AU2009305343A1 - Clarification process at higher cell density - Google Patents

Description

WO 2010/043700 PCT/EP2009/063565 CLARIFICATION PROCESS AT HIGHER CELL DENSITY The present invention relates to a method for the clarification of a cell broth and to a method for the recovery of secreted desired biological substances from 5 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. 10 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, 15 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 20 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. Disadvantages of the use of many flocculation agents are, amongst others, that they may bind the desired biological substances of interest, that they may 25 inactivate the desired biological substances of interest, that the flocculation process takes too long and/or that the flocculation agent may be hard or expensive to prepare in the high quality needed for medical use. In addition the flocculent needs to be removed from the final purified molecule a process that often requires expensive and time consuming analytical assays to verify its removal. 30 The method according to the present invention does not have any of these disadvantages. Furthermore, these existing methods were shown to establish clarification only at relatively low cell densities. Also, most of these methods were not shown to be successfully applied to mammalian cells, in particular not to mammalian 35 cells which produce secreted desired biological substances.

WO 2010/043700 PCT/EP2009/063565 -2 An objective of the present invention is to provide a cost-effective method for the clarification of a cell broth harvested from a bioreactor and which contains mammalian cells as well as secreted desired biological substances. 5 A further objective of the present invention is to provide a method for the clarification of a cell broth containing mammalian cells at a higher cell density. In a specific embodiment, the present invention relates to a method for the clarification of a cell broth containing mammalian cells and culture medium as well as secreted desired biological substances having an overall positive charge in the cell broth 10 wherein the cells are at an initial cell density of at least 15x1 06 cells/ml by the following steps: a. contacting the cell broth with particulate anion exchange material, b. allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, and 15 c. separating the resulting cell pellet from the supernatant layer. In US patent application 2003/170810 a method is described wherein a crude cell lysate is clarified by mixing the lysate with an anion exchange resin in a batch and then separating the insoluble material (including the resin and anything bound thereto, cells and cell debris) from the soluble material via filtration, 20 centrifugation or gravity separation. Unlike the disclosure in this US patent application, according to the present invention cells are not lysed before they are mixed with anion exchange resin, clarification takes place at particularly high cells density and the desired biological substance in the cell broth has an overall positive charge. It is considered surprising that according to the present invention clarification could be 25 attained with a cell broth at high cell density. 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 30 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. With "secreted biological substances" is meant here biological substances which upon the production thereof by the mammalian cells are 35 predominantly released (actively or passively) into the culture medium.

WO 2010/043700 PCT/EP2009/063565 -3 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 5 charged ionogenic groups on the biological substance under the solvent conditions in the cell broth results in a net positive charge. The overall charge of a biological substance is based on the pKa 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 pl (the pH where the net charge is zero) of the 10 substance must be higher than the pH of the cell broth. With "culture medium" is meant here the extracellular environment of the cells, which contains the nutrients and other constituents supporting the growth and production of cells, but may also contain waste products or host cell proteins (HCP) or material from lysed cells. The composition of the culture medium may vary in time 15 during the course of the culturing of cells and at the stage of clarification may be depleted of one or more of the original constituents. With "contacting" is meant here introduction of anion exchange material to cell broth and settling of cells (e.g. under gravity or with centrifugation). With "anion exchange material" is meant here particulate weak or 20 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 metacrylate. Suitable inorganic materials are silica, ceramics and metals. The particles preferably may have a size of between 15 and 150 pm. More preferably their 25 size is between 15 and 70 pm. 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. 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. 30 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 35 cell density significantly lower than the initial cell density.

WO 2010/043700 PCT/EP2009/063565 -4 The initial cell density for the process of the present invention is at least 15x10 6 cells/ ml, more preferably at least 80x10 6 cells/ml. As a practical upper limit, the process according to the present invention may be carried out with cell densities up to 175x10 6 cells/ml, more preferably up to 130x 106 cells/ml. 5 The cell density can be measured using a cell counter such as Vi-CELL T M (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 130x10 6 cells/ml it is advisable to first 10 dilute the cell broth. In practice it is preferred that a cell broth with an initial cell density above 1 00x1 06 cells/ml be first diluted. Dilution preferably may be done to a cell density of not more than 80x10 6 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. 15 According to a further embodiment the present invention relates to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance having an overall positive charge in the cell broth wherein the cells in the cell broth are mammalian cells at an initial cell density of at least 15x1 06 cells/ml by 20 a. contacting the cell broth with particulate anion exchange material b. allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. extracting the secreted desired biological substances from the supernatant layer. 25 With "recovery" is meant here obtaining the desired product essentially free 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 30 cell broth are mammalian cells at an initial cell density of at least 15x1 06 cells/ml, wherein the resulting cell pellet is further processed by e. re-suspending the resulting cell pellet, f. allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, 35 g. separating the resulting cell pellet from the supernatant layer and WO 2010/043700 PCT/EP2009/063565 -5 h. extracting the secreted desired biological substances from the supernatant layer. In a further method according to the present invention step e. through h. of the above process are repeated one or more times. In a further preferred method according to the present invention the 5 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. 10 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, El immortalized HER cells, mouse cells, for example NSO cells. More preferably, El immortalized HER cells are used, most preferably PER.C6 cells. 15 In a preferred embodiment, the cells in the process of the present invention are El-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 20 reference here). The cell broth for clarification may be obtained by any cell culturing method suitable for attaining a cell density of the mammalian cells of at least 15x10 6 cells/ml. Suitable methods in this respect are described in e.g. W02005095578, W02004099396 and W02008006494. The contents thereof are incorporated herein by 25 reference. Biological substances, which may be produced by the cells according to the present invention (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 30 cascade, multifunctional proteins such as for instance erythropoietin, virus or bacterial proteins for instance for use in vaccines; immunoglobulins such as for example IgG or IgM, and the like. Preferably a protein, more preferably an immunoglobulin or a part thereof 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 WO 2010/043700 PCT/EP2009/063565 -6 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 5 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 10 chromatography. In case of immunoglobulins as the desired biological substances affinity chromatography, in particular protein A chromatography, and cation exchange chromatography are especially suitable separation methods. Short description of the figures 15 Figure 1. Recovery of product after initial settling with Si-PEI and subsequent washing steps with PBS (a) Supernatant cell density as a function of time for various anion exchange materials. (b) Supernatant volume as a function of time for various anion 20 exchange materials. EXAMPLES Symbols: 25 Xi = 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) Streamline DEAE = diethylaminoethyl grafted agarose beads (GE Healthcare) 30 PrA HPLC = Analytical Protein A High Pressure Liquid Chromatography. PBS = Phosphate Buffered Saline The following clarification experiments were undertaken with PER.C6* cells at various cell densities and prepared according to the procedure outlined in W02008006494. The PER.C6* cells produced an antibody. The 35 concentration of antibody in the supernatant layers was measured by PrA HPLC. The WO 2010/043700 PCT/EP2009/063565 -7 concentrations were corrected for biomass when necessary, i.e. when the cell density is extremely high, the cells contribute significantly to the working volume. Example 1. Clarification with low cell density. 5 Different amounts of Si-PEI were added to individual vials containing 10 ml of cell culture. Xi = 4.3x1 06 cells/ml. The cells were allowed to settle for 15 minutes. Only 5% (vol) of Si-PEI was needed to settle 97% of the cells. Adding 10% (vol) of Si-PEI settled 99% of the cells. Product recovery was 100%. The addition of Si-PEI greatly reduced the time needed for the cells to 10 settle. Also, the addition of Si-PEI resulted in a more compact pellet in comparison with the control (no Si-PEI added). Example 2 Clarification with intermediate cell density. Different amounts of Si-PEI were added to individual vials containing 15 5 ml of cell culture. Xt = 63.5x10 6 cells/ml. The cells were allowed to settle for 30 minutes. Adding 5% (vol) of Si-PEI settled 87% of the cells. Adding 10% (vol) of Si-PEI settled 89% of the cells. Adding 20% (vol) of Si-PEl settled 85% of the cells. In each case the resulting cell density was below 1 Ox1 06 cells/ml which is a suitable feed for depth filtration. Product recovery was 97%. 20 The addition of Si-PEI greatly reduced the time needed for the cells to settle. Also, the addition of Si-PEI resulted in a more compact pellet in comparison with the control (no Si-PEI added). Example 3 Clarification with hiqh cell density. 25 10% (vol) of Si-PEI was added to 345 ml of cell culture broth. Xi = 123x10 6 cells/ml. Due to the high cell density two hours of settling were allowed. After these two hours the cell density in the resulting supernatant was 13.6x10 6 cells/ml. The pellet volume was 53% of the total volume (93% for the control where no Si-PEI was added). The supernatant was decanted and the pellet was 30 washed twice with isotonic PBS with 1 hour of settling after each wash. Product recovery was 93% after the two washes. The total process time was 4 hours. After pooling the supernatants, the final process volume was 600 ml and the cell density was 9.9x10 6 cells/ml.

WO 2010/043700 PCT/EP2009/063565 -8 Example 4 Maximizinq recovery of product by repeated washings 10% (vol) of Si-PEI was added to 345 ml of cell culture broth. Xt = 78x10 6 or 120x10 6 cells/ml. About 200ml of the supernatant layer were decanted after initial settling. This volume was replaced by an equal volume of PBS, and the cells 5 were allowed to settle for 60 minutes. Again about 200 ml of the supernatant layer was decanted and replaced by an equal volume of PBS followed by settling for 60 minutes. And again 200 ml of the supernatant layer was decanted. The product recovery through the several washing steps is summarized in Figure 1. The results show a very considerable improvement of the 10 product recovery by collecting the product from the supernatants after only two washing steps. Example 5 Clarification with hiqh cell density and with various anion exchange materials 15 10 % (vol) of the anion exchange materials Si-PEI, DEAE Hyper D or Streamline DEAE were added to samples of 20 ml of cell culture broth with initial Xt of 123x10 6 cells/ml. The cells were allowed to settle for 2 hours. Figure 2a shows the cell densities in the supernatants as a function of 20 time for each of the anion exchange materials as well as the control where no anion exchange material was added. Figure 2b shows the supernatant volume as a function of time. The addition of anion exchange material resulted in a more compact pellet, i.e. increased supernatant volume. 25 Example 6 Clarification with extremely hiqh cell density. Clarification was attempted with a harvest at Xt = 150x10 6 cells/ml. Very little settling was observed at this cell density with undiluted harvest. Diluting the cell culture 1:1 with PBS facilitated settling. To this end, 10 ml of media was diluted to a 30 final volume of 20 ml and 10% (total vol) Si-PEI was added (2 ml). Product recovery was 92.6% after the two washes. The concentration of HCPs was reduced by 28%. The total process time was 4 hours. After pooling the supernatants, the final process volume was 48 ml (4.8 x increase) and the cell density was 6.1x10 6 cells/ml. 35 WO 2010/043700 PCT/EP2009/063565 -9 Example 7 Purification of desired biological substance An cell culture harvest with initial cell density of 175x10 6 cells/mL was diluted to - 75 x106 cells/mL with PBS(Initial volume of 1.7 L). Following dilution Si-PEI chromatography media were added to the harvest (0.1 L of Si-PEI resin per L of 5 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 x10 6 cells/ml, and the HCP content is reduced by 59%. 10 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 pim pore size) used for further reduction of the cell mass, followed by a secondary filter (typically 3 or 1 tm pore size) that removes smaller particles and prepares the clarified harvest for sterile filtration typically through a gradient 0.8/0.2 tm filter. The depth filtration train 15 can be Millipore Millistak+HC filters containing media such as DOHC (primary) followed by X0HC (secondary) or CUNO ZetaPlus filters containing such as 10M02 (primary) followed by 60ZA05A (secondary). In either case the clarified harvest is further filtered through 0.8/0.2 D m filters (Supor, Pall). In addition an 85% HCP reduction was observed through the 20 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). 25 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 30 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.

WO 2010/043700 PCT/EP2009/063565 - 10 The overall reduction in Host Cell Proteins from the cell culture harvest through the CEX step is summarized below: HCP (pg/mg MAb) % HCP Clearance Cell Culture Harvest 200 0 Post-PEI Cell Settling 81 59 Post Depth Filtration 12 85 Post CEX Capture 2.8 78 Overall 99

Claims (11)

1. A method for the clarification of a cell broth containing mammalian cells as well as secreted desired biological substances having an overall positive 5 charge in the cell broth wherein the cells are at an initial cell density of at least 15x10 6 cell/ml by the following steps: a. contacting the cell broth with particulate anion exchange material, b. allowing an adequate incubation time to result in the formation of a cell pellet and a supernatant layer, and 10 c. separating the resulting cell pellet from the supernatant layer.

2. A method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance having an overall positive charge in the cell broth wherein the cells in the cell broth are mammalian cells at an initial cell density of at least 15x10 6 cell/ml by 15 a. contacting the cell broth with particulate anion exchange material, b. allowing an adequate incubation time to result in the formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. extracting the secreted desired biological substances from the 20 supernatant layer.

3. Method according to claim 2, wherein the resulting cell pellet is further processed by e. re-suspending the resulting cell pellet, f. allowing an adequate incubation time to result into the formation of a cell 25 pellet and a supernatant layer, g. separating the then resulting cell pellet from the supernatant layer, and h. extracting the secreted desired biological substances from the supernatant layer.

4. Method according to claim 3, wherein step e through h are repeated. 30

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

6. Method according to any of the claims 2 to 5 wherein the desired biological substance is extracted using cation exchange chromatography.

7. Method according to claim 3 or 4, wherein the resulting cell pellet is 35 resuspended in an aqueous salt solution. WO 2010/043700 PCT/EP2009/063565 - 12

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

9. Method according to any of the claims 1 to 8 wherein the initial cell density is less than 130x10 6 cells/ml.

10. Method according to any of the claims 1 to 8 wherein the initial cell density is 5 more than 100x10 6 cells/ml and wherein the cells prior to step "a" are diluted to not more than 80x10 6 cells/ml.

11. Method according to any of the claims above, wherein the desired biological substances are immunoglobulins or parts thereof