KR20110085980A - Clarification process at higher cell density - Google Patents

Abstract

The present invention allows for sufficient incubation time to contact the cell broth with the anion exchange material, to form the cell pellet and the supernatant layer, and to separate the resulting cell pellet from the supernatant layer, thereby producing a desired secretion with overall positive charge in the cell broth. A method for the purification of cell broths containing biological material and mammalian cells, the present invention relates to a method in which said cells are present at high cell density. The present invention further contains mammalian cells at a high cell density which, after performing the above steps, extracts the desired secreted biological material from the supernatant layer, thereby producing the desired secreted biological material with the overall positive charge in the cell broth. To a method for recovering the desired secreted biological material from the cell broth.

Description

CLARIFICATION PROCESS AT HIGHER CELL DENSITY

The present invention relates to a method for purifying cell broth and a method for recovering a desired secreted biological material from a cell broth containing cells producing a desired secreted biological material.

Fermentation production of biological materials, such as pharmaceuticals, in particular monoclonal antibodies, yields complex cell broths in which the biological material must be isolated and purified in multiple steps.

As a first step, solid materials such as cells and cell debris must be separated from the cell broth fluid-this step is called purification. In many cases, the biological material is extracellular and therefore will be present in the cellular broth fluid.

Examples of purification methods used to date include centrifugation, filtration (eg, microfiltration, deep filtration, and filtration through absolute pore size membranes) and expanded bed chromatography. Agglomeration can be used to enhance any of the above purification methods, especially those combined with filtration.

Known flocculants for this purpose can range from simple electrolytes to synthetic poly-electrolytes (eg DEAE dextran, acrylic-based polymers, polyethylene amines) or inorganic materials (eg diatomaceous earth or pearlite). Recently, the use of chitosan has been developed for this purpose.

The disadvantages of using multiple flocculants are, in particular, that they can bind the desired biological material of interest, that they can deactivate the desired biological material of interest, that the flocculation process takes too long and / or that the flocculant is medical It can be difficult or expensive to manufacture with the high quality required for the application. In addition, flocculants must be removed from the final purified molecule, which requires expensive and time-consuming assays to confirm their removal.

The method according to the invention has none of the above disadvantages.

Moreover, this existing method has been shown to achieve clarification only at relatively low cell densities. In addition, most of these methods have not been shown to be successfully applied to mammalian cells, particularly mammalian cells that produce the desired secreted biological material.

It is an object of the present invention to provide a cost-effective purification method of cell broth harvested from a bioreactor and containing mammalian cells and the desired secreted biological material.

It is a further object of the present invention to provide a method for purifying cell broths containing mammalian cells at high cell densities.

In certain embodiments, the present invention provides a method of purifying a cell broth containing the desired secreted biological material having a globally positive charge in the cell broth as well as mammalian cells and culture medium, wherein the cells are 15 × 10. A method of presenting at an initial cell density of at least ⁶ cells / ml:

a. Contacting the cell broth with the particulate anion exchange material,

b. Allowing sufficient incubation time to form cell pellet and supernatant layers, and

c. Separating the resulting cell pellet from the supernatant layer.

U.S. Patent Application 2003/170810 discloses the separation of insoluble materials (including resins and their bounds, cells and cell debris) from soluble materials by batch mixing anion exchange resins and lysates followed by filtration, centrifugation or specific gravity separation. A method of purifying crude cell lysate is described. Contrary to the disclosure of the above U.S. patent application, according to the present invention, after cells are not lysed, they are mixed with an anion exchange resin, clarification occurs at a particularly high cell density, and the desired biological material in the cell broth is responsible for the overall positive charge. Have It is surprisingly believed that according to the present invention purification can be achieved with high cell density cell broth.

In the context of the present invention, "cell broth" means a cell culture inoculated with intact mammalian cells, which may further contain secreted biological material as well as culture medium as defined below. In the process of the invention, it may be desirable to dilute the starting material from the bioreactor to the desired cell density, especially when the cell density is very high. For the purposes of the present invention, such diluted substances are still included in the term cell broth.

By "secreted biological material" is meant herein a biological material that is mainly released (actively or passively) into the culture medium upon production by mammalian cells.

"Want" means herein that the biological material is intentionally produced using mammalian cells.

By "total positive charge" of the desired secreted biological material is meant herein the electrostatic contribution of the positive and negatively charged ionizers on the biological material under solvent conditions in the cell broth to produce a net positive charge. The overall charge of the biological material is based on the pKa of acidic and basic residues and the pH of the solution-in this case the pH of the cell broth. In order for a biological material to have a net positive charge in the cell broth, the pI (the pH at which the net charge is zero) of the material must be higher than the pH of the cell broth.

"Culturing medium" refers herein to the extracellular environment of a cell, which contains nutrients and other ingredients that support the growth and production of cells, but also from waste products or host cell proteins (HCP) or lysed cells It may contain. The composition of the culture medium may vary during the culturing process of the cells, and at least one original component may be greatly reduced in the clarification step.

"Contact" herein means introducing an anion exchange material into a cell broth and settling the cell (eg, under gravity or by centrifugation).

"Anion exchange material" means herein a particulate weak or strong anion exchange chromatography medium. Anion exchange materials generally comprise a carrier, which may be an organic or inorganic material or a mixture of organic and inorganic materials. Suitable organic materials are agarose based media and methacrylates. Suitable inorganic materials are silica, ceramics and metals. The particles may preferably be 15 to 150 μm in size. More preferably its size is from 15 to 70 μm. The particles may have a density suitable to settle cells relatively quickly from the cell broth, but the density should not be too high because it observes that too dense particles do not settle.

“Moderate incubation time” means herein the time for cell precipitation to produce a clear cell pellet volume and supernatant layer.

"Separation" means herein any method for removing the supernatant from cell pellets, such as by decanting or drawing out the supernatant, or withdrawing pellet from a tube, for example, through a port in the bottom.

A "supernatant layer" is a volume of liquid superimposed as a result of sedimentation. The supernatant layer may still contain (usually will) cells at a cell density significantly lower than the initial cell density.

The initial cell density for the method of the present invention is at least 15 × 10 ⁶ cells / ml, more preferably at least 80 × 10 ⁶ cells / ml. As a practical upper limit, the method according to the invention can be carried out at a cell density of 175 × 10 ⁶ cells / ml or less, more preferably 130 × 10 ⁶ cells / ml or less.

Cell density can be measured using a cell counter such as Vi-CELL ™ (using trypan blue exclusion method), but other suitable methods include cell count, compressed cell volume measurement or Coulter counter (using Electrical Sensing Zone Method). do.

If the initial cell density is greater than 130 × 10 ⁶ cells / ml, it is preferred to first dilute the cell broth. Indeed, it is preferred that cell broth with an initial cell density greater than 100 x 10 ⁶ cells / ml be first diluted. Preferably it can be diluted to a cell density of 80 x 10 ⁶ cells / ml or less. The cell broth may be diluted with a solution that does not significantly change the environment of the cell, ie, an isotonic solution such as PBS, so as not to cause lysis of mammalian cells.

According to a further embodiment, the present invention provides a method of recovering a desired secreted biological material from a cell broth containing cells that produce a desired secreted biological material having a total positive charge in the cell broth, the cell comprising: A method of recovery wherein the cells in the broth are mammalian cells with an initial cell density of at least 15 × 10 ⁶ cells / ml:

a. Contacting the cell broth with the particulate anion exchange material,

b. Allowing sufficient incubation time to form the cell pellet and supernatant layer,

c. Separating the resulting cell pellet from the supernatant layer, and

d. Extracting the desired secreted biological material from the supernatant layer.

"Recovery" means herein to obtain the desired product which is essentially free of by-products and wastes.

The present invention further provides a method of recovering a desired secreted biological material from a cell broth containing cells producing a desired secreted biological material as described above, wherein the cells in said cell broth are 15 × 10 ⁶ cells / ml. Mammalian cells of the above initial cell density, and the resulting cell pellet is further processed to the following steps:

e. Resuspending the resulting cell pellet,

f. Allowing sufficient incubation time to form the cell pellet and supernatant layer,

g. Separating the resulting cell pellet from the supernatant layer and

h. Extracting the desired secreted biological material from the supernatant layer.

In a further method according to the invention, steps e to h of the method are repeated one or more times.

In a further preferred method according to the invention, the resulting cell pellets are added to a solution which does not significantly change the environment of the cells such as to cause lysis of mammalian cells, such as an aqueous solution of salt (preferably isotonic), more preferably PBS. Resuspend.

Preferably the supernatant layer is collected and the desired secreted biological material is extracted from the pooled supernatant.

Examples of mammalian cells include CHO (Chinese hamster ovary) cells, hybridomas, BHK (cub hamster kidney) cells, myeloma cells, human cells, eg HEK-293 cells, human lymphoblasts, E1 immortalized HER cells , Mouse cells, such as NS0 cells. More preferably, El immortalized HER cells, most preferably PER.C6 cells, are used.

In a preferred embodiment, the cells of the methods of the invention are E1-immortalized HER cells, more preferably PER.C6 cells (see US Pat. No. 5,994,128, the contents of which are incorporated herein by reference). PER.C6 cells are exemplified as cells deposited under ECACC No. 96022940 (see, eg, US Pat. No. 5,994,128, EP 0833934 B1, the contents of which are incorporated herein by reference).

Cell broth for clarification can be obtained by any cell culture method suitable for reaching cell densities of mammalian cells of at least 15 × 10 ⁶ cells / ml. Suitable methods for this are described for example in WO2005095578, WO2004099396 and WO2008006494. These contents are incorporated herein by reference.

Biological substances that can be produced by the cell (eg, thus expressing (recombinant) gene coding) according to the invention are for example viral or (recombinant) proteins, in particular receptors, enzymes, fusion proteins, blood aggregation cascades. Blood proteins such as proteins from, for example, multifunctional proteins such as erythropoietin, eg viral or bacterial proteins for vaccines; For example immunoglobulins such as IgG or IgM. Preferably the protein, more preferably the immunoglobulin or part thereof, is produced by the cell. Preferably, biological substances such as proteins or vaccines produced by the cells can be used as active ingredients in pharmaceutical preparations. In the context of the present invention, the terms 'product' and 'biological material' are interchangeable.

Suitable methods for extracting the desired secreted biological material from the supernatant layer include, for example, filtration (eg, deep filtration, microfiltration, ultrafiltration, diafiltration), chromatography (eg, 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 material can be extracted very effectively by cation exchange chromatography. For immunoglobulins as desired biological materials, affinity chromatography, in particular Protein A chromatography and cation exchange chromatography, are particularly suitable separation methods.

Figure 1. Recovery of product after initial settling with Si-PEI and subsequent washing with PBS
(a) Supernatant cell density over time for various anion exchange materials.
(b) Supernatant volume over time for various anion exchange materials.

Example

sign:

X _t = total cell density, cell / ml

Si-PEI = Bakerbond Wide-Pore PEI Prep LC Packing Grafted Silica Beads (JT Baker)

DEAE Hyper D = diethylaminoethyl grafted ceramic beads (Pall)

Streamline DEAE = diethylaminoethyl grafted agarose beads (GE Healthcare)

PrA HPLC = Analytical Protein A High Pressure Liquid Chromatography.

PBS = phosphate buffered saline

The following clarification experiments were performed with PER.C6® cells of various cell densities and prepared according to the procedure shown in WO2008006494. PER.C6® cells produced antibodies. The concentration of antibody in the supernatant layer was measured by PrA HPLC. If necessary, ie if the cell density was too high, the concentration was corrected for biomass and the cells contributed significantly to the working volume.

Example  1. Purification with low cell density.

Different amounts of Si-PEI were added to individual vials containing 10 ml of cell culture. X _t = 4.3 × 10 ⁶ cells / ml. The cells were allowed to settle for 15 minutes. Only 5% (vol) of Si-PEI was needed to settle 97% of cells. 10% (vol) of Si-PEI was added to settle 99% of cells. Product recovery was 100%.

The addition of Si-PEI greatly reduced the time required to settle the cells. In addition, addition of Si-PEI produced more dense pellets compared to the control (no addition of Si-PEI).

Example  2. Purification using medium cell density.

Different amounts of Si-PEI were added to individual vials containing 5 ml of cell culture. X _t = 63.5 × 10 ⁶ cells / ml. The cells were allowed to settle for 30 minutes. 5% (vol) of Si-PEI precipitated 87% of cells. 89% of the cells were allowed to settle by adding 10% (vol) of Si-PEI. 85% of the cells were allowed to settle by adding 20% (vol) of Si-PEI. In each case, the resulting cell density was less than 10 × 10 ⁶ cells / ml, which is a suitable feed for deep filtration. Product recovery was 97%.

The addition of Si-PEI greatly reduced the time required to settle the cells. In addition, addition of Si-PEI produced more dense pellets compared to the control (no addition of Si-PEI).

Example  3. Purification using high cell density

10% (vol) of Si-PEI was added to 345 ml of cell culture broth. X _t = 123 x 10 ⁶ cells / ml. It settled for 2 hours due to the high cell density. After 2 hours, the cell density in the resulting supernatant was 13.6 × 10 ⁶ cells / ml. The pellet volume was 53% of the total volume (93% for the control without addition of Si-PEI). The supernatant was decanted and the pellet washed twice with isotonic PBS and allowed to settle for 1 hour after each wash. Product recovery was 93% after two washes. Total treatment time was 4 hours. After pooling the supernatant, the final treatment volume was 600 ml and the cell density was 9.9 × 10 ⁶ cells / ml.

Example  4. Maximize product recovery by repeated washing

10% (vol) of Si-PEI was added to 345 ml of cell culture broth. X _t = 78 × 10 ⁶ or 120 × 10 ⁶ cells / ml. About 200 mL of the supernatant layer was decanted after initial settling. This volume was replaced with an equal volume of PBS and the cells were allowed to settle for 60 minutes. About 200 mL of the supernatant layer was decanted again and replaced with an equal volume of PBS and allowed to settle for 60 minutes. Then, 200 ml of the supernatant layer was decanted again.

Product recovery through several washing steps is summarized in FIG. 1. The results showed a very significant improvement in product recovery by collecting the product from the supernatant after only two wash steps.

Example  5. Purification using high cell density and various anion exchange materials

10% (vol) of anion exchange material Si-PEI, DEAE Hyper D or Streamline DEAE was added to a sample of 20 ml of cell culture broth and the initial X _t was 123 × 10 ⁶ cells / ml.

The cells were allowed to settle for 2 hours.

FIG. 2A shows the cell density in the supernatant over time for each of the anion exchange material and the cell density of the control without an anion exchange material added.

2B shows the supernatant volume over time. The addition of anion exchange material produced more dense pellets, ie increased supernatant volume.

Example  6. Purification with very high cell density

Purification was attempted with a harvest of X _t = 150 × 10 ⁶ cells / ml. Very little settling was observed at this cell density with undiluted harvest. The cell culture was diluted 1: 1 with PBS to facilitate settling. To this end, 10 ml of medium was diluted to a final volume of 20 ml and 10% (total vol) Si-PEI was added (2 ml). Product recovery after 9 washes was 92.6%. The concentration of HCP was reduced by 28%. Total treatment time was 4 hours. After pooling the supernatant, the final treatment volume was 48 ml (4.8 × increase) and cell density was 6.1 × 10 ⁶ cells / ml.

Example  7. Purification of the desired biological material

The initial cell density of 175 × 10 ⁶ cells / ㎖ the cell culture harvest was diluted to ~ 75 × 10 ⁶ cells / ㎖ in PBS (the initial volume of 1.7 ℓ). After dilution Si-PEI chromatography media was added to the harvest (0.1 L of Si-PEI resin per 1 L of diluted harvest). Cells were allowed to settle for ˜60 minutes. The product containing the supernatant was decanted and the precipitated cells were washed twice with PBS. Initial supernatant was pooled with two washes to maximize product recovery (˜95%). The pooled pools contained less than 5 × 10 ⁶ cells / ml and the HCP content was reduced by 59%.

The product recovered after Si-PEI sedimentation was further purified by deep filtration. Deep filtration removes the primary filter (typically 10 or 5 μm pore size) used for further reduction of cell volume, followed by removal of smaller particles and clarified harvest for aseptic filtration, usually through a gradient 0.8 / 0.2 μm filter. Resulting secondary filter (usually 3 or 1 μm pore size). The deep filtration train may be a Millipore Millistak + HC filter containing a medium such as D0HC (primary) followed by X0HC (secondary) or a CUNO ZetaPlus filter containing a medium such as 10M02 (primary) followed by 60ZA05A (secondary). Can be. In this case, the clarified harvest is further filtered through a 0.8 / 0.2 μm filter (Supor, Pall).

A 85% HCP reduction was also observed through the secondary filter during deep filtration. The reduction of HCP through the secondary filter could contribute to the charge characteristics of this filter, see 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 was further purified by cation exchange chromatography such as GigaCap S (Tosoh). Monoclonal antibodies (products) were immobilized to the resin at a volume of chromatographic media of greater than 95 g / l. The conditions used for antibody immobilization were somewhat acidic (pH-5.3) and the conductivity was ˜4.5 mS / cm. After binding the antibodies were washed with equilibration buffer and finally eluted with a buffer step containing 100 mM sodium chloride. A further reduction in HCP content (78%) was obtained with this step.

Eluted antibodies 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 protein from cell culture harvest through the CEX step is summarized below:

Claims (11)

In the following steps, a method of purifying a cell broth containing desired secreted biological material and mammalian cells having an overall positive charge in the cell broth, wherein the cells are present at an initial cell density of at least 15 × 10 ⁶ cells / ml :
a. Contacting the cell broth with the particulate anion exchange material,
b. Allowing sufficient incubation time to form cell pellet and supernatant layers, and
c. Separating the resulting cell pellet from the supernatant layer. In the following steps, a method of recovering a desired secreted biological material from a cell broth containing cells that produce a desired secreted biological material having a total positive charge in the cell broth, wherein the cells in the cell broth are 15 x 10 ⁶ cells. Methods for Mammalian Cells with Initial Cell Density of at least / mL:
a. Contacting the cell broth with the particulate anion exchange material,
b. Allowing sufficient incubation time to form the cell pellet and supernatant layer,
c. Separating the resulting cell pellet from the supernatant layer, and
d. Extracting the desired secreted biological material from the supernatant layer. The method of claim 2, wherein the resulting cell pellet is further processed in the following steps:
e. Resuspending the resulting cell pellet,
f. Allowing sufficient incubation time to form the cell pellet and supernatant layer,
g. Separating the resulting cell pellet from the supernatant layer and
h. Extracting the desired secreted biological material from the supernatant layer. 4. The method of claim 3, wherein steps e through h are repeated. The method of claim 3 or 4, wherein each supernatant layer is harvested prior to extracting the desired biological material. The method of claim 2, wherein the desired biological material is extracted using cation exchange chromatography. The method according to claim 3 or 4, wherein the resulting cell pellet is resuspended in an aqueous salt solution. The method of claim 6 wherein the aqueous salt solution is PBS. The method of claim 1, wherein the initial cell density is no greater than 130 × 10 ⁶ cells / ml. 9. The method of claim 1, wherein the initial cell density is greater than 100 × 10 ⁶ cells / ml and the cells before step “a” are diluted to 80 × 10 ⁶ cells / ml or less. 10. The method according to any one of claims 1 to 10, wherein the desired biological substance is an immunoglobulin or part thereof.

Images