutes to about 72 hours, about 60 minutes to about 66 hours, about 60 minutes to about 60 hours, about 60 minutes to about 54 hours, about 60 minutes to about 48hours, about 60 minutes to about 42 hours, about 60 minutes to about 36 hours, about 60 minutes to about 30 hours, about 60 minutes to about 24 hours, from about 60 minutes to about 18 hours, from about 60 minutes to about 12 hours, from about 60 minutes to about 6 hours, from about 60 minutes to about 5 hours, from about 60 minutes to about 240 minutes, from about 60 minutes to about 210 minutes, from about 60 minutes to about 180 minutes, from about 60 minutes to about 150 minutes, from about 60 minutes to about 120 minutes, from about 60 minutes to about 90 minutes, from about 60 minutes to about 85 minutes, from about 60 minutes to about 80 minutes, from about 60 minutes to about 75 minutes, from about 60 minutes to about 70 minutes; about 70 minutes to about 2 weeks, about 70 minutes to about 168 hours, about 70 minutes to about 162 hours, about 70 minutes to about 156 hours, about 70 minutes to about 150 hours, about 70 minutes to about 144 hours, about 70 minutes to about 138 hours, about 70 minutes to about 132 hours, about 70 minutes to about 126 hours, about 70 minutes to about 120 hours, about 70 minutes to about 114 hours, about 70 minutes to about 108 hours, about 70 minutes to about 102 hours, about 70 minutes to about 96 hours, about 70 minutes to about 90 hours, about 70 minutes to about 84 hours, about 70 minutes to about 78 hours, about 70 minutes to about 72 hours, about 70 minutes to about 66 hours, about 70 minutes to about 60 hours, about 70 minutes to about 54 hours, about 70 minutes to about 48hours, about 70 minutes to about 42 hours, about 70 minutes to about 36 hours, about 70 minutes to about 30 hours, about 70 minutes to about 24 hours, about 70 minutes to about 18 hours, about 70 minutes to about 12 hours, about 70 minutes to about 6 hours, about 70 minutes to about 5 hours, about 70 minutes to about 240 minutes, about 70 minutes to about 210 minutes, about 70 minutes to about 180 minutes, about 70 minutes to about 150 minutes, about 70 minutes to about 120 minutes, about 70 minutes to about 90 minutes, about 70 minutes to about 85 minutes, about 70 minutes to about 80 minutes; from about 75 minutes to about 80 minutes, from about 75 minutes to about 85 minutes, from about 75 minutes to about 90 minutes; about 90 minutes to about 2 weeks, about 90 minutes to about 168 hours, about 90 minutes to about 162 hours, about 90 minutes to about 156 hours, about 90 minutes to about 150 hours, about 90 minutes to about 144 hours, about 90 minutes to about 138 hours, about 90 minutes to about 132 hours, about 90 minutes to about 126 hours, about 90 minutes to about 120 hours, about 90 minutes to about 114 hours, about 90 minutes to about 108 hours, about 90 minutes to about 102 hours, about 90 minutes to about 96 hours, about 90 minutes to about 90 hours, about 90 minutes to about 84 hours, about 90 minutes to about 78 hours, about 90 minutes to about 72 hours, about 90 minutes to about 66 hours, about 90 minutes to about 60 hours, about 90 minutes to about 54 hours, about 90 minutes to about 48hours, about 90 minutes to about 42 hours, about 90 minutes to about 36 hours, about 90 minutes to about 30 hours, about 90 minutes to about 24 hours, about 90 minutes to about 18 hours, about 90 minutes to about 12 hours, about 90 minutes to about 6 hours, about 90 minutes to about 5 hours, about 90 minutes to about 240 minutes, about 90 minutes to about 210 minutes, about 90 minutes to about 180 minutes, about 90 minutes to about 150 minutes, about 90 minutes to about 120 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 110 minutes, about 100 minutes to about 120 minutes; about 120 minutes to about 2 weeks, about 120 minutes to about 168 hours, about 120 minutes to about 162 hours, about 120 minutes to about 156 hours, about 120 minutes to about 150 hours, about 120 minutes to about 144 hours, about 120 minutes to about 138 hours, about 120 minutes to about 132 hours, about 120 minutes to about 126 hours, about 120 minutes to about 120 hours, about 120 minutes to about 114 hours, about 120 minutes to about 108 hours, about 120 minutes to about 102 hours, about 120 minutes to about 96 hours, about 120 minutes to about 90 hours, about 120 minutes to about 84 hours, about 120 minutes to about 78 hours, about 120 minutes to about 72 hours, about 120 minutes to about 66 hours, about 120 minutes to about 60 hours, about 120 minutes to about 54 hours, about 120 minutes to about 48hours, about 120 minutes to about 42 hours, about 120 minutes to about 36 hours, about 120 minutes to about 30 hours, about 120 minutes to about 24 hours, from about 120 minutes to about 18 hours, from about 120 minutes to about 12 hours, from about 120 minutes to about 6 hours, from about 120 minutes to about 5 hours, from about 120 minutes to about 240 minutes, from about 120 minutes to about 210 minutes, from about 120 minutes to about 180 minutes, from about 120 minutes to about 150 minutes, from about 120 minutes to about 130 minutes, from about 120 minutes to about 140 minutes, from about 130 minutes to about 150 minutes, from about 140 minutes to about 150 minutes; about 150 minutes to about 2 weeks, about 150 minutes to about 168 hours, about 150 minutes to about 162 hours, about 150 minutes to about 156 hours, about 150 minutes to about 150 hours, about 150 minutes to about 144 hours, about 150 minutes to about 138 hours, about 150 minutes to about 132 hours, about 150 minutes to about 126 hours, about 150 minutes to about 120 hours, about 150 minutes to about 114 hours, about 150 minutes to about 108 hours, about 150 minutes to about 102 hours, about 150 minutes to about 96 hours, about 150 minutes to about 90 hours, about 150 minutes to about 84 hours, about 150 minutes to about 78 hours, about 150 minutes to about 72 hours, about 150 minutes to about 66 hours, about 150 minutes to about 60 hours, about 150 minutes to about 54 hours, about 150 minutes to about 48hours, about 150 minutes to about 42 hours, about 150 minutes to about 36 hours, about 150 minutes to about 30 hours, about 150 minutes to about 24 hours, from about 150 minutes to about 18 hours, from about 150 minutes to about 12 hours, from about 150 minutes to about 6 hours, from about 150 minutes to about 5 hours, from about 150 minutes to about 240 minutes, from about 150 minutes to about 210 minutes, from about 150 minutes to about 180 minutes; about 180 minutes to about 2 weeks, about 180 minutes to about 168 hours, about 180 minutes to about 162 hours, about 180 minutes to about 156 hours, about 180 minutes to about 150 hours, about 180 minutes to about 144 hours, about 180 minutes to about 138 hours, about 180 minutes to about 132 hours, about 180 minutes to about 126 hours, about 180 minutes to about 120 hours, about 180 minutes to about 114 hours, about 180 minutes to about 108 hours, about 180 minutes to about 102 hours, about 180 minutes to about 96 hours, about 180 minutes to about 90 hours, about 180 minutes to about 84 hours, about 180 minutes to about 78 hours, about 180 minutes to about 72 hours, about 180 minutes to about 66 hours, about 180 minutes to about 60 hours, about 180 minutes to about 54 hours, about 180 minutes to about 48hours, about 180 minutes to about 42 hours, about 180 minutes to about 36 hours, about 180 minutes to about 30 hours, about 180 minutes to about 24 hours, about 180 minutes to about 18 hours, about 180 minutes to about 12 hours, about 180 minutes to about 6 hours, about 180 minutes to about 5 hours, about 180 minutes to about 240 minutes, about 180 minutes to about 210 minutes, about 180 minutes to about 200 minutes, about 180 minutes to about 190 minutes, about 190 minutes to about 200 minutes, about 190 minutes to about 210 minutes; about 210 minutes to about 2 weeks, about 210 minutes to about 168 hours, about 210 minutes to about 162 hours, about 210 minutes to about 156 hours, about 210 minutes to about 150 hours, about 210 minutes to about 144 hours, about 210 minutes to about 138 hours, about 210 minutes to about 132 hours, about 210 minutes to about 126 hours, about 210 minutes to about 120 hours, about 210 minutes to about 114 hours, about 210 minutes to about 108 hours, about 210 minutes to about 102 hours, about 210 minutes to about 96 hours, about 210 minutes to about 90 hours, about 210 minutes to about 84 hours, about 210 minutes to about 78 hours, about 210 minutes to about 72 hours, about 210 minutes to about 66 hours, about 210 minutes to about 60 hours, about 210 minutes to about 54 hours, about 210 minutes to about 48hours, about 210 minutes to about 42 hours, about 210 minutes to about 36 hours, about 210 minutes to about 30 hours, about 210 minutes to about 24 hours, from about 210 minutes to about 18 hours, from about 210 minutes to about 12 hours, from about 210 minutes to about 6 hours, from about 210 minutes to about 5 hours, from about 210 minutes to about 240 minutes, from about 210 minutes to about 230 minutes, from about 210 minutes to about 220 minutes; about 240 minutes to about 2 weeks, about 240 minutes to about 168 hours, about 240 minutes to about 162 hours, about 240 minutes to about 156 hours, about 240 minutes to about 150 hours, about 240 minutes to about 144 hours, about 240 minutes to about 138 hours, about 240 minutes to about 132 hours, about 240 minutes to about 126 hours, about 240 minutes to about 120 hours, about 240 minutes to about 114 hours, about 240 minutes to about 108 hours, about 240 minutes to about 102 hours, about 240 minutes to about 96 hours, about 240 minutes to about 90 hours, about 240 minutes to about 84 hours, about 240 minutes to about 78 hours, about 240 minutes to about 72 hours, about 240 minutes to about 66 hours, about 240 minutes to about 60 hours, about 240 minutes to about 54 hours, about 240 minutes to about 48hours, about 240 minutes to about 42 hours, about 240 minutes to about 36 hours, about 240 minutes to about 30 hours, about 240 minutes to about 24 hours, about 240 minutes to about 18 hours, about 240 minutes to about 12 hours, about 240 minutes to about 6 hours, about 240 minutes to about 5 hours, about 240 minutes to about 280 minutes, about 240 minutes to about 270 minutes, about 240 minutes to about 260 minutes, about 240 minutes to about 250 minutes; from about 270 minutes to about 280 minutes, from about 270 minutes to about 290 minutes, from about 270 minutes to about 5 hours; about 5 hours to about 2 weeks, about 5 hours to about 168 hours, about 5 hours to about 162 hours, about 5 hours to about 156 hours, about 5 hours to about 150 hours, about 5 hours to about 144 hours, about 5 hours to about 138 hours, about 5 hours to about 132 hours, about 5 hours to about 126 hours, about 5 hours to about 120 hours, about 5 hours to about 114 hours, about 5 hours to about 108 hours, about 5 hours to about 102 hours, about 5 hours to about 96 hours, about 5 hours to about 90 hours, about 5 hours to about 84 hours, about 5 hours to about 78 hours, about 5 hours to about 72 hours, about 5 hours to about 66 hours, about 5 hours to about 60 hours, about 5 hours to about 54 hours, about 5 hours to about 48hours, about 5 hours to about 42 hours, about 5 hours to about 36 hours, about 5 hours to about 30 hours, about 5 hours to about 24 hours, about 5 hours to about 18 hours, about 5 hours to about 12 hours, about 5 hours to about 11 hours, about 5 hours to about 10 hours, about 5 hours to about 9 hours, about 5 hours to about 8hours, about 5 hours to about 7 hours, about 5 hours to about 6 hours; from about 6 hours to about 12 hours, from about 6 hours to about 8hours, from about 6 hours to about 10 hours, from about 8hours to about 12 hours; about 8hours to about 2 weeks, about 8hours to about 168 hours, about 8hours to about 162 hours, about 6 hours to about 156 hours, about 6 hours to about 150 hours, about 6 hours to about 144 hours, about 6 hours to about 138 hours, about 6 hours to about 132 hours, about 6 hours to about 126 hours, about 6 hours to about 120 hours, about 6 hours to about 114 hours, about 6 hours to about 108 hours, about 6 hours to about 102 hours, about 6 hours to about 96 hours, about 6 hours to about 90 hours, about 6 hours to about 84 hours, about 6 hours to about 78 hours, about 6 hours to about 72 hours, about 6 hours to about 66 hours, about 6 hours to about 60 hours, about 6 hours to about 54 hours, about 6 hours to about 48hours, about 6 hours to about 42 hours, about 6 hours to about 36 hours, about 6 hours to about 30 hours, about 6 hours to about 24 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 11 hours, about 6 hours to about 10 hours, about 6 hours to about 9 hours, about 6 hours to about 8hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours; about 12 hours to about 2 weeks, about 12 hours to about 168 hours, about 12 hours to about 162 hours, about 12 hours to about 156 hours, about 12 hours to about 150 hours, about 12 hours to about 144 hours, about 12 hours to about 138 hours, about 12 hours to about 132 hours, about 12 hours to about 126 hours, about 12 hours to about 120 hours, about 12 hours to about 114 hours, about 12 hours to about 108 hours, about 12 hours to about 102 hours, about 12 hours to about 96 hours, about 12 hours to about 90 hours, about 12 hours to about 84 hours, about 12 hours to about 78 hours, about 12 hours to about 72 hours, about 12 hours to about 66 hours, about 12 hours to about 60 hours, about 12 hours to about 54 hours, about 12 hours to about 48hours, about 12 hours to about 42 hours, about 12 hours to about 36 hours, about 12 hours to about 30 hours, about 12 hours to about 24 hours, about 12 hours to about 18 hours, about 12 hours to about 14 hours, about 12 hours to about 16 hours, about 14 hours to about 18 hours, about 14 hours to about 24 hours, about 14 hours to about 36 hours, about 14 hours to about 48hours, about 14 hours to about 36 hours, about 16 hours to about 18 hours, about 16 hours to about 20 hours, about 16 hours to about 24 hours, about 16 hours to about 36 hours, about 16 hours to about 48hours, about 18 hours to about 24 hours, about 18 hours to about 36 hours, about 18 hours to about 48 hours; about 24 hours to about 2 weeks, about 24 hours to about 168 hours, about 24 hours to about 162 hours, about 24 hours to about 156 hours, about 24 hours to about 150 hours, about 24 hours to about 144 hours, about 24 hours to about 138 hours, about 24 hours to about 132 hours, about 24 hours to about 126 hours, about 24 hours to about 120 hours, about 24 hours to about 114 hours, about 24 hours to about 108 hours, about 24 hours to about 102 hours, about 24 hours to about 96 hours, about 24 hours to about 90 hours, about 24 hours to about 84 hours, about 24 hours to about 78 hours, about 24 hours to about 72 hours, about 24 hours to about 66 hours, about 24 hours to about 60 hours, about 24 hours to about 54 hours, about 24 hours to about 48hours, about 24 hours to about 42 hours, about 24 hours to about 36 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours; about 26 hours to about 28 hours, about 26 hours to about 30 hours, about 26 hours to about 36 hours, about 26 hours to about 48hours, about 28 hours to about 30 hours, about 28 hours to about 36 hours, about 28 hours to about 48hours, about 30 hours to about 36 hours, about 30 hours to about 48 hours; about 36 hours to about 2 weeks, about 36 hours to about 168 hours, about 36 hours to about 162 hours, about 36 hours to about 156 hours, about 36 hours to about 150 hours, about 36 hours to about 144 hours, about 36 hours to about 138 hours, about 36 hours to about 132 hours, about 36 hours to about 126 hours, about 36 hours to about 120 hours, about 36 hours to about 114 hours, about 36 hours to about 108 hours, about 36 hours to about 102 hours, about 36 hours to about 96 hours, about 36 hours to about 90 hours, about 36 hours to about 84 hours, about 36 hours to about 78 hours, about 36 hours to about 72 hours, about 36 hours to about 66 hours, about 36 hours to about 60 hours, about 36 hours to about 54 hours, about 36 hours to about 48hours, about 36 hours to about 42 hours, about 36 hours to about 40 hours, about 40 hours to about 42 hours; about 48hours to about 2 weeks, about 48hours to about 168 hours, about 48hours to about 162 hours, about 48hours to about 156 hours, about 48hours to about 150 hours, about 48hours to about 144 hours, about 48hours to about 138 hours, about 48hours to about 132 hours, about 48hours to about 126 hours, about 48hours to about 120 hours, about 48hours to about 114 hours, about 48hours to about 108 hours, about 48hours to about 102 hours, about 48hours to about 96 hours, about 48hours to about 90 hours, about 48hours to about 84 hours, about 48hours to about 78 hours, about 48hours to about 72 hours, about 48hours to about 66 hours, about 48hours to about 60 hours, about 48hours to about 54 hours; about 60 hours to about 2 weeks, about 60 hours to about 168 hours, about 60 hours to about 162 hours, about 60 hours to about 156 hours, about 60 hours to about 150 hours, about 60 hours to about 144 hours, about 60 hours to about 138 hours, about 60 hours to about 132 hours, about 60 hours to about 126 hours, about 60 hours to about 120 hours, about 60 hours to about 114 hours, about 60 hours to about 108 hours, about 60 hours to about 102 hours, about 60 hours to about 96 hours, about 60 hours to about 90 hours, about 60 hours to about 84 hours, about 60 hours to about 78 hours, about 60 hours to about 72 hours, about 60 hours to about 66 hours; about 72 hours to about 2 weeks, about 72 hours to about 168 hours, about 72 hours to about 162 hours, about 72 hours to about 156 hours, about 72 hours to about 150 hours, about 72 hours to about 144 hours, about 72 hours to about 138 hours, about 72 hours to about 132 hours, about 72 hours to about 126 hours, about 72 hours to about 120 hours, about 72 hours to about 114 hours, about 72 hours to about 108 hours, about 72 hours to about 102 hours, about 72 hours to about 96 hours, about 72 hours to about 90 hours, about 72 hours to about 84 hours, about 72 hours to about 78 hours; about 84 hours to about 2 weeks, about 84 hours to about 168 hours, about 84 hours to about 162 hours, about 84 hours to about 156 hours, about 84 hours to about 150 hours, about 84 hours to about 144 hours, about 84 hours to about 138 hours, about 84 hours to about 132 hours, about 84 hours to about 126 hours, about 84 hours to about 120 hours, about 84 hours to about 114 hours, about 84 hours to about 108 hours, about 84 hours to about 102 hours, about 84 hours to about 96 hours, about 84 hours to about 90 hours; about 96 hours to about 2 weeks, about 96 hours to about 168 hours, about 96 hours to about 162 hours, about 96 hours to about 156 hours, about 96 hours to about 150 hours, about 96 hours to about 144 hours, about 96 hours to about 138 hours, about 96 hours to about 132 hours, about 96 hours to about 126 hours, about 96 hours to about 120 hours, about 96 hours to about 114 hours, about 96 hours to about 108 hours, about 96 hours to about 102 hours; about 108 hours to about 2 weeks, about 108 hours to about 168 hours, about 108 hours to about 162 hours, about 108 hours to about 156 hours, about 108 hours to about 150 hours, about 108 hours to about 144 hours, about 108 hours to about 138 hours, about 108 hours to about 132 hours, about 108 hours to about 126 hours, about 108 hours to about 120 hours, about 108 hours to about 114 hours; about 120 hours to about 2 weeks, about 120 hours to about 168 hours, about 120 hours to about 162 hours, about 120 hours to about 156 hours, about 120 hours to about 150 hours, about 120 hours to about 144 hours, about 120 hours to about 138 hours, about 120 hours to about 132 hours, about 120 hours to about 126 hours; about 132 hours to about 2 weeks, about 132 hours to about 168 hours, about 132 hours to about 162 hours, about 132 hours to about 156 hours, about 132 hours to about 150 hours, about 132 hours to about 144 hours, about 132 hours to about 138 hours; about 144 hours to about 2 weeks, about 144 hours to about 168 hours, about 144 hours to about 162 hours, about 144 hours to about 156 hours, about 144 hours to about 150 hours; about 156 hours to about 2 weeks, about 156 hours to about 168 hours, about 156 hours to about 162 hours; or about 168 hours to about 2 weeks.

In some embodiments of the contacting step of any of the methods provided herein, the period of time occurs when the liquid cell culture is in its exponential growth phase. In some embodiments of the contacting step of any of the methods provided herein, the period of time occurs while the liquid cell culture is in its quiescent growth stage. In some embodiments, the contacting can occur while the liquid cell culture is in its death phase. In some embodiments of the contacting step of any of the methods provided herein, the period of time occurs between day 4 and day 5 of the cell culture.

In some embodiments of any of the methods described herein, the contacting step can comprise contacting one or more pods (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) sequentially or simultaneously with the liquid cell culture for a period of time (e.g., any period of time described herein).

Recombinant proteins

Recombinant proteins described herein include, but are not limited to, receptors, antibodies, antigen-binding antibody fragments, fusion proteins, ligands, secreted proteins, enzymes, recombinant protein drug products, and any recombinant protein with or without a tag. Non-limiting examples of tags include: fluorescent proteins (e.g., green fluorescent protein, red fluorescent protein, cyan fluorescent protein, yellow fluorescent protein), radioisotopes, His tags, FLAG tags, c-myc tags, Maltose Binding Protein (MBP) tags, Albumin Binding Protein (ABP) tags, bacteriophage T7 epitopes (T7 tags), Biotin Carboxyl Carrier Protein (BCCP) tags, bluetongue virus tags (B tags), calmodulin binding peptide tags,a cellulose binding domain tag, a chitin binding domain tag, a choline binding domain tag, a galactose binding protein tag, a Glu-Glu epitope tag, a human influenza hemagglutinin tag,

histidine Affinity TAG (HAT), polyarginine TAG (Asp-TAG), polyphenylalanine TAG (Phe-TAG), S-TAG, streptavidin-binding peptide, streptavidin TAG, streptomyces TAG, enzymes (e.g., alkaline phosphatase, Chloramphenicol Acetyltransferase (CAT), glutathione S-transferase). In some embodiments, the recombinant protein is a multimeric protein (e.g., a heterodimeric protein, a homodimeric protein, or a trimeric protein).

Exemplary recombinant antibodies and recombinant antibody fragments that can be expressed in any of the cells described herein can include recombinant IgG1 (e.g., recombinant human IgG1), recombinant IgG2 (e.g., recombinant human IgG2), recombinant IgG3 (e.g., recombinant human IgG3), recombinant IgG4 (e.g., recombinant human IgG4), recombinant IgM (e.g., recombinant human IgM), recombinant IgE (e.g., recombinant human IgE), recombinant IgD (e.g., recombinant human IgD), recombinant IgA (e.g., recombinant human IgA), recombinant Fab, recombinant scFv, recombinant VNAR, recombinant VHH, recombinant diabody (e.g., recombinant bispecific diabody), recombinant scFv-Fc, recombinant IgNAR, recombinant bispecific diabody, recombinant light chain variable domain, recombinant heavy chain variable domain, recombinant triabody, recombinant tetrabody, tandem recombinant scFv, recombinant tandem scFv-Fc, recombinant ant, ant-Fc polylinker, recombinant scFv-Fc, recombinant F (ab ') 2, recombinant Fab-scFv, recombinant (Fab' scFv)₂Recombinant scDiabody-Fc, recombinant scDiabody-CH3, recombinant IgG-scFv, recombinant V-IgG, recombinant IgG-V, recombinant two-in-one IgG, recombinant double V domain IgG and recombinant multiple-junctions pore with common light chain.

In some embodiments, the recombinant protein is an antibody. As used herein, the term "antibody" refers to an intact or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule. Antibodies are tetrameric in nature, comprising two identical heavy chain protein portions and two identical light chain protein portions joined together to adopt a "Y" shaped conformation linked together by disulfide bonds in the hinge region. The term "antibody" includes monoclonal, chimeric or chimeric antibodies, humanized antibodies, deimmunized human antibodies and fully human antibodies. Antibodies can be made in, or derived from, a variety of species, e.g., mammals, e.g., humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. In some embodiments, the antibodies may be produced synthetically, e.g., via synthetically produced oligonucleotides, as described, e.g., Fuh et al, j.mol.biol.340:1073-1093, 2004 and Knappik et al, j.mol.biol.296(11):57-86, 2000, the disclosures of each of which are incorporated herein by reference in their entirety. In some embodiments, antibodies can be produced by antibody phage display in yeast and/or human cells.

As used herein, the term "antibody fragment," "antigen-binding fragment," or similar terms refer to a fragment of an antibody that retains the ability to bind an antigen, e.g., a single chain antibody (scFv), an Fd fragment, an Fab 'fragment, or an F (ab')₂And (3) fragment. An scFv is a single polypeptide chain comprising both the heavy chain variable region and the light chain variable region of an antibody from which the scFv is derived. In addition, diabodies (Poljak, Structure 2 (12): 1121-1123, 1994; Hudson et al, J.Immunol. methods 23 (1-2): 177-189, 1999, the disclosures of both of which are incorporated herein by reference in their entirety), miniantibodies, triabodies (Schonooghe et al, BMC Biotechnol.9:70, 2009), domain antibodies (also known as "heavy chain immunoglobulins" or camelids; Holt et al, Trends Biotechnol.21(11):484-490, 2003); and in vivo antibodies (Huston et al, Humanantibodies 10 (3-4): 127-142, 2001; Wheeler et al, mol. Ther.8(3):355-366, 2003; Stocks, Drug Discov. today 9(22):960-966, 2004, the disclosure of each of which is incorporated herein by reference in its entirety) are encompassed by the definition of antibody fragments and can be incorporated into compositions and used in the methods described herein.

In some embodiments, the recombinant protein is a bispecific antibody. Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, having binding specificity for at least two different antigens.

Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy/light chain pairs, wherein the two heavy/light chain pairs have different specificities (Milstein and Cuello, Nature 305: 537-539, 1983). Antibody variable domains (antibody-antigen binding sites) with the desired binding specificity can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably to an immunoglobulin heavy chain constant domain, comprising at least a portion of the hinge, CH2 and CH3 regions. For further details of exemplary currently known Methods for generating bispecific antibodies, see, e.g., Suresh et al, Methods enzymol.121:210, 1986; WO 96/27011; brennan et al, Science 229: 81, 1985; shalaby et al, J.Exp.Med.175:217-225, 1992; kostelny et al, J.Immunol.148(5):1547-1553, 1992; hollinger et al, Proc.Natl.Acad.Sci.U.S.A.90:6444-6448, 1993; gruber et al, J.Immunol.152:5368, 1994; and Tutt et al, J.Immunol.147:60, 1991.

Various techniques have also been described for the preparation and isolation of bispecific antibody fragments directly from recombinant cell cultures. For example, bispecific antibodies have been produced using leucine zippers. See, for example, Kostelny et al, J.Immunol.148(5):1547-1553, 1992. Leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448, 1993 provides an alternative mechanism for the preparation of bispecific antibody fragments. Fragments include a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al, J.Immunol.152:5368, 1994. Alternatively, the antibody may be a "linear antibody" as described, for example, in Zapata et al, ProteinEng.8(10):1057-1062, 1995. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies having more than two valency states (e.g., trispecific antibodies) are contemplated and described, for example, in Tutt et al, J.Immunol.147:60, 1991.

The present disclosure also includes variant forms of multispecific antibodies, such as the double variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al, Nat Biotechnol.25(11):1290-1297, 2007. DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are connected together in tandem either directly or through a short linker by recombinant DNA technology, followed by a light chain constant domain. Similarly, a heavy chain comprises two different heavy chain variable domains (VH) connected in series, followed by a constant domain CH1 and an Fc region. Methods for preparing DVD-Ig molecules from two parent antibodies are further described, for example, in WO 08/024188 and WO 07/024715. In some embodiments, the bispecific antibody is a tandem Fabs immunoglobulin in which a light chain variable region with the second specificity is fused to a heavy chain variable region of a whole antibody. Such antibodies are described, for example, in WO 2015/103072.

In some embodiments, the recombinant protein is a nanobody, such as a camelid or dromedary antibody (e.g., an antibody derived from Camelus bactrianus, Calelus hydromaderius, or Lama paccos). Unlike typical two-chain (fragment) or four-chain (whole antibody) antibodies of most mammals, such antibodies often lack a light chain. See U.S. patent nos. 5,759,808; stijlemans et al, J.biol.chem.279:1256-1261, 2004; dumoulin et al, Nature 424:783-788, 2003 and Pleschberger et al, Bioconjugate chem.14:440-448, 2003. Like other antibodies of non-human origin, the amino acid sequence of camelid antibodies can be recombinantly altered to obtain sequences more similar to human sequences, i.e., nanobodies can be "humanized" to further reduce the potential immunogenicity of the antibody.

In some embodiments, the recombinant protein is a non-antibody scaffold protein. Typically, these proteins are obtained by modification of existing antigen binding proteins based on combinatorial chemistry. For example, the binding sites of human transferrin to human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. Ali et al, J.biol.chem.274:24066-24073, 1999. The portion of human transferrin not involved in binding to the receptor remains unchanged and serves as a scaffold, such as the framework regions of antibodies, to present variant binding sites.

One skilled in the art will appreciate that the scaffold portion of a non-antibody scaffold protein may include, for example, all or part of: s. aureus protein a Z domain, human transferrin, human tenth fibronectin type III domain, kunitz human trypsin inhibitor, human CTLA-4, ankyrin repeat protein, human lipocalins (e.g., anticalins), human crystallin, human ubiquitin or trypsin inhibitor from e.

In some embodiments, the recombinant protein is a fusion protein. Non-limiting examples of fusion proteins that can be produced by any of the cells described herein include chimeric antibodies (e.g., humanized antibodies), labeled antibodies (e.g., antibodies labeled with an enzyme or fluorescent protein tag), protein ligands that specifically bind to cancer cell surface receptors, wherein the protein ligands are labeled with a protein cytotoxin, antibodies with fusion partners that can increase the half-life of the antibody in vivo (e.g., antibodies fused to human albumin), bispecific antibodies in which one light chain variable domain and one heavy chain variable domain bind to an antigen on an immune cell (e.g., T cell, macrophage, neutrophil, or monocyte) and the other light chain variable domain and the other heavy chain variable domain bind to an antigen or pathogen on a target cell (e.g., cancer cell or virus-infected cell) (e.g., viruses, mycobacteria or parasites).

Other examples of recombinant proteins include, but are not limited to, ligands, enzymes, peptides, scfvs, non-antibody scaffold proteins, chimeric antigen receptor molecules, etc., wherein a diversity element is fused in frame with one or more constant elements. In some embodiments, the recombinant protein is a multimeric protein, such as, but not limited to, a Fab, a whole antibody, an MHC class I molecule, a T cell receptor molecule, a bispecific antibody, a trispecific antibody, an integrin, a tumor growth factor receptor dimer, a G protein-coupled receptor, and the like.

Cells

The liquid cell culture may comprise any cell, such as a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having distinct membrane-bound nuclei. Such cells can include, for example, mammalian (e.g., rodent, non-human primate or human), insect, fungal or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as a mammalian, avian, plant, or insect cell.

Non-limiting examples of mammalian cells that can produce recombinant proteins include, for example, rodent (e.g., mouse, rat, or hamster) or human cells. In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, a human retinal per.c6 cell, a human ammonia cell CAP cell, a mouse melanoma NS0 or Sp2/0 cell, a duck EB66 cell, a hamster BHK cell, a human embryonic kidney (HEK 293) cell, a COS cell, or a chicken derived B cell line DT 40. In some embodiments, the HEK293 cell is a freestyle HEK293-F cell, HEK 2936F, HEK293FT, HEK 293T cell, or Expi293F cell. CHO cells are epithelial cells that can grow as an attached monolayer or in suspension. CHO cell lines derived from primary cells isolated from Tjio and Puck (Tjio et al, J.Exp.Med.108:259-271, 1958) may include point mutations, deletions or integrations of nucleic acid sequences relative to the primary CHO cell line. A subgroup of CHO cells may include, but is not limited to, CHO DP-12 cells, CHO-K1 cells, CHO/dhfr^-Cells, CHO-S cells, CHO-GS cells, CHO-K1 DUX B11 cells, CHO pro3^-Cells, CHO-DG44 cells, CHO GAT^-Cells or freestyle CHO-S cells. In some embodiments, the eukaryotic cells areThe cell is a primary cell or cell line. In some embodiments, the mammalian cell is a cancer cell. In some embodiments, the mammalian cell is a human cancer cell line, including those listed in the "cancer cell line encyclopedia" (Barretina et al, Nature 483(7391):603-607, 2012 or F or bes et al, curr. In some embodiments, the eukaryotic cell is a T lymphocyte lineage cell (e.g., a primary T cell or T cell line), a T cell derived cell line lacking TCR expression (see, e.g., T or res et al, j.immunol.2003) or a B lymphocyte lineage cell, such as a pro-B cell (see, e.g., Urashima et al, am.j.hematol.1994), pre-B cell (see, e.g., Adachi et al, dnacellbiol.2006) and B cell, as well as cell lines derived therefrom. In some embodiments, the B lineage cell line is a murine pre-B cell line, such as 1624-5(Holms et al, J.Exp.Med.1986; Principato et al, mol.cell biol.1990), or a pro-B cell line, such as Ba/F3 (commercially available from Thermo Scientific). Mammalian cells can express secreted forms of recombinant proteins. In some embodiments, the secreted form of the recombinant protein is transiently expressed. In some embodiments, a secreted form of the recombinant protein is stably expressed.

In some embodiments, the liquid cell culture can include insect cells (e.g., baculovirus-insect cells, drosophila cells (e.g., drosophila S2 cells), trichomonas cells, lepidopteran cells), yeast cells (e.g., saccharomyces cerevisiae cells, pichia pastoris cells), plant cells, or bacterial cells (e.g., escherichia coli cells, bacillus brevis cells, bacillus megaterium cells, bacillus subtilis cells, bacillus crescentus cells).

The skilled artisan will appreciate that the proteins and vectors described herein can be introduced into a cell (e.g., a mammalian cell, an insect cell, a yeast cell, a bacterial cell, or a plant cell). Non-limiting examples of vectors and methods for introducing vectors into cells are described herein.

Method for culturing cells

Methods of culturing cells are well known in the art. Cells can be maintained in vitro under conditions conducive to growth, proliferation, sustained viability and differentiation. In general, cells can be cultured by contacting the cells with a liquid medium that includes the necessary growth factors and supplements to support viability and growth of the cells. The term "liquid medium" refers to a fluid containing sufficient nutrients to allow cells (e.g., any of the cells described herein) to grow or proliferate in vitro. For example, the liquid medium may comprise one or more of: amino acids (e.g., 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, pyridoxine, riboflavin, thymine, cyanocobalamin, pyruvic acid, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.

In some embodiments, the liquid culture medium may comprise serum from a mammal. In some embodiments, the liquid culture medium does not comprise serum or another extract from a mammal (defined liquid culture medium). In some embodiments, the liquid culture medium may comprise trace metals, mammalian growth hormones and/or mammalian growth factors. Another example of a liquid medium is a minimal medium (e.g., a medium comprising only inorganic salts, a carbon source, and water). Non-limiting examples of liquid media are described herein. Other examples of liquid media are known in the art and are commercially available. The liquid cell culture medium can comprise any density of any cell described herein (e.g., any density of mammalian cells).

As used herein, the term "animal-derived component-free liquid medium" refers to a liquid medium that does not contain any components (e.g., proteins or serum) derived from a mammal. As used herein, the term "serum-free liquid medium" refers to a liquid medium that does not contain mammalian serum. As used herein, the term "serum-containing liquid medium" refers to a liquid medium comprising mammalian serum. As used herein, the term "chemically defined liquid medium" is a term of art and refers to a liquid medium in which all chemical components are known. For example, chemically-defined liquid media do not contain fetal bovine serum, bovine serum albumin, or human serum albumin, as these formulations typically contain a complex mixture of albumin and lipids. As used herein, the term "protein-free liquid medium" refers to a liquid medium that does not contain any protein (e.g., any detectable protein).

In some embodiments, the cells may be cultured at a temperature of about 20 ℃ to about 40 ℃ (e.g., about 39 ℃, about 38 ℃, about 37 ℃, about 36 ℃, or about 35 ℃ (inclusive), about 34 ℃ to about 40 ℃, about 39 ℃, about 38 ℃, about 37 ℃, about 36 ℃, about 34 ℃, about 32 ℃, about 30 ℃, about 28 ℃, about 26 ℃, about 24 ℃, about 22 ℃, about 20 ℃ (inclusive), about 20 ℃ to about 40 ℃, about 20 ℃ to about 38 ℃, about 20 ℃ to about 36 ℃, about 20 ℃ to about 34 ℃, about 20 ℃ to about 32 ℃, about 20 ℃ to about 30 ℃, about 20 ℃ to about 28 ℃, about 20 ℃ to about 26 ℃, about 20 ℃ to about 24 ℃, about 20 ℃ to about 22 ℃ (inclusive), about 22 ℃ to about 38 ℃, about 22 ℃ to about 36 ℃, about 22 ℃ to about 34 ℃, about 22 ℃ to about 32 ℃, about 22 ℃ to about 30 ℃, about 22 ℃ to about 28 ℃, about 22 ℃ to about 26 ℃, about 22 ℃ to about 24 ℃ (inclusive); about 24 ℃ to about 40 ℃, about 24 ℃ to about 38 ℃, about 24 ℃ to about 36 ℃, about 24 ℃ to about 34 ℃, about 24 ℃ to about 32 ℃, about 24 ℃ to about 30 ℃, about 24 ℃ to about 28 ℃, about 24 ℃ to about 26 ℃ (inclusive); about 26 ℃ to about 40 ℃, about 26 ℃ to about 38 ℃, about 26 ℃ to about 36 ℃, about 26 ℃ to about 34 ℃, about 26 ℃ to about 32 ℃, about 26 ℃ to about 30 ℃, about 26 ℃ to about 28 ℃ (inclusive); from about 28 ℃ to about 40 ℃, from about 28 ℃ to about 38 ℃, from about 28 ℃ to about 36 ℃, from about 28 ℃ to about 34 ℃, from about 28 ℃ to about 32 ℃, from about 28 ℃ to about 30 ℃, from about 30 ℃ to about 40 ℃, from about 30 ℃ to about 38 ℃, from about 30 ℃ to about 36 ℃, from about 30 ℃ to about 34 ℃, from about 30 ℃ to about 32 ℃ (inclusive); from about 32 ℃ to about 40 ℃, from about 32 ℃ to about 38 ℃, from about 32 ℃ to about 36 ℃, from about 32 ℃ to about 34 ℃, from about 34 ℃ to about 40 ℃, from about 34 ℃ to about 38 ℃, from about 34 ℃ to about 36 ℃, from about 36 ℃ to about 40 ℃, from about 36 ℃ to about 38 ℃, from about 38 ℃ to about 40 ℃ (inclusive); about 35 ℃ to about 40 ℃, about 39 ℃, about 38 ℃, or about 37 ℃ (inclusive); about 36 ℃ to about 40 ℃, about 39 ℃, or about 38 ℃ (inclusive); about 37 ℃ to about 40 DEG COr about 39 ℃ (inclusive); or about 38 ℃ to about 40 ℃ (inclusive)), at about 5% to about 12% (e.g., about 5% to about 10%, about 5% to about 8%, about 5% to about 7%, about 6% to about 8%, about 6% to about 10%, or about 8% to about 10%) of CO₂For longer periods of time, e.g., from about 1 day to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, about 6 days, about 4 days, or about 2 days (inclusive); from about 2 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, about 6 days, or about 4 days (inclusive); from about 4 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, or about 6 days (inclusive); from about 6 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, or about 8 days (inclusive); from about 8 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, or about 10 days (inclusive); from about 10 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, or about 15 days (inclusive); about 15 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, or about 20 days (inclusive); about 20 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days,about 20 days, or about 25 days (inclusive); about 25 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, or about 30 days (inclusive); about 30 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, or about 35 days (inclusive); about 35 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, or about 40 days (inclusive); about 40 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, or about 50 days (inclusive); from about 50 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, or about 55 days (inclusive); from about 55 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, or about 60 days (inclusive); from about 60 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, or about 65 days (inclusive); from about 65 days to about 90 days, about 85 days, about 80 days, about 75 days, or about 70 days (inclusive); from about 70 days to about 90 days, about 85 days, about 80 days, or about 75 days (inclusive); from about 75 days to about 90 days, about 85 days, or about 80 days (inclusive); from about 80 days to about 90 days, or about 85 days (inclusive); or from about 85 days to about 90 days inclusive. In some embodiments, the liquid cell culture is agitated for a period of time during the culturing (e.g., continuously, periodically). The term "agitation" refers to stirring or otherwise moving a portion of a liquid culture medium in a bioreactor. This is done, for example, to increase the dissolved oxygen concentration in the liquid culture medium in the bioreactor. Stirring can be performed using any method known in the art, such as an instrument (e.g., a rocking, oscillating, or inclined platform) or propeller. Exemplary devices and methods that can be used to agitate a portion of the liquid culture medium in a bioreactor are known in the art.

In some embodiments of any of the methods described herein, the liquid cell culture is a perfusion culture. The term "perfusion culture" is a term of art and refers to culturing cells present in a culture dish (e.g., any of the culture dishes described herein) by periodically or continuously removing a first liquid culture medium and simultaneously or shortly thereafter adding a substantially equal volume of a second liquid culture medium to the culture dish. In some examples, there is an incremental change (e.g., increase or decrease) in the volume of the first liquid culture medium removed and added over an incremental period of time (e.g., about a 24 hour period, a period of about 1 minute to about a 24 hour period, or a period of greater than 24 hours) during the culture (e.g., a medium feed rate per day). The proportion of medium removed and replaced daily can vary depending on the particular cells being cultured, the initial seeding density and the cell density over a particular period of time.

In some embodiments of any of the methods described herein, the liquid cell culture is a fed-batch culture. The term "fed-batch culture" is a term of art and refers to culturing cells present in a culture dish by periodically or continuously adding a second liquid culture medium to a first liquid culture medium without substantially removing the first liquid culture medium or the second liquid culture medium from the liquid cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of fed-batch cultures, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch cultures, the second liquid culture medium is added as a dry powder.

The liquid cell culture may be placed in a culture dish. Non-limiting examples of culture dishes include: shake flasks, shake tubes, wave bags, T-flasks, triple flasks, spinner flasks, roller bottles, bioreactors (e.g., perfusion bioreactors, batch bioreactors) or multi-well plates (e.g., 4-well cell culture plates, 6-well cell culture plates, 8-well cell culture plates, 12-well cell culture plates, 24-well cell culture plates).

In some embodiments, the total internal volume of the culture dish is about 0.2mL to10,000L (e.g., about 0.2mL to about 9,000L, about 0.2mL to about 8,000L, about 0.2mL to about 7,000L, about 0.2mL to about 6,000L, about 0.2mL to about 5,000L, about 0.2mL to about 4,000L, about 0.2mL to about 3,000L, about 0.2mL to about 2,000L, about 0.2mL to about 1,000L, about 0.2mL to about 950L, about 0.2mL to about 900L, about 0.2mL to about 850L, about 0.2mL to about 800L, about 0.2mL to about 750mL, about 0.2mL to about 700L, about 0.2mL to about 650mL, about 0.2mL to about 600L, about 0.2mL to about 800L, about 0.2mL to about 500mL, about 2mL to about 200L, about 0.2mL to about 200mL, about 2mL to about 200L, about 200mL to about 200mL, about 0.2mL to about 130L, about 0.2mL to about 120L, about 0.2mL to about 110L, about 0.2mL to about 100L, about 0.2mL to about 90L, about 0.2mL to about 85L, about 0.2mL to about 80L, about 0.2mL to about 75L, about 0.2mL to about 70L, about 0.2mL to about 65L, about 0.2mL to about 60L, about 0.2mL to about 55L, about 0.2mL to about 50L, about 0.2mL to about 45L, about 0.2mL to about 40L, about 0.2mL to about 35L, about 0.2mL to about 30L, about 0.2mL to about 25L, about 0.2mL to about 20L, about 0.2mL to about 15L, about 0.2mL to about 10L, about 0.2mL to about 5L, about 0.2mL to about 4L, about 0.2mL to about 2mL to about 4L, about 0.2mL to about 1L; about 0.2mL to about 900mL, about 0.2mL to about 800mL, about 0.2mL to about 700mL, about 0.2mL to about 600mL, about 0.2mL to about 500mL, about 0.2mL to about 400mL, about 0.2mL to about 300mL, about 0.2mL to about 250mL, about 0.2mL to about 100mL, about 0.2mL to about 90mL, about 0.2mL to about 80mL, about 0.2mL to about 70mL, about 0.2mL to about 60mL, about 0.2mL to about 50mL, about 0.2mL to about 40mL, about 0.2mL to about 30mL, about 0.2mL to about 25mL, about 0.2mL to about 20mL, about 0.2mL to about 15mL, about 0.2mL to about 10mL, about 0.2mL to about 5mL, about 0.2mL to about 4mL, about 0.2mL to about 2mL, about 0.2mL to about 1 mL; about 0.4mL to10,000L, about 0.4mL to about 9,000L, about 0.4mL to about 8,000L, about 0.4mL to about 7,000L, about 0.4mL to about 6,000L, about 0.4mL to about 5,000L, about 0.4mL to about 4,000L, about 0.4mL to about 3,000L, about 0.4mL to about 2,000L, about 0.4mL to about 1,000L, about 0.4mL to about 950L, about 0.4mL to about 900L, about 0.4mL to about 850L, about 0.4mL to about 800L, about 0.4mL to about 750L, about 0.4mL to about 700L, about 0.4mL to about 650L, about 0.4mL to about 600L, about 0.4mL to about 550L, about 0.4mL to about 0.500L, about 0.4mL to about 500L, about 0.4mL to about 200L, about 0.4mL to about 0.180L, about 0mL to about 4mL to about 200L, about 0.4mL to about 200L, about 0.4mL to about 130L, about 0.4mL to about 120L, about 0.4mL to about 110L, about 0.4mL to about 100L, about 0.4mL to about 90L, about 0.4mL to about 85L, about 0.4mL to about 80L, about 0.4mL to about 75L, about 0.4mL to about 70L, about 0.4mL to about 65L, about 0.4mL to about 60L, about 0.4mL to about 55L, about 0.4mL to about 50L, about 0.4mL to about 45L, about 0.4mL to about 40L, about 0.4mL to about 35L, about 0.4mL to about 30L, about 0.4mL to about 25L, about 0.4mL to about 20L, about 0.4mL to about 15L, about 0.4mL to about 10L, about 0.4mL to about 5L, about 0.4mL to about 4L, about 0.4mL to about 4mL to about 1L; about 0.4mL to about 900mL, about 0.4mL to about 800mL, about 0.4mL to about 700mL, about 0.4mL to about 600mL, about 0.4mL to about 500mL, about 0.4mL to about 400mL, about 0.4mL to about 300mL, about 0.4mL to about 250mL, about 0.4mL to about 100mL, about 0.4mL to about 90mL, about 0.4mL to about 80mL, about 0.4mL to about 70mL, about 0.4mL to about 60mL, about 0.4mL to about 50mL, about 0.4mL to about 40mL, about 0.4mL to about 30mL, about 0.4mL to about 25mL, about 0.4mL to about 20mL, about 0.4mL to about 15mL, about 0.4mL to about 10mL, about 0.4mL to about 5mL, about 0.4mL to about 4mL, about 1mL, about 0.4mL to about 2 mL; about 0.6mL to10,000L, about 0.6mL to about 9,000L, about 0.6mL to about 8,000L, about 0.6mL to about 7,000L, about 0.6mL to about 6,000L, about 0.6mL to about 5,000L, about 0.6mL to about 4,000L, about 0.6mL to about 3,000L, about 0.6mL to about 2,000L, about 0.6mL to about 1,000L, about 0.6mL to about 950L, about 0.6mL to about 900L, about 0.6mL to about 850L, about 0.6mL to about 800L, about 0.6mL to about 750L, about 0.6mL to about 700L, about 0.6mL to about 650L, about 0.6mL to about 600L, about 0.6mL to about 550L, about 0.6mL to about 0.500L, about 0.6mL to about 500L, about 0.6mL to about 6mL to about 200L, about 0.6mL to about 200L, about 6mL to about 200L, about 0.6mL to about 120L, about 0.6mL to about 110L, about 0.6mL to about 100L, about 0.6mL to about 90L, about 0.6mL to about 85L, about 0.6mL to about 80L, about 0.6mL to about 75L, about 0.6mL to about 70L, about 0.6mL to about 65L, about 0.6mL to about 60L, about 0.6mL to about 55L, about 0.6mL to about 50L, about 0.6mL to about 45L, about 0.6mL to about 40L, about 0.6mL to about 35L, about 0.6mL to about 30L, about 0.6mL to about 25L, about 0.6mL to about 20L, about 0.6mL to about 15L, about 0.6mL to about 10L, about 0.6mL to about 5L, about 0.6mL to about 4L, about 0.6mL to about 1L, about 0.6mL to about 6L; about 0.6mL to about 900mL, about 0.6mL to about 800mL, about 0.6mL to about 700mL, about 0.6mL to about 600mL, about 0.6mL to about 500mL, about 0.6mL to about 400mL, about 0.6mL to about 300mL, about 0.6mL to about 250mL, about 0.6mL to about 100mL, about 0.6mL to about 90mL, about 0.6mL to about 80mL, about 0.6mL to about 70mL, about 0.6mL to about 60mL, about 0.6mL to about 50mL, about 0.6mL to about 40mL, about 0.6mL to about 30mL, about 0.6mL to about 25mL, about 0.6mL to about 20mL, about 0.6mL to about 15mL, about 0.6mL to about 10mL, about 0.6mL to about 5mL, about 0.6mL to about 4mL, about 0.6mL to about 6mL, about 0.6mL to about 1mL, about 0.6mL to about 2 mL; about 0.8mL to10,000L, about 0.8mL to about 9,000L, about 0.8mL to about 8,000L, about 0.8mL to about 7,000L, about 0.8mL to about 6,000L, about 0.8mL to about 5,000L, about 0.8mL to about 4,000L, about 0.8mL to about 3,000L, about 0.8mL to about 2,000L, about 0.8mL to about 1,000L, about 0.5mL to about 950L, about 0.8mL to about 900L, about 0.8mL to about 850L, about 0.8mL to about 800L, about 0.8mL to about 750L, about 0.8mL to about 700L, about 0.8mL to about 650L, about 0.8mL to about 600L, about 0.8mL to about 550L, about 0.8mL to about 500L, about 0.8mL to about 200L, about 0.8mL to about 8mL to about 200L, about 0.8mL to about 200L, about 8mL to about 200L, about 0.8mL to about 120L, about 0.8mL to about 110L, about 0.8mL to about 100L, about 0.8mL to about 90L, about 0.8mL to about 85L, about 0.8mL to about 80L, about 0.8mL to about 75L, about 0.8mL to about 70L, about 0.8mL to about 65L, about 0.8mL to about 60L, about 0.8mL to about 55L, about 0.8mL to about 50L, about 0.8mL to about 45L, about 0.8mL to about 40L, about 0.8mL to about 35L, about 0.8mL to about 30L, about 0.8mL to about 25L, about 0.8mL to about 20L, about 0.8mL to about 15L, about 0.8mL to about 10L, about 0.8mL to about 5L, about 0.8mL to about 4L, about 0.8mL to about 1L, about 0.8mL to about 8L; about 0.8mL to about 900mL, about 0.8mL to about 800mL, about 0.8mL to about 700mL, about 0.8mL to about 600mL, about 0.8mL to about 500mL, about 0.8mL to about 400mL, about 0.8mL to about 300mL, about 0.8mL to about 250mL, about 0.8mL to about 100mL, about 0.8mL to about 90mL, about 0.8mL to about 80mL, about 0.8mL to about 70mL, about 0.8mL to about 60mL, about 0.8mL to about 50mL, about 0.8mL to about 40mL, about 0.8mL to about 30mL, about 0.8mL to about 25mL, about 0.8mL to about 20mL, about 0.8mL to about 15mL, about 0.8mL to about 10mL, about 0.8mL to about 5mL, about 0.8mL to about 4mL, about 0.8mL to about 8mL, about 0.8mL to about 1mL, about 0.8mL to about 2 mL; about 1mL to10,000L, about 1mL to about 9,000L, about 1mL to about 8,000L, about 1mL to about 7,000L, about 1mL to about 6,000L, about 1mL to about 5,000L, about 1mL to about 4,000L, about 1mL to about 3,000L, about 1mL to about 2,000L, about 1mL to about 1,000L, about 1mL to about 950L, about 1mL to about 900L, about 1mL to about 850L, about 1mL to about 800L, about 1mL to about 750L, about 1mL to about 700L, about 1mL to about 650L, about 1mL to about 600L, about 1mL to about 550L, about 1mL to about 500L, about 1mL to about 450L, about 1mL to about 400L, about 1mL to about 350L, about 1mL to about 300L, about 1mL to about 250L, about 1mL to about 200L, about 1mL to about 1L, about 1mL to about 180L, about 1mL to about 180L, about 1mL to about 130L, about 1mL to about 120L, about 1mL to about 110L, about 1mL to about 100L, about 1mL to about 90L, about 1mL to about 80L, about 1mL to about 70L, about 1mL to about 60L, about 1mL to about 50L, about 1mL to about 40L, about 1mL to about 30L, about 1mL to about 20L, about 1mL to about 10L, about 1mL to about 5L, about 1mL to about 4L, about 1mL to about 3L, about 1mL to about 2L, about 1mL to about 1L, about 1mL to about 900mL, about 1mL to about 800mL, about 1mL to about 700mL, about 1mL to about 600mL, about 1mL to about 500mL, about 1mL to about 400mL, about 1mL to about 300mL, about 1mL to about 250mL, about 1mL to about 100mL, about 1mL to about 90mL to about 60mL, about 1mL to about 40mL, about 1mL to about 1mL, about 1mL to about 30mL, about 1mL to about 25mL, about 1mL to about 20mL, about 1mL to about 15mL, about 1mL to about 10mL, about 1mL to about 5 mL; about 1mL to about 4mL, about 1mL to about 3mL, about 1mL to about 2 mL; about 2mL to10,000L, about 2mL to about 9,000L, about 2mL to about 8,000L, about 2mL to about 7,000L, about 2mL to about 6,000L, about 2mL to about 5,000L, about 2mL to about 4,000L, about 2mL to about 3,000L, about 2mL to about 2,000L, about 2mL to about 1,000L, about 2mL to about 950L, about 2mL to about 900L, about 2mL to about 850L, about 2mL to about 800L, about 2mL to about 750L, about 2mL to about 700L, about 2mL to about 650L, about 2mL to about 600L, about 2mL to about 550L, about 2mL to about 500L, about 2mL to about 450L, about 2mL to about 400L, about 2mL to about 350L, about 2mL to about 300L, about 2mL to about 250L, about 2mL to about 200L; about 2mL to about 190L, about 2mL to about 180L, about 2mL to about 170L, about 2mL to about 160L, about 2mL to about 150L, about 2mL to about 140L, about 2mL to about 130L, about 2mL to about 120L, about 2mL to about 110L, about 2mL to about 100L, about 2mL to about 90L, about 2mL to about 80L, about 2mL to about 70L, about 2mL to about 60L, about 2mL to about 50L, about 2mL to about 40L, about 2mL to about 30L, about 2mL to about 20L, about 2mL to about 10L, about 2mL to about 5L, about 2mL to about 4L, about 2mL to about 3L, about 2mL to about 2L, about 2mL to about 1L, about 2mL to about 900mL, about 2mL to about 800mL, about 2mL to about 700mL, about 2mL to about 600mL to about 500mL, about 2mL to about 400mL, about 2mL to about 500mL to about 100mL, about 2mL to about 90mL, about 2mL to about 80mL, about 2mL to about 70mL, about 2mL to about 60mL, about 2mL to about 50mL, about 2mL to about 40mL, about 2mL to about 30mL, about 2mL to about 25mL, about 2mL to about 20mL, about 2mL to about 15mL, about 2mL to about 10mL, about 2mL to about 5 mL; about 5mL to10,000L, about 5mL to about 9,000L, about 5mL to about 8,000L, about 5mL to about 7,000L, about 5mL to about 6,000L, about 5mL to about 5,000L, about 5mL to about 4,000L, about 5mL to about 3,000L, about 5mL to about 2,000L, about 5mL to about 1,000L, about 5mL to about 950L, about 5mL to about 900L, about 5mL to about 850L, about 5mL to about 800L, about 5mL to about 750L, about 5mL to about 700L, about 5mL to about 650L, about 5mL to about 600L, about 5mL to about 550L, about 5mL to about 500L, about 5mL to about 450L, about 5mL to about 400L, about 5mL to about 350L, about 5mL to about 300L, about 5mL to about 250L, about 5mL to about 200L; about 5mL to about 190L, about 5mL to about 180L, about 5mL to about 170L, about 5mL to about 160L, about 5mL to about 150L, about 5mL to about 140L, about 5mL to about 130L, about 5mL to about 120L, about 5mL to about 110L, about 5mL to about 100L, about 5mL to about 90L, about 5mL to about 80L, about 5mL to about 70L, about 5mL to about 60L, about 5mL to about 50L, about 5mL to about 40L, about 5mL to about 30L, about 5mL to about 20L, about 5mL to about 10L, about 5mL to about 5L, about 5mL to about 4L, about 5mL to about 3L, about 5mL to about 5L, about 5mL to about 1L, about 5mL to about 900mL, about 5mL to about 800mL, about 5mL to about 700mL, about 5mL to about 600mL, about 5mL to about 500mL, about 5mL to about 400mL, about 5mL to about 5mL, about 5mL to about 90mL, about 5mL to about 80mL, about 5mL to about 70mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 25mL, about 5mL to about 20mL, about 5mL to about 15mL, about 5mL to about 10 mL; about 10mL to10,000L, about 10mL to about 9,000L, about 10mL to about 8,000L, about 10mL to about 7,000L, about 10mL to about 6,000L, about 10mL to about 5,000L, about 10mL to about 4,000L, about 10mL to about 3,000L, about 10mL to about 2,000L, about 10mL to about 1,000L, about 10mL to about 950L, about 10mL to about 900L, about 10mL to about 850L, about 10mL to about 800L, about 10mL to about 750L, about 10mL to about 700L, about 10mL to about 650L, about 10mL to about 600L, about 10mL to about 550L, about 10mL to about 500L, about 10mL to about 450L, about 10mL to about 400L, about 10mL to about 350L, about 10mL to about 300L, about 10mL to about 10L, about 10mL to about 250L, about 10mL to about 180L, about 10mL to about 120L, about 10mL to about 110L, about 10mL to about 100L, about 10mL to about 90L, about 10mL to about 80L, about 10mL to about 70L, about 10mL to about 60L, about 10mL to about 50L, about 10mL to about 40L, about 10mL to about 30L, about 10mL to about 20mL, about 10mL to about 10L, about 10mL to about 5L, about 10mL to about 4L, about 10mL to about 3L, about 10mL to about 5L, about 10mL to about 1L, about 10mL to about 900mL, about 10mL to about 800mL, about 10mL to about 700mL, about 10mL to about 600mL, about 10mL to about 500mL, about 10mL to about 400mL, about 10mL to about 300mL, about 10mL to about 250mL, about 10mL to about 100mL, about 10mL to about 90mL, about 10mL to about 80mL to about 10mL, about 10mL to about 70mL, about 10mL to about 10mL, about 10mL to about 25mL, about 10mL to about 20mL, about 10mL to about 15 mL; about 25mL to10,000L, about 25mL to about 9,000L, about 25mL to about 8,000L, about 25mL to about 7,000L, about 25mL to about 6,000L, about 25mL to about 5,000L, about 25mL to about 4,000L, about 25mL to about 3,000L, about 25mL to about 2,000L, about 25mL to about 1,000L, about 25mL to about 950L, about 25mL to about 900L, about 25mL to about 850L, about 25mL to about 800L, about 25mL to about 750L, about 25mL to about 700L, about 25mL to about 650L, about 25mL to about 600L, about 25mL to about 550L, about 25mL to about 500L, about 25mL to about 450L, about 25mL to about 400L, about 25mL to about 350L, about 25mL to about 300L, about 25mL to about 25L, about 25mL to about 250L, about 25mL to about 25L, about 25mL to about 180L, about 25mL to about 25L, about 25mL to about 180L, about 25mL to about 130L, about 25mL to about 120L, about 25mL to about 110L, about 25mL to about 100L, about 25mL to about 90L, about 25mL to about 80L, about 25mL to about 70L, about 25mL to about 60L, about 25mL to about 50L, about 25mL to about 40L, about 25mL to about 30L, about 25mL to about 20L, about 25mL to about 10L, about 25mL to about 5L, about 25mL to about 4L, about 25mL to about 3L, about 25mL to about 5L, about 25mL to about 1L, about 25mL to about 900mL, about 25mL to about 800mL, about 25mL to about 700mL, about 25mL to about 600mL, about 25mL to about 500mL, about 25mL to about 400mL, about 25mL to about 300mL, about 25mL to about 250mL, about 25mL to about 100mL, about 25mL to about 80mL, about 25mL to about 50mL, about 25mL to about 25mL, about 25mL to about 30 mL; about 50mL to10,000L, about 50mL to about 9,000L, about 50mL to about 8,000L, about 50mL to about 7,000L, about 50mL to about 6,000L, about 50mL to about 5,000L, about 50mL to about 4,000L, about 50mL to about 3,000L, about 50mL to about 2,000L, about 50mL to about 1,000L, about 50mL to about 950L, about 50mL to about 900L, about 50mL to about 850L, about 50mL to about 800L, about 50mL to about 750L, about 50mL to about 700L, about 50mL to about 650L, about 50mL to about 600L, about 50mL to about 550L, about 50mL to about 500L, about 50mL to about 450L, about 50mL to about 400L, about 50mL to about 350L, about 50mL to about 300L, about 50mL to about 250L, about 50mL to about 200L, about 50mL to about 50L, about 50mL to about 180L, about 50mL to about 180L, about 50mL to about 130L, about 50mL to about 120L, about 50mL to about 110L, about 50mL to about 100L, about 50mL to about 90L, about 50mL to about 80L, about 50mL to about 70L, about 50mL to about 60L, about 50mL to about 50L, about 50mL to about 40L, about 50mL to about 30L, about 50mL to about 20L, about 50mL to about 10L, about 50mL to about 5L, about 50mL to about 4L, about 50mL to about 3L, about 50mL to about 5L, about 50mL to about 1L, about 50mL to about 900mL, about 50mL to about 800mL, about 50mL to about 700mL, about 50mL to about 600mL, about 50mL to about 500mL, about 50mL to about 400mL, about 50mL to about 300mL, about 50mL to about 250mL, about 50mL to about 100mL, about 50mL to about 90mL, about 50mL to about 70 mL; about 75mL to10,000L, about 75mL to about 9,000L, about 75mL to about 8,000L, about 75mL to about 7,000L, about 75mL to about 6,000L, about 75mL to about 5,000L, about 75mL to about 4,000L, about 75mL to about 3,000L, about 75mL to about 2,000L, about 75mL to about 1,000L, about 75mL to about 950L, about 75mL to about 900L, about 75mL to about 850L, about 75mL to about 800L, about 75mL to about 750L, about 75mL to about 700L, about 75mL to about 650L, about 75mL to about 600L, about 75mL to about 550L, about 75mL to about 500L, about 75mL to about 450L, about 75mL to about 400L, about 75mL to about 350L, about 75mL to about 300L, about 75mL to about 250L, about 75mL to about 200L, about 75mL to about 180L, about 75mL to about 75L, about 75mL to about 180L, about 75mL to about 130L, about 75mL to about 120L, about 75mL to about 110L, about 75mL to about 100L, about 75mL to about 90L, about 75mL to about 80L, about 75mL to about 70L, about 75mL to about 60L, about 75mL to about 50L, about 75mL to about 40L, about 75mL to about 30L, about 75mL to about 20L, about 75mL to about 10L, about 75mL to about 5L, about 75mL to about 4L, about 75mL to about 3L, about 75mL to about 5L, about 75mL to about 1L, about 75mL to about 900mL, about 75mL to about 800mL, about 75mL to about 700mL, about 75mL to about 600mL, about 75mL to about 500mL, about 75mL to about 400mL, about 75mL to about 300mL, about 75mL to about 250mL, about 75mL to about 100mL, about 75mL to about 90 mL; about 100mL to10,000L, about 100mL to about 9,000L, about 100mL to about 8,000L, about 100mL to about 7,000L, about 100mL to about 6,000L, about 100mL to about 5,000L, about 100mL to about 4,000L, about 100mL to about 3,000L, about 100mL to about 2,000L, about 100mL to about 1,000L, about 100mL to about 950L, about 100mL to about 900L, about 100mL to about 850L, about 100mL to about 800L, about 100mL to about 750L, about 100mL to about 700L, about 100mL to about 650L, about 100mL to about 600L, about 100mL to about 550L, about 100mL to about 500L, about 100mL to about 450L, about 100mL to about 400L, about 100mL to about 350L, about 100mL to about 300L, about 100mL to about 100L, about 100mL to about 250L, about 100mL to about 100L, about 100mL to about 100L, about 100mL to about 120L, about 100mL to about 110L, about 100mL to about 100L, about 100mL to about 90L, about 100mL to about 80L, about 100mL to about 70L, about 100mL to about 60L, about 100mL to about 50L, about 100mL to about 40L, about 100mL to about 30L, about 100mL to about 20L, about 100mL to about 10L, about 100mL to about 5L, about 100mL to about 4L, about 100mL to about 3L, about 100mL to about 5L, about 100mL to about 1L, about 100mL to about 900mL, about 100mL to about 800mL, about 100mL to about 700mL, about 100mL to about 600mL, about 100mL to about 500mL, about 100mL to about 400mL, about 100mL to about 300mL, about 100mL to about 250 mL; about 250mL to10,000L, about 250mL to about 9,000L, about 250mL to about 8,000L, about 250mL to about 7,000L, about 250mL to about 6,000L, about 250mL to about 5,000L, about 250mL to about 4,000L, about 250mL to about 3,000L, about 250mL to about 2,000L, about 250mL to about 1,000L, about 250mL to about 950L, about 250mL to about 900L, about 250mL to about 850L, about 250mL to about 800L, about 250mL to about 750L, about 250mL to about 700L, about 250mL to about 650L, about 250mL to about 600L, about 250mL to about 550L, about 250mL to about 500L, about 250mL to about 450L, about 250mL to about 400L, about 250mL to about 350L, about 250mL to about 300L, about 250mL to about 250L, about 250mL to about 200L, about 250mL to about 250L, about 250mL to about 180L, about 250mL to about 250L, about 250mL to about 180L, about 250mL to about 250L, about 250mL to about 130L, about 250mL to about 120L, about 250mL to about 110L, about 250mL to about 100L, about 250mL to about 90L, about 250mL to about 80L, about 250mL to about 70L, about 250mL to about 60L, about 250mL to about 50L, about 250mL to about 40L, about 250mL to about 30L, about 250mL to about 20L, about 250mL to about 10L, about 250mL to about 5L, about 250mL to about 4L, about 250mL to about 3L, about 250mL to about 5L, about 250mL to about 1L, about 250mL to about 900mL, about 250mL to about 800mL, about 250mL to about 700mL, about 250mL to about 600mL, about 250mL to about 500mL, about 250mL to about 400mL, about 250mL to about 300 mL; about 300mL to10,000L, about 300mL to about 9,000L, about 300mL to about 8,000L, about 300mL to about 7,000L, about 300mL to about 6,000L, about 300mL to about 5,000L, about 300mL to about 4,000L, about 300mL to about 3,000L, about 300mL to about 2,000L, about 300mL to about 1,000L, about 300mL to about 950L, about 300mL to about 900L, about 300mL to about 850L, about 300mL to about 800L, about 300mL to about 750L, about 300mL to about 700L, about 300mL to about 650L, about 300mL to about 600L, about 300mL to about 550L, about 300mL to about 500L, about 300mL to about 450L, about 300mL to about 400L, about 300mL to about 350L, about 300mL to about 300L, about 300mL to about 300L, about 300mL to about 120L, about 300mL to about 110L, about 300mL to about 100L, about 300mL to about 90L, about 300mL to about 80L, about 300mL to about 70L, about 300mL to about 60L, about 300mL to about 50L, about 300mL to about 40L, about 300mL to about 30L, about 300mL to about 20L, about 300mL to about 10L, about 300mL to about 5L, about 300mL to about 4L, about 300mL to about 3L, about 300mL to about 5L, about 300mL to about 1L, about 300mL to about 900mL, about 300mL to about 800mL, about 300mL to about 700mL, about 300mL to about 600mL, about 300mL to about 500mL, about 300mL to about 400 mL; about 450mL to10,000L, about 450mL to about 9,000L, about 450mL to about 8,000L, about 450mL to about 7,000L, about 450mL to about 6,000L, about 450mL to about 5,000L, about 450mL to about 4,000L, about 450mL to about 3,000L, about 450mL to about 2,000L, about 450mL to about 1,000L, about 450mL to about 950L, about 450mL to about 900L, about 450mL to about 850L, about 450mL to about 800L, about 450mL to about 750L, about 450mL to about 700L, about 450mL to about 650L, about 450mL to about 600L, about 450mL to about 550L, about 450mL to about 500L, about 450mL to about 450L, about 450mL to about 400L, about 450mL to about 350L, about 450mL to about 300L, about 450mL to about 450L, about 450L to about 450L, about 450mL to about 450L, about 450L to about 450L, about 450mL to about 450L, about 450L to about 450L, about 450mL to about 130L, about 450mL to about 120L, about 450mL to about 110L, about 450mL to about 100L, about 450mL to about 90L, about 450mL to about 80L, about 450mL to about 70L, about 450mL to about 60L, about 450mL to about 50L, about 450mL to about 40L, about 450mL to about 30L, about 450mL to about 20L, about 450mL to about 10L, about 450mL to about 5L, about 450mL to about 4L, about 450mL to about 3L, about 450mL to about 5L, about 450mL to about 1L, about 450mL to about 900mL, about 450mL to about 800mL, about 450mL to about 700mL, about 450mL to about 600 mL; about 500mL to10,000L, about 500mL to about 9,000L, about 500mL to about 8,000L, about 500mL to about 7,000L, about 500mL to about 6,000L, about 500mL to about 5,000L, about 500mL to about 4,000L, about 500mL to about 3,000L, about 500mL to about 2,000L, about 500mL to about 1,000L, about 500mL to about 950L, about 500mL to about 900L, about 500mL to about 850L, about 500mL to about 800L, about 500mL to about 750L, about 500mL to about 700L, about 500mL to about 650L, about 500mL to about 600L, about 500mL to about 550L, about 500mL to about 500L, about 500mL to about 450L, about 500mL to about 400L, about 500mL to about 350L, about 500mL to about 300L, about 500mL to about 250L, about 500mL to about 500L, about 500mL to about 180L, about 500mL to about 500L, about 500mL to about 180L, about 500mL to about 120L, about 500mL to about 110L, about 500mL to about 100L, about 500mL to about 90L, about 500mL to about 80L, about 500mL to about 70L, about 500mL to about 60L, about 500mL to about 50L, about 500mL to about 40L, about 500mL to about 30L, about 500mL to about 20L, about 500mL to about 10L, about 500mL to about 5L, about 500mL to about 4L, about 500mL to about 3L, about 500mL to about 5L, about 500mL to about 1L, about 500mL to about 900mL, about 500mL to about 800mL, about 500mL to about 700mL, about 500mL to about 600 mL; about 750mL to10,000L, about 750mL to about 9,000L, about 750mL to about 8,000L, about 750mL to about 7,000L, about 750mL to about 6,000L, about 750mL to about 5,000L, about 750mL to about 4,000L, about 750mL to about 3,000L, about 750mL to about 2,000L, about 750mL to about 1,000L, about 750mL to about 950L, about 750mL to about 900L, about 750mL to about 850L, about 750mL to about 800L, about 750mL to about 750L, about 750mL to about 700L, about 750mL to about 650L, about 750mL to about 600L, about 750mL to about 550L, about 750mL to about 500L, about 750mL to about 450L, about 750mL to about 400L, about 750mL to about 350L, about 750mL to about 300L, about 750mL to about 250L, about 750mL to about 200L, about 750mL to about 750L, about 750L to about 750L, about 750mL to about 750L, about 750L to about 750L, about 750mL to about 750L, about 750mL to about 130L, about 750mL to about 120L, about 750mL to about 110L, about 750mL to about 100L, about 750mL to about 90L, about 750mL to about 80L, about 750mL to about 70L, about 750mL to about 60L, about 750mL to about 50L, about 750mL to about 40L, about 750mL to about 30L, about 750mL to about 20L, about 750mL to about 10L, about 750mL to about 5L, about 750mL to about 4L, about 750mL to about 3L, about 750mL to about 5L, about 750mL to about 1L, about 750mL to about 900mL, about 750mL to about 800 mL; about 1L to10,000L, about 1L to about 9,000L, about 1L to about 8,000L, about 1L to about 7,000L, about 1L to about 6,000L, about 1L to about 5,000L, about 1L to about 4,000L, about 1L to about 3,000L, about 1L to about 2,000L, about 1L to about 1,000L, about 1L to about 950L, about 1L to about 900L, about 1L to about 850L, about 1L to about 800L, about 1L to about 750L, about 1L to about 700L, about 1L to about 650L, about 1L to about 600L, about 1L to about 550L, about 1L to about 500L, about 1L to about 450L, about 1L to about 400L, about 1L to about 350L, about 1L to about 300L, about 1L to about 250L, about 1L to about 200L, about 1L to about 180L, about 1L to about 1L, about 1L to about 180L, about 1L to about 130L, about 1L to about 120L, about 1L to about 110L, about 1L to about 100L, about 1L to about 90L, about 1L to about 80L, about 1L to about 70L, about 1L to about 60L, about 1L to about 50L, about 1L to about 40L, about 1L to about 30L, about 1L to about 20L, about 1L to about 10L, about 1L to about 5L, about 1L to about 4L, about 1L to about 3L, about 1L to about 2L; about 2L to10,000L, about 2L to about 9,000L, about 2L to about 8,000L, about 2L to about 7,000L, about 2L to about 6,000L, about 2L to about 5,000L, about 2L to about 4,000L, about 2L to about 3,000L, about 2L to about 2,000L, about 2L to about 1,000L, about 2L to about 950L, about 2L to about 900L, about 2L to about 850L, about 2L to about 800L, about 2L to about 750L, about 2L to about 700L, about 2L to about 650L, about 2L to about 600L, about 2L to about 550L, about 2L to about 500L, about 2L to about 450L, about 2L to about 400L, about 2L to about 350L, about 2L to about 300L, about 2L to about 250L, about 2L to about 200L, about 2L to about 150L, about 2L to about 180L, about 2L to about 120L, about 2L to about 110L, about 2L to about 100L, about 2L to about 90L, about 2L to about 80L, about 2L to about 70L, about 2L to about 60L, about 2L to about 50L, about 2L to about 40L, about 2L to about 30L, about 2L to about 20L, about 2L to about 10L, about 2L to about 5L, about 2L to about 4L, about 2L to about 3L; about 4L to10,000L, about 4L to about 9,000L, about 4L to about 8,000L, about 4L to about 7,000L, about 4L to about 6,000L, about 4L to about 5,000L, about 4L to about 4,000L, about 4L to about 3,000L, about 4L to about 2,000L, about 4L to about 1,000L, about 4L to about 950L, about 4L to about 900L, about 4L to about 850L, about 4L to about 800L, about 4L to about 750L, about 4L to about 700L, about 4L to about 650L, about 4L to about 600L, about 4L to about 550L, about 4L to about 500L, about 4L to about 450L, about 4L to about 400L, about 4L to about 350L, about 4L to about 300L, about 4L to about 250L, about 4L to about 200L, about 4L to about 180L, about 4L to about 180L, about 4L to about 130L, about 4L to about 120L, about 4L to about 110L, about 4L to about 100L, about 4L to about 90L, about 4L to about 80L, about 4L to about 70L, about 4L to about 60L, about 4L to about 50L, about 4L to about 40L, about 4L to about 30L, about 4L to about 20L, about 4L to about 10L, about 4L to about 5L; about 5L to10,000L, about 5L to about 9,000L, about 5L to about 8,000L, about 5L to about 7,000L, about 5L to about 6,000L, about 5L to about 5,000L, about 5L to about 4,000L, about 5L to about 3,000L, about 5L to about 2,000L, about 5L to about 1,000L, about 5L to about 950L, about 5L to about 900L, about 5L to about 850L, about 5L to about 800L, about 5L to about 750L, about 5L to about 700L, about 5L to about 650L, about 5L to about 600L, about 5L to about 550L, about 5L to about 500L, about 5L to about 450L, about 5L to about 400L, about 5L to about 350L, about 5L to about 300L, about 5L to about 250L, about 5L to about 200L, about 5L to about 180L, about 5L to about 5L, about 5L to about 180L, about 5L to about 5L, about 5L to about 130L, about 5L to about 120L, about 5L to about 110L, about 5L to about 100L, about 5L to about 90L, about 5L to about 80L, about 5L to about 70L, about 5L to about 60L, about 5L to about 50L, about 5L to about 40L, about 5L to about 30L, about 5L to about 20L, about 5L to about 10L, about 6L to about 8L, about 8L to about 10L; about 7.5L to10,000L, about 7.5L to about 9,000L, about 7.5L to about 8,000L, about 7.5L to about 7,000L, about 7.5L to about 6,000L, about 7.5L to about 5,000L, about 7.5L to about 4,000L, about 7.5L to about 3,000L, about 7.5L to about 2,000L, about 7.5L to about 1,000L, about 7.5L to about 950L, about 7.5L to about 900L, about 7.5L to about 850L, about 7.5L to about 800L, about 7.5L to about 750L, about 7.5L to about 700L, about 7.5L to about 650L, about 7.5L to about 600L, about 7.5L to about 550L, about 7.5L to about 500L, about 7.5L to about 5L to about 200L, about 7.5L to about 5L, about 400L to about 5L, about 7.180L, about 7.5L to about 7.5L, about 7.5L to about 5L to about 400L, about 7.5L to about 5L, about 5L to about 400L, about 5L, about 7.180L to about 7.180L, about 7.5L to about 7.180L to about 7.5L, about 400L, about 7.5L, about 7.5L to about 130L, about 7.5L to about 120L, about 7.5L to about 110L, about 7.5L to about 100L, about 7.5L to about 90L, about 7.5L to about 80L, about 7.5L to about 70L, about 7.5L to about 60L, about 7.5L to about 50L, about 7.5L to about 40L, about 7.5L to about 30L, about 7.5L to about 20L, about 7.5L to about 10L; about 10L to10,000L, about 10L to about 9,000L, about 10L to about 8,000L, about 10L to about 7,000L, about 10L to about 6,000L, about 10L to about 5,000L, about 10L to about 4,000L, about 10L to about 3,000L, about 10L to about 2,000L, about 10L to about 1,000L, about 10L to about 950L, about 10L to about 900L, about 10L to about 850L, about 10L to about 800L, about 10L to about 750L, about 10L to about 700L, about 10L to about 650L, about 10L to about 600L, about 10L to about 550L, about 10L to about 500L, about 10L to about 450L, about 10L to about 400L, about 10L to about 350L, about 10L to about 300L, about 10L to about 250L, about 10L to about 200L, about 10L to about 150L, about 10L to about 180L, about 10L to about 10L, about 10L to about 130L, about 10L to about 120L, about 10L to about 110L, about 10L to about 100L, about 10L to about 90L, about 10L to about 80L, about 10L to about 70L, about 10L to about 60L, about 10L to about 50L, about 10L to about 40L, about 10L to about 30L, about 10L to about 20L, about 10L to about 15L, about 15L to about 20L, about 15L to about 25L, about 15L to about 30L; about 15L to about 40L, about 15L to about 50L, about 15L to about 60L, about 15L to about 70L, about 15L to about 80L, about 15L to about 90L, about 15L to about 100L, about 15L to about 110L, about 15L to about 120L, about 15L to about 130L, about 15L to about 140L, about 15L to about 150L, about 15L to about 160L, about 15L to about 170L, about 15L to about 180L, about 15L to about 190L; about 15L to about 200L, about 15L to about 250L, about 15L to about 300L, about 15L to about 350L, about 15L to about 400L, about 15L to about 450L, about 15L to about 500L, about 15L to about 550L, about 15L to about 600L, about 15L to about 650L, about 15L to about 700L, about 15L to about 750L, about 15L to about 800L, about 15L to about 850L, about 15L to about 900L, about 15L to about 950L, about 15L to about 1,000L, about 15L to about 2,000L, about 15L to about 3,000L, about 15L to about 4,000L, about 15L to about 5,000L, about 15L to about 6,000L, about 15L to about 7,000L, about 15L to about 8,000L, about 15L to about 9,000L, about 15L to about 10,000L; about 20L to about 25L, about 20L to about 30L, about 20L to about 40L, about 20L to about 50L, about 20L to about 60L, about 20L to about 70L, about 20L to about 80L, about 20L to about 90L, about 20L to about 100L, about 20L to about 110L, about 20L to about 120L, about 20L to about 130L, about 20L to about 140L, about 20L to about 150L, about 20L to about 160L, about 20L to about 170L, about 20L to about 180L, about 20L to about 190L, about 20L to about 200L, about 20L to about 250L, about 20L to about 300L, about 20L to about 350L, about 20L to about 400L, about 20L to about 450L, about 20L to about 500L, about 20L to about 550L, about 20L to about 600L, about 20L to about 650L, about 20L to about 800L, about 850L to about 800L, about 20L to about 800L, about 20L to about 950L, about 20L to about 1,000L, about 20L to about 2,000L, about 20L to about 3,000L, about 20L to about 4,000L, about 20L to about 5,000L, about 20L to about 6,000L, about 20L to about 7,000L, about 20L to about 8,000L, about 20L to about 9,000L, about 20L to10,000L; about 25L to about 30L; about 25L to about 40L, about 25L to about 50L, about 25L to about 60L, about 25L to about 70L, about 25L to about 80L, about 25L to about 90L, about 25L to about 100L, about 25L to about 110L, about 25L to about 120L, about 25L to about 130L, about 25L to about 140L, about 25L to about 150L, about 25L to about 160L, about 25L to about 170L, about 25L to about 180L, about 25L to about 190L, about 25L to about 200L, about 25L to about 250L, about 25L to about 300L, about 25L to about 350L, about 25L to about 400L, about 25L to about 450L, about 25L to about 500L, about 25L to about 550L, about 25L to about 600L, about 25L to about 650L, about 25L to about 700L, about 25L to about 25L, about 25L to about 800L, about 25L to about 900L, about 25L to about 800L, about 25L to about 900L, about 25L to about 2,000L, about 25L to about 3,000L, about 25L to about 4,000L, about 25L to about 5,000L, about 25L to about 6,000L, about 25L to about 7,000L, about 25L to about 8,000L, about 25L to about 9,000L, about 25L to10,000L; about 30L to about 50L, about 30L to about 75L, about 30L to about 100L, about 30L to about 150L, about 30L to about 200L, about 30L to about 250L, about 30L to about 300L, about 30L to about 350L, about 30L to about 400L, about 30L to about 450L, about 30L to about 500L, about 30L to about 550L, about 30L to about 600L, about 30L to about 650L, about 30L to about 700L, about 30L to about 750L, about 30L to about 800L, about 30L to about 850L, about 30L to about 900L, about 30L to about 950L, about 30L to about 1,000L, about 30L to about 2,000L, about 30L to about 3,000L, about 30L to about 4,000L, about 30L to about 5,000L, about 30L to about 6,000L, about 30L to about 30,000L, about 30L to about 10,000L, about 30,000L, about 30L to about 800L, about 800L; about 40L to about 75L, about 40L to about 100L, about 40L to about 150L, about 40L to about 200L; about 40L to about 250L, about 40L to about 300L, about 40L to about 350L, about 40L to about 400L, about 40L to about 450L, about 40L to about 500L, about 40L to about 550L, about 40L to about 600L, about 40L to about 650L, about 40L to about 700L, about 40L to about 750L, about 40L to about 800L, about 40L to about 850L, about 40L to about 900L, about 40L to about 950L, about 40L to about 1,000L, about 40L to about 2,000L, about 40L to about 3,000L, about 40L to about 4,000L, about 40L to about 5,000L, about 40L to about 6,000L, about 40L to about 7,000L, about 40L to about 8,000L, about 40L to about 9,000L, about 40L to10,000L; about 50L to about 60L, about 50L to about 70L, about 50L to about 80L, about 50L to about 90L, about 50L to about 100L, about 50L to about 110L, about 50L to about 120L, about 50L to about 130L, about 50L to about 140L, about 50L to about 150L, about 50L to about 160L, about 50L to about 170L, about 50L to about 180L, about 50L to about 190L, about 50L to about 200L, about 50L to about 250L, about 50L to about 300L, about 50L to about 350L, about 50L to about 400L, about 50L to about 450L, about 50L to about 500L, about 50L to about 550L, about 50L to about 600L, about 50L to about 650L, about 50L to about 700L, about 50L to about 750L, about 50L to about 800L, about 50L to about 900L, about 50L to about 850L, about 50L to about 900L, about 2L to about 900, about 3L, about 2L to about 900L, about 50L to about 4,000L, about 50L to about 5,000L, about 50L to about 6,000L, about 50L to about 7,000L, about 50L to about 8,000L, about 50L to about 9,000L, about 50L to10,000L; about 75L to about 100L, about 75L to about 110L, about 75L to about 120L, about 75L to about 130L, about 75L to about 140L, about 75L to about 150L, about 75L to about 160L, about 75L to about 170L, about 75L to about 180L, about 75L to about 190L, about 75L to about 200L, about 75L to about 250L, about 75L to about 300L, about 75L to about 350L, about 75L to about 400L, about 75L to about 450L, about 75L to about 500L, about 75L to about 550L, about 75L to about 600L, about 75L to about 650L, about 75L to about 700L, about 75L to about 750L, about 75L to about 800L, about 75L to about 850L, about 75L to about 900L, about 75L to about 950L, about 75L to about 1,000L, about 75L to about 2L to about 75,000L, about 75L to about 5,000L, about 75L to about 75,000L, about 75,000L to about 75,000L, about 75L to about 8,000L, about 75L to about 9,000L, about 75L to10,000L; about 100L to about 110L, about 100L to about 120L, about 100L to about 130L, about 100L to about 140L, about 100L to about 150L, about 100L to about 160L, about 100L to about 170L, about 100L to about 180L, about 100L to about 190L, about 100L to about 200L, about 100L to about 250L, about 100L to about 300L, about 100L to about 350L, about 100L to about 400L, about 100L to about 450L, about 100L to about 500L, about 100L to about 550L, about 100L to about 600L, about 100L to about 650L, about 100L to about 700L, about 100L to about 750L, about 100L to about 800L, about 100L to about 850L, about 100L to about 900L, about 100L to about 950L, about 100L to about 1,000L, about 100L to about 2,000L, about 100L to about 2L to about 800L, about 100L to about 5,000L, about 100L to about 100,000L, about 100L to about 100,000L, about 100L to about 9,000L, about 100L to10,000L; about 120L to about 130L, about 120L to about 140L, about 120L to about 150L, about 120L to about 160L, about 120L to about 170L, about 120L to about 180L, about 120L to about 190L, about 120L to about 200L, about 120L to about 250L, about 120L to about 300L, about 120L to about 350L, about 120L to about 400L, about 120L to about 450L, about 120L to about 500L, about 120L to about 550L, about 120L to about 600L, about 120L to about 650L, about 120L to about 700L, about 120L to about 750L, about 120L to about 800L, about 120L to about 850L, about 120L to about 900L, about 120L to about 950L, about 120L to about 1,000L, about 120L to about 2,000L, about 120L to about 3,000L, about 120L to about 120L, about 120L to about 4L, about 120L to about 120L, about 120L to about 5,000L, about 10,000L, about 120L to about 10,000L, about 10,000L; about 150L to about 160L, about 150L to about 170L, about 150L to about 180L, about 150L to about 190L, about 150L to about 200L, about 150L to about 250L, about 150L to about 300L, about 150L to about 350L, about 150L to about 400L, about 150L to about 450L, about 150L to about 500L, about 150L to about 550L, about 150L to about 600L, about 150L to about 650L, about 150L to about 700L, about 150L to about 750L, about 150L to about 800L, about 150L to about 850L, about 150L to about 900L, about 150L to about 950L, about 150L to about 1,000L, about 150L to about 2,000L, about 150L to about 3,000L, about 150L to about 4,000L, about 150L to about 5,000L, about 150L to about 6,000L, about 150L to about 150,000L, about 150L to about 7,000L, about 10,000L, about 150L to about 150,000L, about 150L, about 8,000L; about 200L to about 250L, about 200L to about 300L, about 200L to about 350L, about 200L to about 400L, about 200L to about 450L, about 200L to about 500L, about 200L to about 550L, about 200L to about 600L, about 200L to about 650L, about 200L to about 700L, about 200L to about 750L, about 200L to about 800L, about 200L to about 850L, about 200L to about 900L, about 200L to about 950L, about 200L to about 1,000L, about 200L to about 2,000L, about 200L to about 3,000L, about 200L to about 4,000L, about 200L to about 5,000L, about 200L to about 6,000L, about 200L to about 7,000L, about 200L to about 8,000L, about 200L to about 9,000L, about 200L to about 10,000L; about 250L to about 300L, about 250L to about 350L, about 250L to about 400L, about 250L to about 450L, about 250L to about 500L, about 250L to about 550L, about 250L to about 600L, about 250L to about 650L, about 250L to about 700L, about 250L to about 750L, about 250L to about 800L, about 250L to about 850L, about 250L to about 900L, about 250L to about 950L, about 250L to about 1,000L, about 250L to about 2,000L, about 250L to about 3,000L, about 250L to about 4,000L, about 250L to about 5,000L, about 250L to about 6,000L, about 250L to about 7,000L, about 250L to about 8,000L, about 250L to about 9,000L, about 250L to about 10,000l; about 300L to about 350L, about 300L to about 400L, about 300L to about 450L, about 300L to about 500L, about 300L to about 550L, about 300L to about 600L, about 300L to about 650L, about 300L to about 700L, about 300L to about 750L, about 300L to about 800L, about 300L to about 850L, about 300L to about 900L, about 300L to about 950L, about 300L to about 1,000L, about 300L to about 2,000L, about 300L to about 3,000L, about 300L to about 4,000L, about 300L to about 5,000L, about 300L to about 6,000L, about 300L to about 7,000L, about 300L to about 8,000L, about 300L to about 9,000L, about 300L to10,000L; about 350L to about 400L, about 350L to about 450L, about 350L to about 500L, about 350L to about 550L, about 350L to about 600L, about 350L to about 650L, about 350L to about 700L, about 350L to about 750L, about 350L to about 800L, about 350L to about 850L, about 350L to about 900L, about 350L to about 950L, about 350L to about 1,000L, about 350L to about 2,000L, about 350L to about 3,000L, about 350L to about 4,000L, about 350L to about 5,000L, about 350L to about 6,000L, about 350L to about 7,000L, about 350L to about 8,000L, about 350L to about 9,000L, about 350L to10,000L; about 400L to about 450L, about 400L to about 500L, about 400L to about 550L, about 400L to about 600L, about 400L to about 650L, about 400L to about 700L, about 400L to about 750L, about 400L to about 800L, about 400L to about 850L, about 400L to about 900L, about 400L to about 950L, about 400L to about 1,000L, about 400L to about 2,000L, about 400L to about 3,000L, about 400L to about 4,000L, about 400L to about 5,000L, about 400L to about 6,000L, about 400L to about 7,000L, about 400L to about 8,000L, about 400L to about 9,000L, about 400L to10,000L; about 450L to about 500L, about 450L to about 550L, about 450L to about 600L, about 450L to about 650L, about 450L to about 700L, about 450L to about 750L, about 450L to about 800L, about 450L to about 850L, about 450L to about 900L, about 450L to about 950L, about 450L to about 1,000L, about 450L to about 2,000L, about 450L to about 3,000L, about 450L to about 4,000L, about 450L to about 5,000L, about 450L to about 6,000L, about 450L to about 7,000L, about 450L to about 8,000L, about 450L to about 9,000L, about 450L to10,000l; about 500L to about 550L, about 500L to about 600L, about 500L to about 650L, about 500L to about 700L, about 500L to about 750L, about 500L to about 800L, about 500L to about 850L, about 500L to about 900L, about 500L to about 950L, about 500L to about 1,000L, about 500L to about 2,000L, about 500L to about 3,000L, about 500L to about 4,000L, about 500L to about 5,000L, about 500L to about 6,000L, about 500L to about 7,000L, about 500L to about 8,000L, about 500L to about 9,000L, about 500L to10,000l; about 600L to about 650L, about 600L to about 700L, about 600L to about 750L, about 600L to about 800L, about 600L to about 850L, about 600L to about 900L, about 600L to about 950L, about 600L to about 1,000L, about 600L to about 2,000L, about 600L to about 3,000L, about 600L to about 4,000L, about 600L to about 5,000L, about 600L to about 6,000L, about 600L to about 7,000L, about 600L to about 8,000L, about 600L to about 9,000L, about 600L to10,000L; about 700L to about 750L, about 700L to about 800L, about 700L to about 850L, about 700L to about 900L, about 700L to about 950L, about 700L to about 1,000L, about 700L to about 2,000L, about 700L to about 3,000L, about 700L to about 4,000L, about 700L to about 5,000L, about 700L to about 6,000L, about 700L to about 7,000L, about 700L to about 8,000L, about 700L to about 9,000L, about 700L to about 10,000L; about 800L to about 850L, about 800L to about 900L, about 800L to about 950L, about 800L to about 1,000L, about 800L to about 2,000L, about 800L to about 3,000L, about 800L to about 4,000L, about 800L to about 5,000L, about 800L to about 6,000L, about 800L to about 7,000L, about 800L to about 8,000L, about 800L to about 9,000L, about 800L to10,000L; about 900L to about 950L, about 900L to about 1,000L, about 900L to about 2,000L, about 900L to about 3,000L, about 900L to about 4,000L, about 900L to about 5,000L, about 900L to about 6,000L, about 900L to about 7,000L, about 900L to about 8,000L, about 900L to about 9,000L, about 900L to10,000L; about 950L to about 10,000L).

Carrier

Provided herein are cells in liquid cell culture, which can include a nucleic acid or vector encoding a recombinant protein (e.g., any of the recombinant proteins described herein). The nucleic acid encoding any of the recombinant proteins described herein can be contained in an expression vector. The skilled artisan will be able to select an appropriate expression vector to prepare the recombinant protein described herein and use that expression vector to express the recombinant protein in a cell. For example, in selecting an expression vector, the cell must be considered, as the vector may need to be capable of replication therein and/or capable of integration into the chromosome of the cell. Exemplary expression vectors that can be used to produce the recombinant proteins are also described below.

A "vector" according to the invention is a polynucleotide capable of inducing expression of a recombinant protein (e.g., any of the recombinant proteins described herein) in a cell. The vector functions similarly to a "molecular vector" to deliver the nucleic acid fragment into a host cell (e.g., any of the cells described herein). It may include an expression cassette with regulatory sequences. The exogenous polynucleotide may be inserted into an expression cassette of a vector nucleic acid for expression therefrom. The support according to the invention may be present in circular or linearized form. The term "vector" also includes an artificial chromosome or similar corresponding polynucleotide that allows transfer of a foreign nucleic acid fragment. For the production of recombinant proteins as described herein in cells, the respective vectors may be used as expression vectors.

Methods for constructing suitable vectors carrying DNA encoding the recombinant proteins described herein and suitable for transformation (e.g., eukaryotic cells, mammalian cells, insect cells, yeast cells, bacterial cells) are well known in the art. See, e.g., Ausubel et al, eds. "Current Protocols in Molecular Biology" Current Protocols, 1993; and Sambrook et al, eds, "Molecular Cloning: A Lab or at or y Manual," 2nd d., Cold Spring Harb or Press, 1989.

Method for introducing nucleic acid into cells

Any of the nucleic acids and expression vectors disclosed herein can be introduced into a cell (e.g., eukaryotic cell, plant cell) using a variety of different methods known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell extrusion, ultrasonic perforation, transformation, optical transfection, infection, hydrodynamic delivery, magnetic transfection, viral transduction (e.g., adenovirus and lentivirus transduction), and nanoparticle transfection. These and other methods of introducing nucleic acids into cells are well known in the art. In some embodiments, the nucleic acid introduced into the cell is transiently expressed. In some embodiments, the nucleic acid introduced into the cell is integrated into the genome of the cell.

Contacting the pod with a liquid cell culture

The contacting step results in at least a portion (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%) of the at least one region being contacted with the liquid cell culture. In some embodiments, the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin. In some embodiments, the contacting is performed under sterile conditions. In some embodiments, the contacting can be performed during agitation of the liquid cell culture (e.g., by shaking, by tilting, by rotational agitation (e.g., using a propeller), by vortexing, and/or aeration). In some embodiments, the contacting may occur when the liquid cell culture is in its exponential growth phase or resting growth phase. In some embodiments, the contacting may occur while the liquid cell culture is in its death phase. In some embodiments of any of the methods described herein, the contacting can occur in a clarified supernatant of the liquid cell culture.

In some embodiments, the contacting is performed at a temperature of about 20 ℃ to about 40 ℃ (e.g., about 20 ℃ to about 40 ℃, about 20 ℃ to about 25 ℃, about 20 ℃ to about 30 ℃, about 20 ℃ to about 35 ℃, about 20 ℃ to about 37 ℃, about 20 ℃ to about 38 ℃, about 20 ℃ to about 39 ℃, about 27 ℃ to about 30 ℃, about 25 ℃ to about 37 ℃, about 25 ℃ to about 38 ℃, about 25 ℃ to about 39 ℃, about 25 ℃ to about 40 ℃, about 27 ℃ to about 30 ℃, about 27 ℃ to about 35 ℃, about 27 ℃ to about 37 ℃, about 27 ℃ to about 38 ℃, about 27 ℃ to about 39 ℃, about 30 ℃ to about 35 ℃, about 30 ℃ to about 37 ℃, about 30 ℃ to about 38 ℃, about 30 ℃ to about 40 ℃, about 35 ℃ to about 38 ℃, or about 32 ℃ to about 38 ℃).

In some embodiments, the contacting is performed at a temperature of about 4 ℃ to about 18 ℃ (e.g., about 4 ℃ to about 16 ℃, about 4 ℃ to about 14 ℃, about 4 ℃ to about 12 ℃, about 4 ℃ to about 10 ℃, about 4 ℃ to about 8 ℃, about 4 ℃ to about 6 ℃, about 6 ℃ to about 18 ℃, about 6 ℃ to about 16 ℃, about 6 ℃ to about 14 ℃, about 6 ℃ to about 12 ℃, about 6 ℃ to about 10 ℃, about 6 ℃ to about 8 ℃, about 8 ℃ to about 18 ℃, about 8 ℃ to about 16 ℃, about 8 ℃ to about 14 ℃, about 8 ℃ to about 12 ℃, about 8 ℃ to about 10 ℃, about 10 ℃ to about 18 ℃, about 10 ℃ to about 16 ℃, about 10 ℃ to about 14 ℃, about 10 ℃ to about 12 ℃, about 12 ℃ to about 18 ℃, about 12 ℃ to about 16 ℃, about 12 ℃ to about 14 ℃, about 14 ℃ to about 18 ℃, about 14 ℃ to about 16 ℃, or about 16 ℃ to about 18 ℃).

In some embodiments, the contacting can occur at a pod 90 ° ± 50 ° (e.g., at a 50 ° to 140 °, 50 ° to 135 °, 50 ° to 130 °, 50 ° to 125 °, 50 ° to 120 °, 50 ° to 115 °, 50 ° to 110 °, 50 ° to 105 °, 50 ° to 100 °, 50 ° to 95 °, 50 ° to 90 °, 50 ° to 85 °, 50 ° to 80 °, 50 ° to 75 °, 50 ° to 70 °, 50 ° to 65 °, 50 ° to 60 °, 55 ° to 140 °, 55 ° to 135 °, 55 ° to 130 °, 55 ° to 125 °, 55 ° to 120 °, 115 ° to 110 ° to 105 °, 55 ° to 55 °,105 °,55 ° to 55 °,55 ° to 125 °,55 °, at an angle of 55 ° to 100 °, at an angle of 55 ° to 95 °, at an angle of 55 ° to 90 °, at an angle of 55 ° to 85 °, at an angle of 55 ° to 80 °, at an angle of 55 ° to 75 °, at an angle of 55 ° to 70 °, at an angle of 55 ° to 65 °, at an angle of 55 ° to 60 °, at an angle of 60 ° to 140 °, at an angle of 60 ° to 135 °, at an angle of 60 ° to 130 °, at an angle of 60 ° to 125 °, at an angle of 60 ° to 120 °, at an angle of 60 ° to 115 °, at an angle of 60 ° to 110 °, at an angle of 60 ° to 105 °, at an angle of 60 ° to 100 °, at an angle of 60 ° to 95 °, at an angle of 60 ° to 90 °, at an angle of 60 ° to 85 °, at an angle of 60 ° to 80 °, at an angle of 60 ° to 75 °, angle of 60 ° to 70 °, at an angle of 60 ° to 65 °, an angle of 65 ° to 140 °, an angle of 65 ° to 135 °, an angle of 65 ° to 130 °, an angle of 130 ° to 125 °, an angle of 60 ° to 105 °, an angle of 60 ° to 105 ° to 100 ° to 115 °, an angle, at an angle of 65 ° to 100 °, at an angle of 65 ° to 95 °, at an angle of 65 ° to 90 °, at an angle of 65 ° to 85 °, at an angle of 65 ° to 80 °, at an angle of 65 ° to 75 °, at an angle of 65 ° to 70 °, at an angle of 70 ° to 140 °, at an angle of 70 ° to 135 °, at an angle of 70 ° to 130 °, at an angle of 70 ° to 125 °, at an angle of 70 ° to 120 °, at an angle of 70 ° to 115 °, at an angle of 70 ° to 110 °, at an angle of 70 ° to 105 °, at an angle of 70 ° to 100 °, at an angle of 70 ° to 95 °, at an angle of 70 ° to 90 °, at an angle of 70 ° to 85 °, at an angle of 70 ° to 80 °, at an angle of 70 ° to 75 °, at an angle of 75 ° to 140 °, at an angle of 75 ° to 135 °, angle of 75 ° to 130 °, angle of 75 ° to 125 °, angle of 75 ° to 120 °, angle of 75 ° to 115 °, angle of 105 ° to 110 °, angle of 75 ° to 75 °, angle of 75 ° to 95 ° to 75 °, angle, at an angle of 75 ° to 80 °, at an angle of 80 ° to 140 °, at an angle of 80 ° to 135 °, at an angle of 80 ° to 130 °, at an angle of 80 ° to 125 °, at an angle of 80 ° to 120 °, at an angle of 80 ° to 115 °, at an angle of 80 ° to 110 °, at an angle of 80 ° to 105 °, at an angle of 80 ° to 100 °, at an angle of 80 ° to 95 °, at an angle of 80 ° to 90 °, at an angle of 80 ° to 85 °, at an angle of 85 ° to 140 °, at an angle of 85 ° to 135 °, at an angle of 85 ° to 130 °, at an angle of 85 ° to 125 °, at an angle of 85 ° to 120 °, at an angle of 85 ° to 115 °, at an angle of 85 ° to 110 °, at an angle of 85 ° to 105 °, at an angle of 85 ° to 100 °, angle of 85 ° to 95 °, angle of 85 ° to 90 °, at an angle of 90 ° to 140 °, at an angle of 90 ° to 135 °, angle of 90 ° to 130 °, angle of 90 ° to 125 °, angle of 90 ° to 100 °, at an angle of 90 ° to 100 ° angle, at an angle of 90 ° to 100 °, an angle of 85 ° to 125 ° angle, at an angle of 90 ° to 95 °, at an angle of 95 ° to 140 °, at an angle of 95 ° to 135 °, at an angle of 95 ° to 130 °, at an angle of 95 ° to 125 °, at an angle of 95 ° to 120 °, at an angle of 95 ° to 115 °, at an angle of 95 ° to 110 °, at an angle of 95 ° to 105 °, at an angle of 95 ° to 100 °, at an angle of 100 ° to 140 °, at an angle of 100 ° to 135 °, at an angle of 100 ° to 130 °, at an angle of 100 ° to 125 °, at an angle of 100 ° to 120 °, at an angle of 100 ° to 115 °, at an angle of 100 ° to 110 °, at an angle of 105 ° to 140 °, at an angle of 105 ° to 135 °, at an angle of 105 ° to 130 °, at an angle of 105 ° to 125 °, at an angle of 105 ° to 120 °, at an angle of 105 ° to 115 °, at an angle of 105 ° to 110 ° to 140 °, at an angle of 110 ° to 135 °, an angle of 110 ° to 130 °, at an angle of 110 ° to 125 °, at an angle of 110 ° to 140 °, at an angle of 100 ° to 115 °, an angle of 100 ° to 105 ° to 115 °, an angle, at an angle of 115 ° to 130 °, at an angle of 115 ° to 125 °, at an angle of 115 ° to 120 °, at an angle of 120 ° to 140 °, at an angle of 120 ° to 135 °, at an angle of 120 ° to 130 °, at an angle of 120 ° to 125 °, at an angle of 125 ° to 140 °, at an angle of 125 ° to 135 °, at an angle of 125 ° to 130 °, at an angle of 130 ° to 140 °, at an angle of 130 ° to 135 °, at an angle of 135 ° to 140 °, at an angle of 50 °, at an angle of 55 °, at an angle of 60 °, at an angle of 65 °, at an angle of 70 °, at an angle of 75 °, at an angle of 80 °, at an angle of 85 °, at an angle of 90 °, at an angle of 95 °, at an angle of 100 ° to 105 °, at an angle of 110 °, at an angle of 115 °, at an angle of 120 °, at an angle of 125 °, at an angle of 130 °, at an angle of 135 °, at an angle of 140 °.

In some embodiments, the contacting can occur at a pod angle of ± 35 ° from horizontal (e.g., from the bottom of a culture dish (e.g., any of the culture dishes described herein)) (e.g., at an angle of 145 ° to 180 °, 145 ° to 175 °, 145 ° to 170 °, 145 ° to 165 °, 145 ° to 160 °, 145 ° to 155 °, 145 ° to 150 °, 150 ° to 180 °, 150 ° to 175 °, 150 ° to 170 °, 150 ° to 165 °, 150 ° to 160 °, 150 ° to 155 °, 155 ° to 180 °, 155 ° to 175 °, 155 ° to 170 °, 155 ° to 165 °, 155 ° to 160 °, 160 ° to 180 °, 160 ° to 175 °, 160 ° to 170 °, 160 ° to 165 °, 165 ° to 165 °, 165 ° to 175 °,165 °, 175 ° to 170 °, 160 ° to 175 °,165 ° to 165 °, 165 ° to 165 °,165, at an angle of 170 ° to 180 °, at an angle of 170 ° to 175 °, at an angle of 175 ° to 180 °, at an angle of 145 °, at an angle of 150 °, at an angle of 155 °, at an angle of 160 °, at an angle of 165 °, at an angle of 170 °, at an angle of 175 °, at an angle of 180 °).

Sterilization of liquid media and/or pods containing chromatography resins

In some embodiments, the pods comprising the chromatography resin may be sterilized. Non-limiting examples of sterilization methods include: filtration, gamma irradiation, electron beam irradiation, steam sterilization, autoclaving and chemical sterilization (e.g., hydrogen chloride, sodium N-lauroyl sarcosinate, sodium hydroxide or ethanol).

In some embodiments, the chromatography resin is sterile. As used herein, the term "sterility assurance level" refers to the likelihood that an individual unit (e.g., a chromatography resin) becomes non-sterile after undergoing a sterilization process. In some embodiments, the sterility assurance level of the chromatography resin is 1x10^-5To about 1x10^-15(e.g., about 1x10^-5To about 1x10^-10About 1x10^-5To about 1x10^-15About 1x10^-6To about 1x10^-10About 1x10^-6To about 1x10^-12About 1x10^-6To about 1x10^-15About 1x10^-5About 1x10^-6About 1x10^-7About 1x10^-7About 1x10^-8About 1x10^-9About 1x10^-10About 1x10^-11About 1x10^-12About 1x10^-13About 1x10^-14About 1x10^-15)。

In some embodiments, the liquid cell culture medium present in the liquid cell culture can be sterile and/or aseptic.

Washing chromatography resin

In some embodiments of any of the methods described herein, the method may further comprise a washing step. In some embodiments, the washing step is performed between the contacting step and the eluting step. In some embodiments, the washing step comprises one or more washes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) with a wash buffer. In some embodiments, the washing step removes or substantially removes contaminants and/or non-specific proteins. In some embodiments, the washing step equilibrates the chromatography resin of the pod prior to contact with the liquid cell culture.

In some embodiments, the washing step is performed prior to removing the pods from the liquid cell culture and/or the culture dish. In some embodiments, the washing step is performed after removing the pods from the liquid cell culture and/or the culture dish. In some embodiments, the washing step is performed after removing the pod from the liquid cell culture and/or the culture dish, and may include moving a wash buffer directly into the interior of the pod.

In some embodiments, the washing step is performed at about 2 ℃ to about 25 ℃ (e.g., 2 ℃ to about 4 ℃, about 2 ℃ to about 6 ℃, about 2 ℃ to about 8 ℃, about 2 ℃ to about 10 ℃, about 2 ℃ to about 12 ℃, about 4 ℃ to about 6 ℃, about 4 ℃ to about 8 ℃, about 4 ℃ to about 10 ℃, about 4 ℃ to about 12 ℃, about 6 ℃ to about 8 ℃, about 6 ℃ to about 10 ℃, about 6 ℃ to about 12 ℃, about 10 ℃ to about 25 ℃, about 10 ℃ to about 22 ℃, about 10 ℃ to about 20 ℃, about 10 ℃ to about 18 ℃, about 10 ℃ to about 14 ℃, about 12 ℃ to about 16 ℃, about 16 ℃ to about 18 ℃, about 16 ℃ to about 20 ℃, about 16 ℃ to about 25 ℃, about 20 ℃ to about 22 ℃, or about 22 ℃ to about 25 ℃).

In some embodiments, the washing step is performed using a wash buffer comprising about 2mM to about 6mM (e.g., about 2mM to about 5mM, about 2mM to about 4mM, about 2mM to about 3mM, about 3mM to about 6mM, about 3mM to about 5mM, about 3mM to about 4mM, about 4mM to about 6mM, about 4mM to about 5mM, about 5mM to about 6mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM) ammonium acetate. In some embodiments, the wash buffer may further comprise a salt and/or a detergent. As known in the art, wash buffer does not imply dissociation of the recombinant protein from the chromatography resin.

In some embodiments, about 0.05mL to about 1,000mL, about 0.05mL to about 950mL, about 0.05mL to about 900mL, about 0.05mL to about 850mL, about 0.05mL to about 800mL, about 0.05mL to about 750mL, about 0.05mL to about 700mL, about 0.05mL to about 650mL, about 0.05mL to about 600mL, about 0.05mL to about 550mL, about 0.05mL to about 500mL, about 0.05mL to about 450mL, about 0.05mL to about 400mL, about 0.05mL to about 350mL, about 0.05mL to about 300mL, about 0.05mL to about 250mL, about 0.05mL to about 200mL, about 0.05mL to about 150mL, about 0.05mL to about 100mL, about 0.05mL to about 90mL, about 0.05mL to about 80mL, about 0.05mL to about 200mL, about 0.05mL to about 0mL, about 0.05mL to about 20mL, about 0mL to about 0.05mL, about 0mL to about 20mL, about 0.05mL to about 20mL, about 0mL to about 0.05mL, about 0.05mL to about 0mL, about 20mL to about 0mL, about 0.05mL to about 10mL, about 0.05mL to about 8mL, about 0.05mL to about 6mL, about 0.05mL to about 5mL, about 0.05mL to about 4mL, about 0.05mL to about 3mL, about 0.05mL to about 2mL, about 0.05mL to about 1mL, about 0.05mL to about 0.5mL, about 0.05mL to about 0.1mL, about 0.1mL to about 1,000mL, about 0.1mL to about 950mL, about 0.1mL to about 900mL, about 0.1mL to about 850mL, about 0.1mL to about 800mL, about 0.1mL to about 750mL, about 0.1mL to about 700mL, about 0.1mL to about 650mL, about 0.1mL to about 600mL, about 0.1mL to about 550mL, about 0.1 to about 500mL, about 0.1mL to about 1mL to about 200mL, about 0.1mL to about 1mL to about 400mL, about 0.1mL to about 1mL, about 0.1mL to about 100mL, about 0mL to about 1mL, about 0.1mL to about 100mL, about 0.1mL to about 50mL, about 0.1mL to about 45mL, about 0.1mL to about 40mL, about 0.1mL to about 35mL, about 0.1mL to about 30mL, about 0.1mL to about 25mL, about 0.1mL to about 20mL, about 0.1mL to about 15mL, about 0.1mL to about 10mL, about 0.1mL to about 8mL, about 0.1mL to about 6mL, about 0.1mL to about 5mL, about 0.1mL to about 4mL, about 0.1mL to about 3mL, about 0.1mL to about 2mL, about 0.1mL to about 1mL, about 0.1mL to about 0.5mL, about 0.5mL to about 1,000mL, about 0.5mL to about 950mL, about 0.5mL to about 900mL, about 0.5mL to about 850mL, about 0.5mL to about 5mL, about 0.5mL to about 400mL, about 0.5mL to about 500mL, about 400mL to about 400mL, about 0.5mL to about 400mL, about 500mL, about 400mL to about 400mL, about 0.5mL to about 200mL, about 0.5mL to about 150mL, about 0.5mL to about 100mL, about 0.5mL to about 90mL, about 0.5mL to about 80mL, about 0.5mL to about 70mL, about 0.5mL to about 60mL, about 0.5mL to about 50mL, about 0.5mL to about 45mL, about 0.5mL to about 40mL, about 0.5mL to about 35mL, about 0.5mL to about 30mL, about 0.5mL to about 25mL, about 0.5mL to about 20mL, about 0.5mL to about 15mL, about 0.5mL to about 10mL, about 0.5mL to about 8mL, about 0.5mL to about 6mL, about 0.5mL to about 5mL, about 0.5mL to about 4mL, about 0.5mL to about 3mL, about 0.5mL to about 2mL, about 0.5mL to about 1mL, about 1mL to about 400mL, about 1mL to about 1mL, about 1mL to about 250mL, about 1mL to about 200mL, about 1mL to about 150mL, about 1mL to about 100mL, about 1mL to about 90mL, about 1mL to about 80mL, about 1mL to about 70mL, about 1mL to about 60mL, about 1mL to about 50mL, about 1mL to about 40mL, about 1mL to about 30mL, about 1mL to about 20mL, about 1mL to about 10mL, about 1mL to about 5 mL; about 2mL to about 1L, about 2mL to about 900mL, about 2mL to about 800mL, about 2mL to about 700mL, about 2mL to about 600mL, about 2mL to about 500mL, about 2mL to about 250mL, about 2mL to about 400mL, about 2mL to about 350mL, about 2mL to about 300mL, about 2mL to about 250mL, about 2mL to about 200mL, about 2mL to about 150mL, about 2mL to about 100mL, about 2mL to about 90mL, about 2mL to about 80mL, about 2mL to about 70mL, about 2mL to about 60mL, about 2mL to about 50mL, about 2mL to about 40mL, about 2mL to about 30mL, about 2mL to about 20mL, about 2mL to about 10mL, about 2mL to about 5 mL; about 5mL to about 1L, about 5mL to about 900mL, about 5mL to about 800mL, about 5mL to about 700mL, about 5mL to about 600mL, about 5mL to about 500mL, about 5mL to about 250mL, about 5mL to about 400mL, about 5mL to about 350mL, about 5mL to about 300mL, about 5mL to about 250mL, about 5mL to about 200mL, about 5mL to about 150mL, about 5mL to about 100mL, about 5mL to about 90mL, about 5mL to about 80mL, about 5mL to about 70mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 20mL, about 5mL to about 10 mL; about 10mL to about 1L, about 10mL to about 900mL, about 10mL to about 800mL, about 10mL to about 700mL, about 10mL to about 600mL, about 10mL to about 500mL, about 10mL to about 450mL, about 10mL to about 400mL, about 10mL to about 350mL, about 10mL to about 300mL, about 10mL to about 250mL, about 10mL to about 200mL, about 10mL to about 150mL, about 10mL to about 100mL, about 10mL to about 90mL, about 10mL to about 80mL, about 10mL to about 70mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 40mL, about 10mL to about 30mL, about 10mL to about 20 mL; about 20mL to about 1L, about 20mL to about 900mL, about 20mL to about 800mL, about 20mL to about 700mL, about 20mL to about 600mL, about 20mL to about 500mL, about 20mL to about 450mL, about 20mL to about 400mL, about 20mL to about 350mL, about 20mL to about 300mL, about 20mL to about 250mL, about 20mL to about 200mL, about 20mL to about 150mL, about 20mL to about 100mL, about 20mL to about 90mL, about 20mL to about 80mL, about 20mL to about 70mL, about 20mL to about 60mL, about 20mL to about 50mL, about 20mL to about 40mL, about 20mL to about 30 mL; about 50mL to about 1L, about 50mL to about 900mL, about 50mL to about 800mL, about 50mL to about 700mL, about 50mL to about 600mL, about 50mL to about 500mL, about 50mL to about 450mL, about 50mL to about 400mL, about 50mL to about 350mL, about 50mL to about 300mL, about 50mL to about 250mL, about 50mL to about 200mL, about 50mL to about 150mL, about 50mL to about 100mL, about 50mL to about 90mL, about 50mL to about 80mL, about 50mL to about 70mL, about 50mL to about 60 mL; about 100mL to about 1L, about 100mL to about 900mL, about 100mL to about 800mL, about 100mL to about 700mL, about 100mL to about 600mL, about 100mL to about 500mL, about 100mL to about 450mL, about 100mL to about 400mL, about 100mL to about 350mL, about 100mL to about 300mL, about 100mL to about 250mL, about 100mL to about 200mL, about 100mL to about 150 mL; about 150mL to about 1L, about 150mL to about 900mL, about 150mL to about 800mL, about 150mL to about 700mL, about 150mL to about 600mL, about 150mL to about 500mL, about 150mL to about 250mL, about 150mL to about 400mL, about 150mL to about 350mL, about 150mL to about 300mL, about 150mL to about 250mL, about 150mL to about 200 mL; about 200mL to about 1L, about 200mL to about 900mL, about 200mL to about 800mL, about 200mL to about 700mL, about 200mL to about 600mL, about 200mL to about 500mL, about 200mL to about 450mL, about 200mL to about 400mL, about 200mL to about 350mL, about 200mL to about 300mL, about 200mL to about 250 mL; about 250mL to about 1L, about 250mL to about 900mL, about 250mL to about 800mL, about 250mL to about 700mL, about 250mL to about 600mL, about 250mL to about 500mL, about 250mL to about 450mL, about 250mL to about 400mL, about 250mL to about 350mL, about 250mL to about 300 mL; about 300mL to about 1L, about 300mL to about 900mL, about 300mL to about 800mL, about 300mL to about 700mL, about 300mL to about 600mL, about 300mL to about 500mL, about 300mL to about 450mL, about 300mL to about 400mL, about 300mL to about 350 mL; about 350mL to about 1L, about 350mL to about 900mL, about 350mL to about 800mL, about 350mL to about 700mL, about 350mL to about 600mL, about 350mL to about 500mL, about 350mL to about 450 mL; about 350mL to about 400 mL; about 400mL to about 1L, about 400mL to about 900mL, about 400mL to about 800mL, about 400mL to about 700mL, about 400mL to about 600mL, about 400mL to about 500mL, about 400mL to about 450 mL; about 500mL to about 1L, about 500mL to about 900mL, about 500mL to about 800mL, about 500mL to about 700mL, about 500mL to about 600 mL; about 550mL to about 1L, about 550mL to about 900mL, about 550mL to about 800mL, about 550mL to about 700mL, about 550mL to about 600 mL; about 600mL to about 1L, about 600mL to about 900mL, about 600mL to about 800mL, about 600mL to about 700 mL; about 600mL to about 650 mL; about 650mL to about 1L, about 650mL to about 900mL, about 650mL to about 800mL, about 650mL to about 700 mL; about 700mL to about 1L, about 700mL to about 900mL, about 700mL to about 800mL, about 700mL to about 750 mL; about 750mL to about 1L, about 750mL to about 900mL, about 750mL to about 800 mL; about 800mL to about 1L, about 800mL to about 900mL, about 800mL to about 950 mL; about 850mL to about 1L, about 850L to about 950mL, about 850mL to about 900mL, about 900mL to about 1L, about 900mL to about 950mL of a wash buffer (e.g., any of the wash buffers described herein).

In some embodiments of any of the methods described herein, the washing step can include one or more wash buffers (e.g., 2, 3, 4, 5, 6, 7, or 8) of any of the wash buffers described herein.

Elution of recombinant proteins

In some embodiments, any of the pods described herein can be removed from the liquid culture medium prior to performing the elution step. In some embodiments, the pod affinity chromatography resin is a protein a resin. In some embodiments where the pod affinity chromatography resin is a protein a resin, elution is performed using an elution buffer having an acidic pH. In some embodiments, the elution buffer comprises arginine or citrate. In some embodiments, the elution buffer comprises from about 0.01M to about 1M (e.g., from about 0.01M to about 1M, from about 0.01M to about 0.8M, from about 0.01M to about 0.6M, from about 0.01M to about 0.4M, from about 0.01M to about 0.2M, from about 0.01M to about 0.1M, from about 0.01M to about 0.08M, from about 0.01M to about 0.06M, from about 0.01M to about 0.05M, from about 0.01M to about 0.04M, from about 0.01M to about 0.02M, from about 0.02M to about 1M, from about 0.02M to about 0.8M, from about 0.02M to about 0.6M, from about 0.02M to about 0.4M, from about 0.02M to about 0.2M, from about 0.02M to about 0.1M, from about 0.02M to about 0.04, from about 0.04 to about 0.04, from about 0.0.04 to about 0.0.04, from about 0.0.0M to about 0.04, from about 0.04 to about 0.0.0.06M, from about 0.04 to about 0.04, from about 0.0.0.04 to about 0.0.0.04, from about 0.0.0.04 to about 0.0.0.0.04, from about 0.0.0.0.0 to about 0.04, from about 0.0.0 to about 0M to about 0M, 0.05M to about 1M, about 0.05M to about 0.8M, about 0.05M to about 0.6M, about 0.05M to about 0.4M, about 0.05M to about 0.2M, about 0.05M to about 0.1M, about 0.05M to about 0.08M, about 0.05M to about 0.06M, 0.06M to about 1M, about 0.06M to about 0.8M, about 0.06M to about 0.6M, about 0.06M to about 0.4M, about 0.06M to about 0.2M, about 0.06M to about 0.1M, about 0.06M to about 0.08M, 0.08M to about 1M, about 0.08M to about 0.8M, about 0.08M to about 0.6M, about 0.08M to about 0.0.08M, about 0.0.0.0 to about 0.0.08M, about 0.0.0.1M, about 0 to about 0.1M, about 0.8M, about 0.0 to about 0.1M, about 0.0.0 to about 0.1M, about 0.0.1M to about 0.0, about 0.0.0.1M to about 0, about 0.8M to about 0.0.0, about 0.2M to about 0, about 0.1M, about 0.2M, about 0, about 0.0.1M to about 0, about 0.4M to about 0.2M to about 0, about 0.4M, about 0., 0.6M to about 1M, about 0.6M to about 0.8M, 0.8M to about 1M, about 0.01M, about 0.02M, about 0.05M, about 0.06M, about 0.08M, about 0.1M, about 0.2M, about 0.4M, about 0.5M, about 0.6M, about 0.8M, or about 1M) arginine or citrate. In some embodiments, the elution buffer comprises about 0.1 to about 1M (e.g., about 0.1 to about 0.9M, about 0.1M to about 0.8M, about 0.1M to about 0.7M, about 0.1M to about 0.6M, about 0.1M to about 0.5M, about 0.1M to about 0.4M, about 0.1M to about 0.3M, about 0.1M to about 0.2M, about 0.2M to about 1M, about 0.2M to about 0.9M, about 0.2M to about 0.8M, about 0.2M to about 0.7M, about 0.2M to about 0.6M, about 0.2M to about 0.5M, about 0.2M to about 0.4M, about 0.2M to about 0.3M, about 0.3M to about 1M, about 0.2M to about 0.3M, about 0.4M to about 0.0M, about 0 to about 0.3M, about 0M to about 0.4M, about 0.3M to about 0, about 0M to about 0.4M to about 0, about 0.3M to about 0, about 0M to about 0.4M to about 0, about 0M to about 0, about 0.4M to about 0, about 0.3M to about 0, about 0M to about 0, about 0M to about 0, about 3M to about 0.4M to, about 0.5M to about 0.9M, about 0.5M to about 0.8M, about 0.5M to about 0.7M, about 0.5M to about 0.6M, about 0.6M to about 1M, about 0.6M to about 0.9M, about 0.6M to about 0.8M, about 0.6M to about 0.7M, about 0.7M to about 1M, about 0.7M to about 0.9M, about 0.7M to about 0.8M, about 0.8M to about 1M, about 0.8M to about 0.9M, about 0.9M to about 1M) acetic acid.

In some embodiments, the elution buffer comprises about 0.1M glycine (pH 3.0) or about 0.1M citric acid (pH 3.0). In some embodiments of any of the chromatography resins used herein, the eluted recombinant protein is neutralized using a buffer comprising about 1M Tris (pH 9.0). In some embodiments of any of the chromatography resins used herein, a chromatography resin comprising about 1M hepes (pH 7.0) (e.g., about 0.01M to about 1M, about 0.01M to about 0.8M, about 0.01M to about 0.6M, about 0.01M to about 0.4M, about 0.01M to about 0.2M, about 0.01 to about 0.1M, about 0.01M to about 0.05M, about 0.02M to about 1M, about 0.02M to about 0.8M, about 0.02M to about 0.6M, about 0.02M to about 0.4M, about 0.02M to about 0.2M, about 0.02 to about 0.1M, about 0.05M to about 1M, about 0.05M to about 0.8M, about 0.05M to about 0.6M, about 0.05M to about 0.4M, about 0.0.05M to about 0.1M, about 0.0 to about 0.0.0, about 0.05M to about 0.0, about 0 to about 1M to about 0, about 0.0 to about 0M to about 0, about 0.1M to about 0, about 0M to about 0, about 0.05M to about 0, about 0 to about 0M to about 0, about 0M to about 0.0, about 0 to about 0M to about 0, about 0M to about 0, about 0.2M to about 0, about 0M to about 0.0, about 0M to, about 0.4M to about 1M, about 0.4M to about 0.8M, about 0.4M to about 0.6M, about 0.6M to about 1M, about 0.6M to about 0.8M, or about 0.8M to about 1M) of buffer.

In some embodiments, about 0.05mL to about 1,000mL, about 0.05mL to about 950mL, about 0.05mL to about 900mL, about 0.05mL to about 850mL, about 0.05mL to about 800mL, about 0.05mL to about 750mL, about 0.05mL to about 700mL, about 0.05mL to about 650mL, about 0.05mL to about 600mL, about 0.05mL to about 550mL, about 0.05mL to about 500mL, about 0.05mL to about 450mL, about 0.05mL to about 400mL, about 0.05mL to about 350mL, about 0.05mL to about 300mL, about 0.05mL to about 250mL, about 0.05mL to about 200mL, about 0.05mL to about 150mL, about 0.05mL to about 100mL, about 0.05mL to about 90mL, about 0.05mL to about 80mL, about 0.05mL to about 200mL, about 0.05mL to about 0mL, about 0.05mL to about 20mL, about 0mL to about 0.05mL, about 0mL to about 20mL, about 0.05mL to about 20mL, about 0mL to about 0.05mL, about 0.05mL to about 0mL, about 20mL to about 0mL, about 0.05mL to about 10mL, about 0.05mL to about 8mL, about 0.05mL to about 6mL, about 0.05mL to about 5mL, about 0.05mL to about 4mL, about 0.05mL to about 3mL, about 0.05mL to about 2mL, about 0.05mL to about 1mL, about 0.05mL to about 0.5mL, about 0.05mL to about 0.1mL, about 0.1mL to about 1,000mL, about 0.1mL to about 950mL, about 0.1mL to about 900mL, about 0.1mL to about 850mL, about 0.1mL to about 800mL, about 0.1mL to about 750mL, about 0.1mL to about 700mL, about 0.1mL to about 650mL, about 0.1mL to about 600mL, about 0.1mL to about 550mL, about 0.1 to about 500mL, about 0.1mL to about 1mL to about 200mL, about 0.1mL to about 1mL to about 400mL, about 0.1mL to about 1mL, about 0.1mL to about 100mL, about 0mL to about 1mL, about 0.1mL to about 100mL, about 0.1mL to about 50mL, about 0.1mL to about 45mL, about 0.1mL to about 40mL, about 0.1mL to about 35mL, about 0.1mL to about 30mL, about 0.1mL to about 25mL, about 0.1mL to about 20mL, about 0.1mL to about 15mL, about 0.1mL to about 10mL, about 0.1mL to about 8mL, about 0.1mL to about 6mL, about 0.1mL to about 5mL, about 0.1mL to about 4mL, about 0.1mL to about 3mL, about 0.1mL to about 2mL, about 0.1mL to about 1mL, about 0.1mL to about 0.5mL, about 0.5mL to about 1,000mL, about 0.5mL to about 950mL, about 0.5mL to about 900mL, about 0.5mL to about 850mL, about 0.5mL to about 5mL, about 0.5mL to about 400mL, about 0.5mL to about 500mL, about 400mL to about 400mL, about 0.5mL to about 400mL, about 500mL, about 400mL to about 400mL, about 0.5mL to about 200mL, about 0.5mL to about 150mL, about 0.5mL to about 100mL, about 0.5mL to about 90mL, about 0.5mL to about 80mL, about 0.5mL to about 70mL, about 0.5mL to about 60mL, about 0.5mL to about 50mL, about 0.5mL to about 45mL, about 0.5mL to about 40mL, about 0.5mL to about 35mL, about 0.5mL to about 30mL, about 0.5mL to about 25mL, about 0.5mL to about 20mL, about 0.5mL to about 15mL, about 0.5mL to about 10mL, about 0.5mL to about 8mL, about 0.5mL to about 6mL, about 0.5mL to about 5mL, about 0.5mL to about 4mL, about 0.5mL to about 3mL, about 0.5mL to about 2mL, about 0.5mL to about 1mL, about 1mL to about 400mL, about 1mL to about 1mL, about 1mL to about 250mL, about 1mL to about 200mL, about 1mL to about 150mL, about 1mL to about 100mL, about 1mL to about 90mL, about 1mL to about 80mL, about 1mL to about 70mL, about 1mL to about 60mL, about 1mL to about 50mL, about 1mL to about 40mL, about 1mL to about 30mL, about 1mL to about 20mL, about 1mL to about 10mL, about 1mL to about 5 mL; about 2mL to about 1L, about 2mL to about 900mL, about 2mL to about 800mL, about 2mL to about 700mL, about 2mL to about 600mL, about 2mL to about 500mL, about 2mL to about 250mL, about 2mL to about 400mL, about 2mL to about 350mL, about 2mL to about 300mL, about 2mL to about 250mL, about 2mL to about 200mL, about 2mL to about 150mL, about 2mL to about 100mL, about 2mL to about 90mL, about 2mL to about 80mL, about 2mL to about 70mL, about 2mL to about 60mL, about 2mL to about 50mL, about 2mL to about 40mL, about 2mL to about 30mL, about 2mL to about 20mL, about 2mL to about 10mL, about 2mL to about 5 mL; about 5mL to about 1L, about 5mL to about 900mL, about 5mL to about 800mL, about 5mL to about 700mL, about 5mL to about 600mL, about 5mL to about 500mL, about 5mL to about 250mL, about 5mL to about 400mL, about 5mL to about 350mL, about 5mL to about 300mL, about 5mL to about 250mL, about 5mL to about 200mL, about 5mL to about 150mL, about 5mL to about 100mL, about 5mL to about 90mL, about 5mL to about 80mL, about 5mL to about 70mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 20mL, about 5mL to about 10 mL; about 10mL to about 1L, about 10mL to about 900mL, about 10mL to about 800mL, about 10mL to about 700mL, about 10mL to about 600mL, about 10mL to about 500mL, about 10mL to about 450mL, about 10mL to about 400mL, about 10mL to about 350mL, about 10mL to about 300mL, about 10mL to about 250mL, about 10mL to about 200mL, about 10mL to about 150mL, about 10mL to about 100mL, about 10mL to about 90mL, about 10mL to about 80mL, about 10mL to about 70mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 40mL, about 10mL to about 30mL, about 10mL to about 20 mL; about 20mL to about 1L, about 20mL to about 900mL, about 20mL to about 800mL, about 20mL to about 700mL, about 20mL to about 600mL, about 20mL to about 500mL, about 20mL to about 450mL, about 20mL to about 400mL, about 20mL to about 350mL, about 20mL to about 300mL, about 20mL to about 250mL, about 20mL to about 200mL, about 20mL to about 150mL, about 20mL to about 100mL, about 20mL to about 90mL, about 20mL to about 80mL, about 20mL to about 70mL, about 20mL to about 60mL, about 20mL to about 50mL, about 20mL to about 40mL, about 20mL to about 30 mL; about 50mL to about 1L, about 50mL to about 900mL, about 50mL to about 800mL, about 50mL to about 700mL, about 50mL to about 600mL, about 50mL to about 500mL, about 50mL to about 450mL, about 50mL to about 400mL, about 50mL to about 350mL, about 50mL to about 300mL, about 50mL to about 250mL, about 50mL to about 200mL, about 50mL to about 150mL, about 50mL to about 100mL, about 50mL to about 90mL, about 50mL to about 80mL, about 50mL to about 70mL, about 50mL to about 60 mL; about 100mL to about 1L, about 100mL to about 900mL, about 100mL to about 800mL, about 100mL to about 700mL, about 100mL to about 600mL, about 100mL to about 500mL, about 100mL to about 450mL, about 100mL to about 400mL, about 100mL to about 350mL, about 100mL to about 300mL, about 100mL to about 250mL, about 100mL to about 200mL, about 100mL to about 150 mL; about 150mL to about 1L, about 150mL to about 900mL, about 150mL to about 800mL, about 150mL to about 700mL, about 150mL to about 600mL, about 150mL to about 500mL, about 150mL to about 250mL, about 150mL to about 400mL, about 150mL to about 350mL, about 150mL to about 300mL, about 150mL to about 250mL, about 150mL to about 200 mL; about 200mL to about 1L, about 200mL to about 900mL, about 200mL to about 800mL, about 200mL to about 700mL, about 200mL to about 600mL, about 200mL to about 500mL, about 200mL to about 450mL, about 200mL to about 400mL, about 200mL to about 350mL, about 200mL to about 300mL, about 200mL to about 250 mL; about 250mL to about 1L, about 250mL to about 900mL, about 250mL to about 800mL, about 250mL to about 700mL, about 250mL to about 600mL, about 250mL to about 500mL, about 250mL to about 450mL, about 250mL to about 400mL, about 250mL to about 350mL, about 250mL to about 300 mL; about 300mL to about 1L, about 300mL to about 900mL, about 300mL to about 800mL, about 300mL to about 700mL, about 300mL to about 600mL, about 300mL to about 500mL, about 300mL to about 450mL, about 300mL to about 400mL, about 300mL to about 350 mL; about 350mL to about 1L, about 350mL to about 900mL, about 350mL to about 800mL, about 350mL to about 700mL, about 350mL to about 600mL, about 350mL to about 500mL, about 350mL to about 450 mL; about 350mL to about 400 mL; about 400mL to about 1L, about 400mL to about 900mL, about 400mL to about 800mL, about 400mL to about 700mL, about 400mL to about 600mL, about 400mL to about 500mL, about 400mL to about 450 mL; about 500mL to about 1L, about 500mL to about 900mL, about 500mL to about 800mL, about 500mL to about 700mL, about 500mL to about 600 mL; about 550mL to about 1L, about 550mL to about 900mL, about 550mL to about 800mL, about 550mL to about 700mL, about 550mL to about 600 mL; about 600mL to about 1L, about 600mL to about 900mL, about 600mL to about 800mL, about 600mL to about 700 mL; about 600mL to about 650 mL; about 650mL to about 1L, about 650mL to about 900mL, about 650mL to about 800mL, about 650mL to about 700 mL; about 700mL to about 1L, about 700mL to about 900mL, about 700mL to about 800mL, about 700mL to about 750 mL; about 750mL to about 1L, about 750mL to about 900mL, about 750mL to about 800 mL; about 800mL to about 1L, about 800mL to about 900mL, about 800mL to about 950 mL; about 850mL to about 1L, about 850L to about 950mL, about 850mL to about 900mL, about 900mL to about 1L, or about 900mL to about 950mL of an elution buffer (e.g., any elution buffer described herein).

In some embodiments, the elution is performed at about 2 ℃ to about 25 ℃ (e.g., 2 ℃ to about 4 ℃, about 2 ℃ to about 6 ℃, about 2 ℃ to about 8 ℃, about 2 ℃ to about 10 ℃, about 2 ℃ to about 12 ℃, about 4 ℃ to about 6 ℃, about 4 ℃ to about 8 ℃, about 4 ℃ to about 10 ℃, about 4 ℃ to about 12 ℃, about 6 ℃ to about 8 ℃, about 6 ℃ to about 10 ℃, about 6 ℃ to about 12 ℃, about 10 ℃ to about 25 ℃, about 10 ℃ to about 22 ℃, about 10 ℃ to about 20 ℃, about 10 ℃ to about 18 ℃, about 10 ℃ to about 14 ℃, about 12 ℃ to about 16 ℃, about 16 ℃ to about 18 ℃, about 16 ℃ to about 20 ℃, about 16 ℃ to about 25 ℃, about 20 ℃ to about 22 ℃, about 22 ℃ to about 25 ℃).

In some embodiments, the total amount of eluted recombinant protein is about 0.05mg to about 80mg (e.g., about 0.05mg to about 0.1mg, about 0.05mg to about 0.2mg, about 0.05mg to about 0.5mg, about 0.05mg to about 0.6mg, about 0.05mg to about 0.8mg, about 0.05mg to about 1mg, about 0.05mg to about 2mg, about 0.05mg to about 5mg, about 0.05mg to about 10mg, about 0.05mg to about 15mg, about 0.05mg to about 20mg, about 0.05mg to about 25mg, about 0.05mg to about 30mg, about 0.05mg to about 35mg, about 0.05mg to about 40mg, about 0.05mg to about 45mg, about 0.05mg to about 50mg, about 0.05mg to about 55mg, about 0.05mg to about 35mg, about 0.05mg to about 0.05mg, about 0.05mg to about 40mg, about 0.05mg to about 0.05mg, about 0.05mg to about 1mg, about 0.05mg to about 0.1mg, about 0.05mg to about 0.1mg, about 0.1mg to about 5mg, about 0.1mg to about 10mg, about 0.1mg to about 15mg, about 0.1mg to about 20mg, about 0.1mg to about 25mg, about 0.1mg to about 30mg, about 0.1mg to about 35mg, about 0.1mg to about 40mg, about 0.1mg to about 45mg, about 0.1mg to about 50mg, about 0.1mg to about 55mg, about 0.1mg to about 60mg, about 0.1mg to about 65mg, about 0.1mg to about 70mg, about 0.1mg to about 75mg, about 0.1mg to about 80mg, about 0.2mg to about 0.5mg, about 0.2mg to about 0.6mg, about 0.2mg to about 0.8mg, about 0.2mg to about 1mg, about 0.2mg to about 2mg, about 0.2mg to about 0.5mg, about 0.2mg to about 0.6mg, about 0.2mg to about 0.8mg, about 0.2mg to about 0.2mg, about 0.2mg, about 0.2mg to about 65mg, about 0.2mg to about 70mg, about 0.2mg to about 75mg, about 0.2mg to about 80mg, about 0.5mg to about 0.6mg, about 0.5mg to about 0.8mg, about 0.5mg to about 1mg, about 0.5mg to about 2mg, about 0.5mg to about 5mg, about 0.5mg to about 10mg, about 0.5mg to about 15mg, about 0.5mg to about 20mg, about 0.5mg to about 25mg, about 0.5mg to about 30mg, about 0.5mg to about 35mg, about 0.5mg to about 40mg, about 0.5mg to about 45mg, about 0.5mg to about 50mg, about 0.5mg to about 55mg, about 0.5mg to about 60mg, about 0.5mg to about 65mg, about 0.5mg to about 70mg, about 0.5mg to about 0.5mg, about 0.6mg to about 0.6mg, about 0.5mg to about 0.5mg, about 0.6mg to about 0.5mg, about 0.5mg to about 0.5mg, about 0.6mg to about 0.6mg, about 0.6mg to about 0.5mg, about 0.6mg to about 35mg, about 0.6mg to about 40mg, about 0.6mg to about 45mg, about 0.6mg to about 50mg, about 0.6mg to about 55mg, about 0.6mg to about 60mg, about 0.6mg to about 65mg, about 0.6mg to about 70mg, about 0.6mg to about 75mg, about 0.6mg to about 80mg, about 0.8mg to about 1mg, about 0.8mg to about 2mg, about 0.8mg to about 5mg, about 0.8mg to about 10mg, about 0.8mg to about 15mg, about 0.8mg to about 20mg, about 0.8mg to about 25mg, about 0.8mg to about 30mg, about 0.8mg to about 35mg, about 0.8mg to about 40mg, about 0.8mg to about 45mg, about 0.8mg to about 50mg, about 0.8mg to about 0.8mg, about 1mg to about 1mg, about 0.8mg to about 1mg, about 8mg, about 0.8mg to about 1mg, from about 1mg to about 25mg, from about 1mg to about 30mg, from about 1mg to about 35mg, from about 1mg to about 40mg, from about 1mg to about 45mg, from about 1mg to about 50mg, from about 1mg to about 55mg, from about 1mg to about 60mg, from about 1mg to about 65mg, from about 1mg to about 70mg, from about 1mg to about 75mg, from about 1mg to about 80mg, from about 2mg to about 5mg, from about 2mg to about 10mg, from about 2mg to about 15mg, from about 2mg to about 20mg, from about 2mg to about 25mg, from about 2mg to about 30mg, from about 2mg to about 35mg, from about 2mg to about 40mg, from about 2mg to about 45mg, from about 2mg to about 50mg, from about 2mg to about 55mg, from about 2mg to about 60mg, from about 2mg to about 65mg, from about 2mg to about 70mg, from about 2mg to about 80mg, from about 2mg to about 5mg, from about 5mg to about 5mg, about 5mg to about 35mg, about 5mg to about 40mg, about 5mg to about 45mg, about 5mg to about 50mg, about 5mg to about 55mg, about 5mg to about 60mg, about 5mg to about 65mg, about 5mg to about 70mg, about 5mg to about 75mg, about 5mg to about 80mg, about 10mg to about 15mg, about 10mg to about 20mg, about 10mg to about 25mg, about 10mg to about 30mg, about 10mg to about 35mg, about 10mg to about 40mg, about 10mg to about 45mg, about 10mg to about 50mg, about 10mg to about 55mg, about 10mg to about 60mg, about 10mg to about 65mg, about 10mg to about 70mg, about 10mg to about 75mg, about 10mg to about 80mg, about 15mg to about 20mg, about 15mg to about 25mg, about 15mg to about 30mg, about 15mg to about 15mg, about 15mg to about 45mg, about 15mg to about 60mg, about 15mg to about 65mg, about 15mg to about 70mg, about 15mg to about 75mg, about 15mg to about 80mg, about 20mg to about 25mg, about 20mg to about 30mg, about 20mg to about 35mg, about 20mg to about 40mg, about 20mg to about 45mg, about 20mg to about 50mg, about 20mg to about 55mg, about 20mg to about 60mg, about 20mg to about 65mg, about 20mg to about 70mg, about 20mg to about 75mg, about 20mg to about 80mg, about 25mg to about 30mg, about 25mg to about 35mg, about 25mg to about 40mg, about 25mg to about 45mg, about 25mg to about 50mg, about 25mg to about 55mg, about 25mg to about 60mg, about 25mg to about 65mg, about 25mg to about 70mg, about 25mg to about 25mg, about 25mg to about 80mg, about 25mg to about 30mg, about 30mg to about 35mg, about 25mg to about 55mg, about 30mg to about 60mg, about 30mg to about 65mg, about 30mg to about 70mg, about 30mg to about 75mg, about 30mg to about 80mg, about 35mg to about 40mg, about 35mg to about 45mg, about 35mg to about 50mg, about 35mg to about 55mg, about 35mg to about 60mg, about 35mg to about 65mg, about 35mg to about 70mg, about 35mg to about 75mg, about 35mg to about 80mg, about 40mg to about 45mg, about 40mg to about 50mg, about 45mg to about 55mg, about 45mg to about 60mg, about 45mg to about 65mg, about 45mg to about 70mg, about 45mg to about 75mg, about 45mg to about 80mg, about 50mg to about 55mg, about 50mg to about 60mg, about 50mg to about 65mg, about 50mg to about 70mg, about 50mg to about 80mg, about 50mg to about 55mg, about 55mg to about 55mg, about 60mg to about 65mg, about 60mg to about 70mg, about 60mg to about 75mg, about 60mg to about 80mg, about 65mg to about 70mg, about 65mg to about 75mg, about 65mg to about 80mg, about 70mg to about 75mg, about 70mg to about 80mg, about 75mg to about 80 mg).

One or more additional unit operations

In some embodiments of any of the methods provided herein, one or more additional unit operations can be performed on the recombinant protein. In some embodiments, the one or more additional unit operations are selected from the group consisting of: viral inactivation, viral filtration, polishing, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, reverse phase chromatography and ultrafiltration/diafiltration. In some embodiments, the one or more additional unit operations include cleaning-in-place (CIP), wherein precipitated and/or denatured material is removed.

In some embodiments of any of the methods described herein, the method can further comprise the step of formulating the recombinant protein. In some embodiments, the recombinant protein is formulated as a recombinant protein drug product.

Examples of the invention

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLE 1 comparative study of traditional purification method of clarified supernatant with "Capture and purification" method

Setting of experiments

The traditional IgG antibody construct C26 was expressed in HEK293 suspension cells on a 1L cell culture scale for 5 days. After harvesting construct C26, the supernatant was clarified by centrifugation and filtration through a filter with a pore size of 0.22 μm. The clear supernatant was divided into four aliquots of 250mL and stored at 4 ℃ until needed for testing. One hundred fifty ml of clarified supernatant (or for

185 ml of purified) were used to test three different purification methods. All three purification methods were performed using the same elution buffer and the same neutralization buffer.

The method used a PBS wash and at least one citrate wash. First purification method

Is a programmed purification method using immobilized resins in a pre-packed column (i.e., a commercial column).

The purification method is based on directed flow. The second purification method is the traditional batch capture method. This traditional batch capture method involves shaking overnight to allow the resin to float freely in the clear supernatant. A third purification method, referred to as "capture and purification", involves the use of chromatography as described hereinThe pod is filtered. The capture and purification process also included the same overnight shaking as the traditional batch capture process.

Results

Experiments showed that the yield and purity of construct C26 were comparable in all three tested methods (table 1).

TABLE 1 summary of the comparison of yields and purities of the three purification methods

The purification method used 5mL of resin, while the traditional batch capture method and the capture and purification method used 3mL of resin. However, based on the observed resin binding capacity, 3-mL of resin was sufficient to capture a significant amount of IgG antibodies in the supernatant. Next, SDS-PAGE was performed to analyze 20 μ L of the Flow Through (FT) sample, 20 μ L of the wash sample (W), 1 μ g of the elution sample (E) during the traditional batch purification method as well as the capture and purification method (fig. 5). Both reduced and non-reduced samples of both purification methods were run on Life Tech 4-12% gels. SDS analysis indicated that both methods were able to accomplish protein capture, as the protein of interest was not lost during either method of flow-through or washing steps.

The experimental schedules for the three methods are shown in fig. 6. As shown in figure 6 of the drawings,

the purification process may take 7 days to complete for transfection, with periods in excess of 5-6 hours being dedicated to clarification and set-up of the experimental equipment. In addition to this, the present invention is,

the purification process is limited by the flow rate, and therefore, for larger volumes (greater than 1L), the completion time may be greatly increased, sometimes taking longer than that shown in fig. 6. In addition, only one protein can be used

The purification method comprises one-time purification. In contrast, the conventional batch purification method and the capture and purification method can capture and purify several proteins simultaneously within the time range as shown in fig. 6. The traditional batch purification method requires 7 days of transfection before it can be completed, which is the most laborious method of the three test methods.

Both the purification process and the traditional batch process require more consumables than the capture and purification process described herein. The capture and purification process is much less labor intensive and requires minimal user manipulation. The capture and purification process can be completed after 6 days of transfection.

Example 2 study of the "Capture and purification" method, the pod used in the method comprises a porous mesh with an average pore size of 20 μm or 40 μm

Setting of experiments

750mL of the HIS-tagged human protein (C27) culture expressed as in example 1 was harvested and divided into two groups for post-harvest processing. The supernatant, labeled "filtered" in table 2, was clarified by centrifugation and then passed through a filter having a pore size of 0.22 microns. The other group was not treated and is labeled "unfiltered" in table 2. Two 125 ml aliquots of "filtered" and "unfiltered" media were added to a 250ml bottle. A capture and purification device with 1ml NI-NTA affinity resin and 20 micron or 40 micron mesh covering the bottom of the device (the only opening in this case) was added to each vial. This experiment did not include traditional batch capture conditions. The experimental conditions and yields are detailed in table 2 below.

TABLE 2 Experimental conditions

As shown in FIG. 7, high purity was observed under all four conditions: (

92%). These data indicate that there is no loss of purity using a 40 μm porous mesh compared to using a 20 μm porous mesh, and that the capture and purification equipment can achieve comparable yields to traditional batch capture methods using HIS affinity tag purification.

Example 3 study of purification method using pod comprising 40 μm porous mesh

Setting of experiments

Different experimental settings were tested for the ability to capture transiently expressed human IgG. In the first setting (conditions #2 and 3), the pod included a porous mesh only at the bottom end of the pod. In the second setting (condition #4), the pod had porous mesh at both the top and bottom of the pod, with vent holes in the pod walls (figure 4).

By placing the pod (in either filtered or unfiltered liquid cell culture) such that its longitudinal axis is approximately vertical, both settings are placed in both filtered (as described in examples 1 and 2) and unfiltered liquid cell culture, about 175mL of media in about a 150mL flask. After the pods were removed from the liquid cell culture, the column was washed and then eluted using PBS and citrate buffer, respectively.

TABLE 3 Capture and purification Experimental conditions for IgG purification

Table 3 shows the yields (mg/L) under all four conditions. Although the HPLC purity was the same under all four conditions (fig. 8), the yields were higher for the third and fourth settings, and lower for the second setting, compared to the conventional batch trap culture.

Example 4 study of purification method using pods with 20 μm porous mesh

In this study, the traditional batch capture culture method using filtered supernatant was compared to the method using unfiltered supernatant and pods as described in example 3 with a porous mesh only at the bottom end of the pod (table 4).

TABLE 4 Experimental conditions

Table 4 shows the yields (mg/L) under the two conditions. Although the HPLC purity was the same under all two test conditions (fig. 9), the yield of the capture and purification process was higher compared to the traditional batch capture culture.

Example 5: study of purification method using pods with 20 μm or 40 μm porous mesh and adding stopcock valve to control flow rate after absorption

In this study, traditional batch capture methods were compared to methods using pods and unfiltered medium using transiently expressed 0.9L IgG and subsequent purification. In this case, the pod has openings on both its vertical sides and base, which are covered by a 20 or 40 micron porous mesh. 0.9L of culture was subdivided into 5 180mL aliquots. One of these aliquots was clarified by centrifugation and filtered through a filter with 0.22 micron pores, and in the other four aliquots, untreated cell culture medium, referred to as "unfiltered". All aliquots were added to 250ml vials. As described above, the pods and 1.5ml of protein a resin added to their interiors were added to the unfiltered conditions, and 1.5ml of protein a resin was added to the filtered conditions. All 250ml bottles were then incubated overnight with shaking. After the pods were removed from the medium after overnight incubation, stopcocks were added to control the flow rate. For experimental conditions and results, see table 5.

Table 5: experimental conditions and results

Yields under all conditions are shown. Although the purity was comparable under all conditions, the pods sometimes showed similar yields to the traditional batch capture method.

Example 6 study of purification method using pod comprising 40 μm porous mesh

Setting of experiments

Different experimental settings were tested for the ability to capture antibodies from filtered or unfiltered but otherwise identical antibody-containing cell culture media. For the set-up using the apparatus shown in FIGS. 27-28, the supernatant was not filtered. For the settings used as positive control, the supernatant was filtered before adding free floating resin.

The first setting used 2mL of free floatin a chromatography resin exposed to filtered supernatant in a free floating batch capture environment with stirring at 150 RPM. Subsequent purification uses a gravity flow column to capture the resin from the supernatant, followed by washing and elution of the antibody from the resin. The gravity column was washed with 25mL PBS and eluted with 20mL elution buffer. The column was loaded with the same filtered cell culture medium as used for contacting the device/pod used in the third setting, which contained the antibody.

The second setting used 2.5mL of free-floating protein a chromatography resin exposed to the filtered supernatant in a free-floating batch capture environment with stirring at 150 RPM. Subsequent purification uses a gravity flow column to capture the resin from the supernatant, followed by washing and elution of the antibody from the resin. The gravity column was washed with 37.5mL of PBS and eluted with 30mL of elution buffer. The column was loaded with the same filtered cell culture medium as was used to contact the device/pod used in the fourth setting.

The third setting used the apparatus shown in FIGS. 27 and 28, which contained 2-mL of protein A chromatography resin. The resin in the pod/device was washed with 75mL PBS and eluted with 30mL elution buffer. After the elution buffer first contacted the resin in the pod/device, a pause of 5 minutes was made before additional elution buffer was flowed through the resin in the pod/device. Use in chromatography

And (4) carrying out the system. The resin in the pod/device is loaded with the same filtered cell culture medium as used to contact the device/pod used in the first setting. More specifically, the pod was contacted with 350mL of filtered cell culture medium in a 500mL flask.

Results

The data are shown in Table 6 below. Figures 32 and 33 show the eluted chromatograms from the device/pod of the third and fourth settings, respectively.

TABLE 6 Capture and purification Experimental conditions for IgG purification

The above data indicate that the pods/apparatus provided herein can provide similar yields of recombinant antibody compared to using standard chromatography columns.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A filtration and chromatography pod comprising:

a container formed by a wall and having a top end and a bottom end;

wherein:

the wall forming a seal with the bottom end; and is

One or more of the walls, the top end and the bottom end include at least one area covered by a semi-porous or porous material that allows liquid to reversibly flow from the exterior of the pod into the interior of the pod.

2. The pod of claim 1, wherein the semi-porous or porous material is characterized by one or both of: (a) not allowing cells to flow from the exterior of the pod into the interior of the pod, and (b) retaining or substantially retaining the resin within the interior of the pod.

3. The pod of claim 2, wherein the cells are mammalian cells.

4. The pod of claim 2, wherein the cells are insect cells or yeast cells.

5. The pod of claim 2, wherein the cells are bacterial cells.

6. The pod of any of claims 1-5, wherein the material is a semi-permeable or porous membrane.

7. The pod of any of claims 1-5, wherein the material is a semi-permeable or porous mesh.

8. The pod of claim 7, wherein the mesh is made of nylon.

9. The pod of any of claims 1-8, wherein the material has a pore size of about 50nm to about 50 μ ι η.

10. The pod of claim 9, wherein the pore size of the material is from about 100nm to about 30 μ ι η.

11. The pod of claim 10, wherein the pore size of the material is from about 500nm to about 20 μ ι η.

12. The pod of claim 11, wherein the pore size of the material is from about 1 μ ι η to about 10 μ ι η.

13. The pod of claim 12, wherein the pore size of the material is about 2 μ ι η to about 5 μ ι η.

14. The pod of any of claims 1-13, wherein the internal volume of the container is about 1mL to about 500 mL.

15. The pod of any of claim 14, wherein the internal volume of the container is about 1mL to about 250 mL.

16. The pod of any of claim 15, wherein the internal volume of the container is about 1mL to about 100 mL.

17. The pod of any of claims 1-16, wherein the bottom end comprises at least one area covered by the material.

18. The pod of any of claims 1-16, wherein the wall comprises at least one area covered by the material.

19. The pod of any of claims 1-16, wherein the bottom end and the wall each comprise at least one area covered by the material.

20. The pod of any of claims 1-16, wherein the tip comprises at least one area covered by the material.

21. The pod of any of claims 1-16, wherein the top end and the bottom end each comprise at least one area covered by the material.

22. The pod of any of claims 1-21, wherein each of the at least one regions has a surface area of about 1mm²To about 25cm²。

23. The pod of claim 22, wherein each of the at least one regions has a surface area of about 1mm²To about 20cm²。

24. The pod of claim 23, wherein each of the at least one areaThe surface area of each of the units is about 1mm²To about 10cm²。

25. The pod of claim 24, wherein each of the at least one regions has a surface area of about 1mm²To about 5cm²。

26. The pod of claim 25, wherein each of the at least one regions has a surface area of about 1mm²To about 2.5cm²。

27. The pod of claim 26, wherein each of the at least one regions has a surface area of about 1mm²To about 1cm²。

28. The pod of any of claims 1-27, wherein the bottom end is flat.

29. The pod of any of claims 1-27, wherein the bottom end is hemispherical.

30. The pod of any of claims 1-27, wherein the bottom end is conical.

31. The pod of any of claims 1-30, wherein the wall is cylindrical.

32. The pod of any of claims 1-30, wherein the wall is conical.

33. The pod of any of claims 1-30, wherein the walls are spherical.

34. The pod of any of claims 1-30, wherein the wall is hemispherical.

35. The pod of any of claims 1-30, wherein the wall is hourglass-shaped.

36. The pod of any of claims 1-35, wherein the pod further comprises a chromatography resin disposed inside the container.

37. The pod of claim 36, wherein the chromatography resin is a commercial chromatography resin.

38. The pod of claim 36 or 37, wherein the chromatography resin is an affinity chromatography resin.

39. The pod of claim 38, wherein the affinity chromatography resin is a protein a resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin.

40. The pod of any of claims 36-39, wherein the chromatography resin has a volume of about 1mL to about 500 mL.

41. The pod of claim 40, wherein the volume of chromatography resin is about 1mL to about 250 mL.

42. The pod of claim 41, wherein the volume of chromatography resin is about 1mL to about 100 mL.

43. The pod of claim 42, wherein the volume of chromatography resin is about 1mL to about 50 mL.

44. The pod of any of claims 36-43, wherein the chromatography resin has a sterility assurance level of about 1x 10^-6To about 1x 10^-10。

45. A kit comprising a pod according to any of claims 1-44.

46. The kit of claim 45, further comprising means for stably positioning the pod inside the culture dish.

47. The kit of claim 46, wherein the culture dish is a shake flask, a shake tube, a bioreactor, or a multi-well plate.

48. The kit of claim 47, wherein the bioreactor is a perfusion bioreactor.

49. The kit of claim 47, wherein the bioreactor is a fed-batch bioreactor.

50. A kit, comprising:

(a) at least one pod of any one of claims 18, 19, and 22-44; and

(b) one or more panels securable to each of the one or more areas in the wall to prevent fluid from leaking from the interior of the pod through the wall to the exterior of the pod.

51. A kit, comprising:

(a) the pod of any of claims 18, 19, and 22-44; and

(b) an outer sleeve securable to the pod to cover at least one area in the wall to prevent fluid from leaking from the interior of the pod through the at least one area in the wall to the exterior of the pod.

52. The kit of claim 51, wherein the sleeve is an empty chromatography column.

53. A kit, comprising:

(a) the pod of any of claims 1-44; and

(b) one or both of:

an elution outlet stably fixed on the bottom end; and

a buffer reservoir stably affixed to the tip.

54. The kit of claim 53, wherein the kit comprises a pod and an elution outlet.

55. The kit of claim 53, wherein the kit comprises a pod and a buffer reservoir.

56. The kit of claim 53, wherein the kit comprises a pod, an elution outlet, and a buffer reservoir.

57. The kit of any one of claims 53, 54, and 56, wherein the elution outlet is a funnel.

58. The kit of any one of claims 53, 54, and 56, wherein the elution outlet comprises a flat surface with a port.

59. The kit of any one of claims 53, 54, and 56-58, wherein the elution outlet is configured to allow for adjustment of a flow rate of fluid through the elution outlet.

60. The kit of any one of claims 53, 54, and 56-58, wherein the elution outlet is not configured to allow adjustment of a flow rate of fluid through the elution outlet.

61. The kit of any one of claims 53, 54 and 56-60, wherein the elution outlet is threadably or snap-fit to a pod.

62. A kit, comprising:

(a) the pod of any of claims 18, 19, and 22-44; and

(b) a movable outer sleeve connected to the wall such that rotation of the movable outer sleeve is capable of covering at least one area in the wall thereby preventing fluid from leaking from the interior of the pod through the at least one area in the wall to the exterior of the pod.

63. The kit of claims 50-62, further comprising means for stably positioning the pod inside the culture dish.

64. A kit, comprising:

(a) the pod of any of claims 17 and 20-44; and

(b) means for stably positioning the long axis of the pod in a horizontal position inside the culture dish.

65. A kit, comprising:

(a) the pod of any of claims 1-19, 22-28, 31, and 36-44; and

(b) an inlet system having a top end, an elongated cylindrical body comprising a fluid conduit, and a bottom end, wherein the top end of the inlet system comprises a fluid inlet and is configured for stable attachment to the top end of the pod and, when stably connected to the pod, allows fluid to flow into the pod through the fluid inlet and the fluid conduit and out the bottom end of the inlet system, and the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod; and

(c) an outlet system comprising a fluid outlet configured for stable attachment to the bottom end of the pod and allowing fluid to flow out of the pod through the fluid outlet when stably connected to the bottom end of the pod.

66. The kit of claim 65, wherein the top end of the inlet system and the top end of the pod comprise threads allowing the top end of the pod to be stably attached to the top end of the inlet system.

67. The kit of claim 65 or 66, wherein the threads in the top end of the inlet system or the threads in the top end of the pod comprise an O-ring.

68. The kit according to any one of claims 65-67, wherein:

the pod comprises a chromatography resin; and is

The inlet system and pod are configured such that the bottom end of the inlet system is proximate to the chromatography resin when the inlet system and pod are stably attached.

69. The kit of claim 68, wherein the inner surface of the pod and the bottom end of the inlet system are configured to allow stable attachment of the pod and the bottom end of the inlet system.

70. The kit of claim 69, wherein the inner surface of the pod and the bottom end of the inlet system comprise threads allowing for stable attachment of the pod and the bottom end of the inlet system.

71. The kit of claim 70, wherein the threads in the inner surface of the pod or the threads in the bottom end of the inlet system comprise an O-ring.

72. The kit of any one of claims 65-71, wherein the outlet system and the bottom end of the pod comprise threads allowing the outlet system to be stably attached to the bottom end of the pod.

73. The kit of claim 72, wherein the threads in the outlet system or the threads in the bottom end of the pod comprise an O-ring.

74. The kit of any one of claims 65-73, wherein the fluid inlet is attachable to a conduit.

75. The kit of any one of claims 65-74, wherein the fluid outlet is connectable to a conduit.

76. The kit according to any one of claims 65-75, further comprising:

(d) means for stably positioning the long axis of the pod in a horizontal position inside the culture dish.

77. A method of capturing a recombinant protein produced by a liquid cell culture, the method comprising contacting a pod of any of claims 36-44 with a liquid cell culture for a period of time,

wherein the contacting step results in at least a portion of the at least one region contacting the liquid cell culture and the contacting is performed under conditions that result in the chromatography resin capturing the recombinant protein.

78. The method of claim 77, wherein the liquid cell culture is disposed in a culture dish.

79. A method of purifying a recombinant protein produced by a liquid cell culture, the method comprising:

(a) contacting the pod of any of claims 36-44 with a liquid cell culture for a period of time, wherein the contacting step results in at least a portion of the at least one region contacting the liquid cell culture and the contacting is under conditions that result in the chromatography resin capturing the recombinant protein; and

(b) eluting the recombinant protein from the chromatography resin after said period of time, thereby purifying the recombinant protein.

80. The method of claim 79, further comprising, after (b), (c) performing one or more additional unit operations on the recombinant protein.

81. The method of claim 80, further comprising, after (c), formulating the recombinant protein into a recombinant protein drug product.

82. The method of any one of claims 77-81, further comprising washing the chromatography resin between step (a) and step (b).

83. The method of any one of claims 77-82, wherein the contacting is performed under sterile conditions.

84. The method of any one of claims 77-83, wherein the period of time is from about 5 minutes to about 2 weeks.

85. The method of claim 84, wherein the period of time is from about 5 minutes to about 168 hours.

86. The method of claim 85, wherein the period of time is from about 5 minutes to about 96 hours.

87. The method of claim 86, wherein the period of time is from about 5 minutes to about 48 hours.

88. The method of claim 87, wherein the period of time is from about 5 minutes to about 24 hours.

89. The method of claim 88, wherein the period of time is from about 5 minutes to about 12 hours.

90. The method of any one of claims 77-89, wherein the period of time occurs when the liquid cell culture is in its exponential growth phase.

91. The method of any one of claims 77-89, wherein the period of time occurs when the liquid cell culture is in its quiescent growth stage.

92. The method of any one of claims 77-91, wherein at least 30% of the total surface area of at least one zone is contacted with the liquid cell culture during the period of time in step (a).

93. The method of claim 92, wherein at least 50% of the total surface area of at least one zone is contacted with liquid cell culture during the period of time in step (a).

94. The method of claim 93, wherein at least 75% of the total surface area of at least one zone is contacted with the liquid cell culture during the period of time in step (a).

95. The method of claim 94, wherein at least 90% of the total surface area of at least one zone is contacted with liquid cell culture during the period of time in step (a).

96. The method of any one of claims 77-95, wherein the liquid cell culture is placed in a culture dish.

97. The method of claim 96, wherein the culture dish is a shake tube, flask, or bioreactor.

98. The method of claim 97, wherein the bioreactor is a perfusion bioreactor.

99. The method of claim 97, wherein the bioreactor is a fed batch bioreactor.

100. The method of any one of claims 77-99, wherein the liquid cell culture comprises bacterial cells, yeast cells, plant cells, or mammalian cells.

101. The method of claim 100, wherein the mammalian cell is a human cell.

102. The method of claim 101, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

103. The method of claim 80, wherein the one or more additional unit operations are selected from the group consisting of virus inactivation, virus filtration, polishing, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and ultrafiltration/diafiltration.

104. The method of any one of claims 77-103, wherein the recombinant protein is a secreted protein.

105. The method of any one of claims 77-104, wherein the recombinant protein is any protein with or without a tag.

106. The method of any one of claims 77-104, wherein the recombinant protein is an antibody, an antigen-binding antibody fragment, an enzyme, or a fusion protein.