DESC:FIELD OF THE INVENTION
The present invention discloses a cell culture process to reduce the amount of host cell protein (HCP) in the production of a glycoprotein.
BACKGROUND OF THE INVENTION
Erythropoiesis stimulating proteins (ESPs), such as erythropoietin and analogs of 5 erythropoietin are glycoprotein hormones that are the principle homeostatic regulators of red blood cell production. Though natural erythropoietin is produced by the kidney, its large scale production for therapeutic purposes is achieved by recombinant DNA methods. Purified recombinant human erythropoietin (rHuEPO) is administered in human patients for the treatment of medical 10 indications associated with inadequate red blood cell supply, e.g., anemia and chronic renal failure (http://pi.amgen.com/united_states/aranesp/ckd/aranesp).
rHuEPO and its analog Darbepoetin alfa, are used for the treatment of anemia and increase red blood cell production associated with various conditions, such as pre-surgery, chronic renal failure, as well in the treatment of side effects associated 15 with HIV, HCV and cancer chemotherapy (http://www.aranesp.com/; Sasaki H, et.al., J. Biol. Chem 1987; 262:12059-76, Rush RS, et.al., Anal. Chem. 1995; 67:1442-52 and Rush RS, et. al., Anal. Chem 1993; 65:1834-42).
Darbepoetin alfa, has five N-linked glycans with up to 22 sialic acids. The in vivo activity of erythropoiesis stimulating proteins has been shown to correlate with the 20 number of N-linked glycans (Elliott et al., Exp Hematol. 2004 32(12):1146-55.). Hence, as compared to rHuEPO with three N-linked carbohydrate chains and a maximum of 14 sialic acids (Egrie JC and Browne JK, Br. J. Cancer, 2001; 84, 3-10), darbepoetin alpha, with additional N-linked glycans, exhibits a three-fold longer serum half-life and increased in vivo activity. 25
However, recombinant therapeutic proteins, including darbepoetin, are expressed in cell culture that are typically associated with impurities such as host cell proteins (HCP), host cell DNA (HCD), endotoxins, variants and aggregates. Particularly,
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HCP can be highly immunogenic and may cause adverse reactions when present at higher levels. Further, HCP in the form of proteases, significantly affect the stability of the protein of interest. Consequently, regulatory agencies, during approval of a recombinant product for therapeutic use require the level of HCP in the product to be in acceptable limit, viz. <100 ppm (Wang X et al., 2009 5 Biotechnology and Bioengineering, Vol. 103, No. 3, 446-458; http://www.bioprocessintl.com/analytical/downstream-validation/host-cellular-protein-quantification-326656/).
Typically, in the manufacturing of a therapeutic product, removal of HCP is dealt with at the “downstream stage” viz., post-harvest of cell culture fluid. WO 10 2007109163A2 teaches inclusion of chaotropic agent(s) in the wash buffer to remove HCP by protein A chromatography. WO 2010151632A1 teaches caprylic acid (octanoic acid) precipitation method to reduce the amount of HCP as a contaminant to the desired glycoprotein.
Other prior-arts describe combination of several chromatographic steps for the 15 removal of HCP. WO2006110277A1 describes a two-step non-affinity chromatography process to remove impurities from the protein of interest, and WO 2013028330A2 teaches a combination of anion-exchange, cation-exchange and hydrophobic interaction chromatography to purify protein of interest from impurities, including HCP. However, it is observed that HCP remain or co-purify 20 even after multiple downstream purification steps.
Thus, the state of the art is largely directed towards optimization of downstream processes for reduction/removal of HCP contents. Moreover, darbepoetin being a complex glycoprotein with several isoforms (Rush RS, et.al., Anal. Chem. 1995; 67:1442-52), the problem of removal of HCP is particularly acute, not least 25 because, the isoelectric point (pI) of darbepoetin, being in the range of pI 3 to pI 5, overlaps with that of HCP, thus rendering their separation involving ion exchange chromatography more difficult.
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The objective of the present invention is to control or reduce the level of HCP at the cell culture/ fermentation stage so as to reduce the amount of bio-burden (HCP) for the ensuing downstream processes. This, in turn, would enhance the efficiency of the downstream processing steps in reducing the HCP level in a therapeutic product. 5
SUMMARY OF THE INVENTION
The present invention discloses a cell culture process to reduce the amount of host cell protein (HCP) in the production of a glycoprotein. The invention results in reduction in the amount of HCP in the harvested cell culture fluid. The method is advantageous in it reduces the HCP content in the harvest, thereby reducing the 10 bio-burden or the HCP load for the ensuing downstream process. In other words, the present invention intends to reduce the HCP content in the harvested cell culture broth, prior to downstream processing.
In addition, the proposed inventive method does not alter any of the critical parameters of the cell culture such as cell viability, titer or quality of the 15 glycoprotein product.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of amount of host cell proteins as discussed in Examples I and II.
Figure 2 is an illustration of viable cell count as described in Examples I and II. 20
Figure 3 is an illustration of viability as described in Examples I and II.
Figure 4 is an illustration of IVCC as described in Examples I and II.
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DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a cell culture process to reduce the amount of host cell protein (HCP) in the harvest of a glycoprotein production.
In an embodiment, the present invention provides a cell culture method of reducing HCP content in a cell culture harvest comprising the steps of; 5
a. culturing the cells expressing the glycoprotein using a culture media comprising wheat hydrolysates in a seed bioreactor,
b. transferring the cells from the seed bioreactor into a production bioreactor,
c. culturing the cells in the production bioreactor using a culture media 10 to express the glycoprotein, wherein, the culture media used is the same as that used in the seed bioreactor, and
d. harvesting the cell culture fluid comprising the said glycoprotein
In a further embodiment, the production cell culture vessel may additionally be supplemented with feed(s). 15
In a further embodiment, the said feed media of the production bioreactor in any of the above-mentioned embodiments may comprise soy or wheat or yeast hydrolysates.
In a further embodiment, the said process results in about 40% reduction in the amount of HCP when compared with the process wherein, the culture media in the 20 seed and production bioreactors are different.
In yet another embodiment, the said cell culture method of reducing HCP content in a glycoprotein harvest may include a temperature shift and/or pH shift etc.
In yet another embodiment, the cell culture characteristics viz., VCC, viability, IVCC of the said process are not altered by the said inventive method. 25
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In another embodiment, the culture media used in the said process is a non-proprietary media comprising DMEM, soy hydrosylates, wheat hydrosylates, amino acid, insulin etc.
The term “glycoprotein” as used herein refers to protein or polypeptide having at least one glycan moiety. 5
As used herein, “IVCC” or “Integral viable cell concentration” refers to cell growth over time or integral of viable cells with respect to culture time that is used for calibration of specific protein production.
The term “Viable cell concentration (VCC)” is defined as number of live cells per unit volume at a given time point. It is usually measured in million cells per ml. 10
The term “viability” is defined as percentage of viable cells versus total cells present in the culture vessel.
The term “bioreactor” or “culture vessel” refers to container(s) used to culture cells.
“Harvest” refers to the cell culture fluid obtained after termination of the cell culture/ fermentation “upstream” process. Harvest comprises the protein of 15 interest and is typically subjected to further “downstream” processing to obtain the protein of interest at high yield and purity.
“Host cell protein” or “HCP” refers to proteins other than the required protein produced by the recombinant cells. They are regarded as impurities.
Certain aspects and embodiments of the invention are more fully defined by 20 reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.
EXAMPLE 1
A glycoprotein, darbepoetin was cloned and expressed in a recombinant CHO cell line as described in U.S. Patent No. 7217689. For generation of seed (inoculum) 25 for the production, cells were grown in PF-CHOTM (Thermo Scientific Hyclone,
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Catalog no. SH30333.3) culture media in seed bioreactor. Recombinant CHO cells from this seed bioreactor (50L) were seeded in the non-proprietary cell culture media, comprising wheat hydrolysates, in the production bioreactor (200L) at a seeding density of 0.2-0.6 million cells/ml, at 37°C and pH 7.05. The cells were supplemented with a feed (CB-2TM; Thermo Scientific Hyclone, Catalogue no SH 5 30596.03) after IVCC of the culture has reached to 1 million cells per ml per day. The culture was further supplemented by feed on 3rd day after first feed and subsequently on third day after second feed. The culture was finally harvested after 240 hours to 288 hours from the day of inoculation or at greater than 50% viability. The values for VCC, viability, IVCC and amount of HCP of the experiment are 10 shown in Figure 1-4. The values of HCP in the product from this culture are shown in Table I.
EXAMPLE 2
A glycoprotein, darbepoetin was cloned and expressed in a recombinant CHO cell 15 line as described in U.S. Patent No. 7217689. For generation of seed (inoculum) for the production, cells were grown in a non-proprietary culture media which comprises of wheat hydrolysate in seed bioreactor (50L). Recombinant CHO cells from this seed bioreactor were seeded in the same cell culture media in the production bioreactor (200L) at a seeding density of 0.2-0.6 million cells/ml at 20 37°C and pH 7.05. The cells were supplemented with a feed (CB-2TM; Thermo Scientific Hyclone, Catalogue no SH 30596.03) after IVCC of the culture has reached to 1 million cells per ml per day. The culture was further supplemented by feed on 3rd day after first feed and subsequently on third day after second feed. The culture was finally harvested after 240 hours to 288 hours from the day of 25 inoculation or at greater than 50% viability. The experiment was run in at least three separate batches and the average values for VCC, viability, IVCC and amount of HCP are shown in Figure 1-4. The average values of HCP in the product from this culture are shown in Table I.
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Table 1: Concentration of HCP in the cell culture harvest
Experiment
HCP (ng/ml)
Example I
43208
Example II
25975 ,CLAIMS:We claim:
1) A cell culture method for reducing HCP content in the cell culture harvest, wherein the method comprises culturing cells in a seed bioreactor in a culture media comprising wheat hydrolysates.
2) A cell culture method for reducing HCP content in the cell culture harvest wherein the method comprises
a) culturing cells in culture media comprising wheat hydrolysates in a seed bioreactor
b) transferring the cells from the seed bioreactor into a production bioreactor
c) culturing the cells in the production bioreactor using a culture media to express the glycoprotein of interest
d) harvesting the cell culture fluid comprising the said glycoprotein.
3) The cell culture method of claim 1 or claim 2 wherein the culture media in seed bioreactor and production bioreactor is substantially similar.
4) The cell culture method of claim 1 or claim 2 wherein the culture media in production bioreactor may further comprise other hydrolysates.
5) The cell culture method of claim 1 or claim 2 wherein reduction in HCP content is at least about 40%.
6) The cell culture method of claim 1 or claim 2 wherein the cell culture method further comprises a temperature shift and/or pH shift.
7) The cell culture method of claim 1 or claim 2 wherein the cell culture method is unaltered wrt VCC, viability and IVCC.
8) The cell culture method of claim 1 or claim 2 wherein the cell are CHO cells.
9) The cell culture method of claim 1 or claim 2 wherein the glycoprotein is a therapeutic glycoprotein.
10) The cell culture method of claim 1 or claim 2 wherein the glycoprotein so produced in Erythropoetin or its variant thereof.