WO2010043703A1

WO2010043703A1 - Removal of host cell proteins

Info

Publication number: WO2010043703A1
Application number: PCT/EP2009/063568
Authority: WO
Inventors: Grigorios Zarbis-Papastoitsis; Michael Christopher Kuczewski; Emily Belcher Schirmer
Original assignee: Dsm Ip Assets B.V.
Priority date: 2008-10-17
Filing date: 2009-10-16
Publication date: 2010-04-22

Abstract

The present invention relates to a method for the removal of host cell protein from a cell broth containing cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by contacting the cell broth with a particulate anion exchanger, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer, and determining the reduction of the HCP content in the supernatant layer as compared to the cell broth. The present invention further relates to a method for the recovery of a secreted desired biological substance from a cell broth containing cells producing the secreted desired biological substance which has an overall positive charge in the cell broth by contacting the cell broth with particulate anion exchanger, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer, determining the reduction of the HCP content in the supernatant layer as compared to the cell broth and extracting the secreted desired biological substance from the supernatant layer.

Description

REMOVAL OF HOST CELL PROTEINS

The present invention relates to a method for the reduction of host cell protein (HCP) in a cell broth and to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance.

Fermentative production of biological substances, such as pharmaceuticals and in particular monoclonal antibodies, delivers a complex cell broth from which the biological substances should be isolated and purified by a great number of steps.

As a first step solid material such as the cells and cell debris is to be separated from the cell broth fluid - a step called clarification. In many instances the biological substances are present extracellularly and will thus be present in the cell broth fluid. Examples of clarification methods used to-date include centrifugation, filtration (such as microfiltration, depth filtration and filtration through absolute pore size membranes) and expanded bed chromatography. Flocculation may be employed in order to enhance any of these clarification methods, in particular in combination with filtration. Known flocculation agents for this purpose can range from simple electrolytes to synthetic poly-electrolytes (such as DEAE dextran, acryl-based polymers, polyethylene amine) or inorganic materials (such as diatomaceous earth or perlites). A recent development is the use of chitosan for this purpose.

An objective of the present invention is to reduce the HCP content of the secreted desired biological substance from a cell culture prior to further downstream processing.

In a specific embodiment the present invention relates to a method for the reduction of host cell protein in a cell broth containing mammalian cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by: a. contacting the cell broth with a particulate anion exchange material, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth.

The international patent application published as WO2007/108955 relates to a protein purification process, in particular to purification of antibodies or antibody-like proteins, from the supernatant of a cell culture, by using cation exchange followed by anion exchange chromatography. This encompasses the removal of HCP. Also, it is described in the document that these chromatography steps may be carried out using a batch process followed by a separation step (separation of supernatant from solid material). The essential difference between the instant invention and the disclosure of WO2007/108955 is that the latter relates to purification from a clarified protein-containing medium, whereas the instant invention relates to a process carried out on a unclarified material containing cells.

The international patent application which is published as WO2008/079280 relates to the purification of biomolecules from a mixture, by adding soluble polymers (e.g. soluble ion exchange polymers) to the mixture and changing the conditions so as to precipitate these and the biomolecules bound to it, thus separating the biomolecules into fractions. The essential difference between the instant invention and the disclosure of WO2008/079280 is that in the latter the ion-exchange polymer is soluble, whereas in the instant the anion exchange material is particulate. Neither of the two documents (WO2007/108955 and

WO2008/079280) suggests a purification process carried out on the complete cell broth. The problem which can be solved by the present invention is that there is a desire in the industry to reduce the number of purification steps in a protein purification process. The present invention solves the problem by combining the removal of HCP with the clarification step of the cell broth.

In the context of the present invention "cell broth" means a cell culture inoculated with intact mammalian cells, and which may further contain culture medium as defined below, as well as secreted biological substances. In the process of the present invention, particularly when the cell density is extremely high, it may also be desirable to dilute the starting material from the bioreactor to a preferred cell density. For the purpose of the present invention the so diluted material is still covered by the term cell broth.

As a practical upper limit, the process according to the present invention may be carried out with cell densities up to 175x10⁶ cells/ml, more preferably up to 130x10⁶ cells/ml. The cell density can be measured using a cell counter such as Vi-CELL™ (with the trypan blue exclusion method) but other suitable methods include cytometry, packed cell volume determination, or Coulter counters (with the Electrical Sensing Zone Method). If the initial cell density is above 130x10⁶ cells/ml it is advisable to first dilute the cell broth. In practice it is preferred that a cell broth with an initial cell density above 100x10⁶ cells/ml be first diluted. Dilution preferably may be done to a cell density of not more than 80x10⁶ cells/ml. The cell broth may be diluted with a solution that does not greatly change the environment of the cell so as to not cause lysis of the mammalian cells, i.e. an isotonic solution such as PBS.

With "secreted biological substances" is meant here biological substances which upon the production thereof by the mammalian cells are predominantly actively or passively released into the extracellular medium.

With "desired" is meant here that the biological substance is intentionally being produced making use of the mammalian cells.

With "overall positive charge" of the secreted desired biological substances is meant here that the electrostatic contribution of positive and negatively charged ionogenic groups on the biological substance under the solvent conditions in the cells broth results in a net positive charge. The overall charge of a biological substance is based on the pK_a of the acidic and basic residues and the pH of the solution - in this case the pH of the cell broth. For the biological substance to have a net positive charge in the cell broth, the pi (the pH where the net charge is zero) of the substance must be higher than the pH of the cell broth.

With "contacting" is meant here introduction of anion exchanger to cell broth and settling under gravity or with centrifugation.

With "anion exchange material" is meant here weak or strong anion exchange chromatography media. The anion exchange material generally comprises a carrier, which may be organic material or inorganic material or a mixture of organic and inorganic material. Suitable organic materials are agarose based media and methacrylate. Suitable inorganic materials are silica, ceramics and metals. The particles preferably may have a size of between 15 and 150 μm. More preferably their size is between 15 and 70 μm. The particles may have a density suitable for effecting relatively rapid sedimentation of the cells from the cell broth, but not too high as it was observed that too dense particles did not effect the sedimentation. - A -

With "adequate incubation time" is meant here the time in which the precipitation of the cells results in a distinct cell pellet volume and a supernatant layer.

With "separating" is meant here any method to remove the supernatant from the cell pellet, such as by decanting or drawing out the supernatant or e.g. by draining the pellet from the vessel through a port at the bottom.

The "supernatant layer" is the liquid overlying volume as a result of the settling. The supernatant layer may (and generally will) still contain cells, be it at a cell density significantly lower than the initial cell density.

The method according to the present invention results in a reduction of the HCP content in the supernatant of at least 10%, preferably at least 20%.

According to a further embodiment the present invention relates to a method for the recovery of a secreted desired biological substance from a cell broth containing cells producing the secreted desired biological substance which has an overall positive charge in the cell broth by a. contacting the cell broth with particulate anion exchanger material, b. allowing an adequate incubation time to result into formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth and e. extracting the secreted desired biological substances from the supernatant layer

With "recovery" is meant here obtaining the desired product from by-products and waste. The present invention further relates to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance as described above and wherein the cells in the cell broth are mammalian cells, wherein the resulting cell pellet is further processed by f. re-suspending the resulting cell pellet, g. allowing an adequate incubation time to result into formation of a cell pellet and a supernatant layer, h. separating the resulting cell pellet from the supernatant layer, and i. extracting the secreted desired biological substances from the supernatant layer. In a further method according to the present invention step f. through i. of the above process are repeated one or more times.

In a further preferred method according to the present invention the resulting cell pellet is re-suspended in a solution that does not greatly change the environment of the cell so as to not cause lysis of the mammalian cells, such as an aqueous (preferably isotonic) salt solution, more preferably in PBS.

Preferably the supernatant layers are collected and the secreted desired biological substance is extracted from the pooled supernatants.

Surprisingly, it has been found that the method according to the present invention enables a considerable reduction of the host cell protein content of a desired biological substance produced by the cells.

The present invention further relates to the use of a particulate anion exchanger for the reduction of host cell protein in a cell broth containing cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by: a. contacting the cell broth with a particulate anion exchanger, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth.

The method according to the present invention is particularly useful for cell broths containing mammalian cells at very high cell density, such as 60x10⁶ cells/ml, but even for cell densities as high as 120x10⁶ cells/mL . Examples of mammalian cells include CHO (Chinese Hamster Ovary) cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, human cells, for example HEK-293 cells, human lymphoblastoid cells, E1 immortalized HER cells, mouse cells, for example NSO cells. More preferably, E1 immortalized HER cells are used, most preferably PER.C6 cells. In a preferred embodiment, the cells in the process of the present invention are E1 -immortalized HER cells, more preferably PER.C6 cells (see U.S. Patent 5,994,128, the content of which is incorporated by reference here). PER.C6 cells are exemplified by cells as deposited under ECACC No. 96022940 (see, e.g., U.S. Patent 5,994,128, EP 0833934 B1 , the contents of which are incorporated by reference here). The cell broth for clarification may be obtained by any cell culturing method suitable for propagation of mammalian cells under conditions wherein a desired biological substance is being made. Particularly suitable methods in this respect are described in e.g. WO2005095578, WO2004099396 and WO2008006494. The contents thereof are incorporated herein by reference. These methods may yield mammalian cells at very high cell densities.

Biological substances, which may be produced by the cells, for example by expressing a (recombinant) gene coding therefore are for example viruses or (recombinant) proteins, in particular receptors, enzymes, fusion proteins, blood proteins such as proteins from the blood coagulation cascade, multifunctional proteins such as for instance erythropoietin, virus or bacterial proteins for instance for use in vaccines; immunoglobulins such as antibodies, for example IgG or IgM, and the like; Preferably a protein, more preferably an antibody is produced by the cells. Preferably, the biological substances such as proteins or vaccines produced by the cells can be used as an active ingredient in a pharmaceutical preparation. In the context of the present invention, the terms 'product' and 'biological substance' are interchangeable.

Suitable methods for extracting the secreted desired biological substances from the supernatant layer are for example filtration (such as depth filtration, microfiltration, ultrafiltration, diafiltration), chromatography (such as size exclusion chromatography, affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography), aqueous two-phase extraction, precipitation or centrifugation.

Advantageously, the desired biological substance can be extracted very efficiently by cation exchange chromatography or in case of immunoglobulins with affinity chromatography, in particular protein A chromatography.

Short description of the figures

Figure 1. Supernatant cell density as a function of time for various resins. The cell densities were measured by Vi-CELL (Beckman-Coulter). Figure 2. Supernatant volume as a function of time for various resins. The total volume in each case was 24 ml.

Figure 3. Shows the SDS-PAGE and agarose gels of samples taken from each of the reactors as described in Example 2. A) SDS-PAGE of reactor 1 and 2 samples B) DNA-Agarose gel of reactor 1 and 2 samples. Samples were pre- treated with protein A to remove the MAb. Lanes: 1-MW standard or 1 kBase ladder; 2-Reactor 1 harvest; 3-Reactor 1 Si-PEI treated partially clarified harvest; 4- Reactor 2 harvest; 5-Reactor 2 Si-PEI treated partially clarified harvest.

EXAMPLES

Symbols and abbreviations:

Xt = Total cell density, cells/mL Si-PEI = Bakerbond Wide-Pore PEI (PolyEthylenelmine) Prep LC Packing grafted silica beads (JT Baker)

DEAE Hyper D = diethylaminoethyl grafted ceramic beads (Pall).

Super Q = quaternary amino functionality on a methacrylate support (Tosoh).

TP DEAE = diethylaminoethyl functionality on a methacrylate support (Tosoh). HCP = Host Cell Protein

PBS = Phosphate Buffered Saline

The following experiments were carried out using PER.C6® cells which were prepared according to the procedure outlined in WO2008006494. These

PER.C6® cells produced an antibody. The first experiment illustrates the removal of HCP from a cell broth with various anion exchangers

The second experiment illustrates the extraction of the desired protein from the cell broth.

Example 1 Removal of HCP

Si-PEI (15 and 40 μm), ToyoPearl Super Q (35 and 65 μm),

ToyoPearl DEAE (35 and 65 μm) and DEAE Hyper D were evaluated with a cell broth containing PER.C6® cells producing a monoclonal antibody (MAb). The X₁ was 98.8^χ10⁶ cells/mL on day 13 of the reactor. The material was diluted to 75^χ10⁶ cells/mL with Dulbecco's PBS (5 mS/cm) to a final volume of 20 mL. The resin was added as a 50/50 slurry as described above (total volume was 24 mL) and the cells were allowed to settle until the pellet volume was constant (60 min).

Aliquots of the harvest and supernatant were analyzed by analytical protein A chromatography to determine the product recovery. The recovery was determined by: Mass of IVlAb in Supernatant

Recovery = C 2 }

Mass of MAb in Harvest

The analyses were corrected for the volume of the biomass when necessary, i.e. when the cell density is extremely high, the cells contribute significantly to the working volume. Aliquots of the harvest and supernatant were analyzed by HCP

ELISA to determine the reduction of impurities. HCP ELISA is an immunological method consisting of polyclonal antibodies raised against host cell proteins, in this case PER.C6^® derived proteins. The polyclonal antibodies are then used to coat micro titer plates which are then incubated with in-process samples. Following incubation the micro titer plates are incubated again with the same polyclonal antibodies, this time the antibodies are conjugated with an enzyme such as alkaline phosphatase. This two step process results in the creation of a "sandwich" complex. The complex is then reacted with a chromogenic substrate and the HCP population is quantified based on the color intensity. The reduction of HCPs was determined by:

„ ., .. .χ _{τ τy}.,_^ { /M of HCP/ms of MAb) in Supernatant Reduction ol HC P = -^? ^: i 3 S i ug of HCP/ina of MAb)io Harvest

The analyses were corrected for the volume of the biomass when necessary.

Figure 1 shows the supernatant cell density as a function of time for each anion exchanger as well as the control where no anion exchange material was added. Accelerated cell settling was observed in each case compared with the control. The smaller particle size appears to decrease the supernatant cell density below 10x10⁶ cells/mL, where as the larger particle size decreases the cell density to 11-15^χ10⁶ cells/mL Figure 2 shows the supernatant volume versus time for each anion exchanger. Addition of the Si-PEI materials results in the largest amount of supernatant volume which corresponds to the most compact pellet. The pellet accounted for 40% of the total volume in this case, whereas the pellet accounted for 63% of the total volume in the control. The Si-PEI materials have a greater density than the methacrylate and agarose based materials, which allows for more compact pellets. The ceramic Hyper D materials have an intermediate density with corresponding intermediate pellet volumes. The product recovery and HCP reduction are summarized in Table 1. The addition of the resin increases the product recovery and significantly reduces the HCP levels in the semi-clarified media.

Table 1 : Summary of product recovery and HCP reduction for various anion exchangers. ^<1) In a control experiment no anion exchanger was added.

The recovery and HCP reduction are determined by eq 2 and 3, respectively.

Figure 3 shows the SDS-PAGE and agarose gels of samples taken from each of the reactors. As evidenced by the gels, the levels of HCP and DNA in the Si-PEI treated samples are much lower than the levels initially in the harvest.

Equivalent volumes were loaded into each lane for both gels. Additionally, the samples were pre treated with protein A to remove the monoclonal antibody.

Table 2: Summary of product recovery, HCP reduction (measured by ELISA assay), and DNA reduction (measured by real time PCR) from Si-PEI treated cell culture broths.

Example 3 Purification of desired biological substance

An cell culture harvest with initial cell density of 175 x 10⁶ cells/mL was diluted to ~ 75 x 10⁶ cells/mL with PBS(lnitial volume of 1.7 L). Following dilution Si-PEI chromatography media was added to the harvest (0.1 L of Si-PEI resin per L of diluted harvest). The cells were allowed to settle for ~ 60 minutes. The product containing supernatant was decanted and the settled cells were washed twice with PBS. The initial supernatant was pooled together with the two washes to maximize product recovery (~ 95%). The combined pool contains less than 5 x 10⁶ cells/ml, and the HCP content is reduced by 59%.

The product recovered after the Si-PEI settling is further purified by depth filtration. Depth filtration consist of a primary filter (typically 10 or 5 μm pore size) used for further reduction of the cell mass, followed by a secondary filter (typically 3 or 1 μm pore size) that removes smaller particles and prepares the clarified harvest for sterile filtration typically through a gradient 0.8/0.2 μm filter. The depth filtration train can be Millipore Millistak+HC filters containing media such as DOHC (primary) followed by XOHC (secondary) or CUNO ZetaPlus filters containing media such as 10M02 (primary) followed by 60ZA05A (secondary). In either case the clarified harvest is further filtered through 0.8/0.2 μm filters (Supor, Pall). In addition an 85% HCP reduction was observed through the secondary filter during depth filtration. Reduction in HCP through the secondary filter could be attributed to the charged nature of these filters and has been previously reported in the literature (Yigzaw Y, Piper R, Tran M, Shukla AA. 2006. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Monoclonal Antibody Purification. Biotechnology Progress 22(1 ):288-296.).

The clarified material is further purified by Cation Exchange

Chromatography such as GigaCap S (Tosoh). The monoclonal antibody (product) is immobilized on the resin at a capacity of > 95 g/L of chromatography media. The conditions used for immobilizing the antibody are slightly acidic (pH ~ 5.3) and conductivity of ~ 4.5 mS/cm. After binding, the antibody is washed with equilibration buffer and finally eluted with a buffer step containing 100 mM sodium chloride. An additional reduction in HCP content (78%) is obtained by this step.

The eluted antibody can be further purified by a combination of chromatography and filtration techniques until the required purity specifications are met.

The overall reduction in Host Cell Proteins from the cell culture harvest through the CEX step is summarized below:

Table 3: Removal of HCP during purification of MAb from a cell broth.

Claims

1. A method for the reduction of host cell protein (HCP) in a cell broth containing cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by: a. contacting the cell broth with a particulate anion exchanger, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth.

2. A method for the recovery of a secreted desired biological substance from a cell broth containing cells producing the secreted desired biological substance which has an overall positive charge in the cell broth by a. contacting the cell broth with particulate anion exchanger, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth and e. extracting the secreted desired biological substances from the supernatant layer.

3. Method according to claim 2, wherein the resulting cell pellet is further processed by f. re-suspending the resulting cell pellet, g. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, h. separating the resulting cell pellet from the supernatant layer and i. extracting the secreted desired biological substances from the supernatant layer.

4. Method according to claim 3, wherein step f through i are repeated.

5. Method according to claim 3 or 4, wherein the resulting cell pellet is re-suspended in an aqueous salt solution.

6. Method according to claim 5, wherein the aqueous salt solution is PBS.

7. Method according to claim 3 or 4 wherein the respective supernatant layers are combined prior to extracting the desired biological substance.

8. Method according to any of claims 2 to 7 wherein the biological substance is extracted using cation exchange chromatography.

9. Use of a particulate anion exchanger for the reduction of host cell protein (HCP) in a cell broth containing cells, culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by: a. contacting the cell broth with a particulate anion exchanger, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. determining the reduction of the HCP content in the supernatant layer as compared to the cell broth.