Available online on 15.09.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2023 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                       Research Article

An Improved Cell Culture Process for Production of Omalizumab

Kaumil Bhavsar¹, *Kaushal Joshi², Ghanshyam Patel³, Parth Vaishnav⁴, Om Narayan⁵, Abhishek Sharma⁶, Hetal Katrodiya⁷, Vivek Dave⁸, Chandramauli Rawal⁹

¹ Associate Director- R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

² Senior Director – IPR and R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

³ Scientist II- R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

⁴ Scientist I- R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

⁵ Senior Vice President- R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

⁶ Senior Executive -IPR, Kashiv BioSciences Private Limited, Ahmedabad, India

⁷ Assistant General Manager- R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

⁸ Senior Scientist- R&D, Kashiv BioSciences Private Limited, Ahmedabad, India

⁹ Chief Operating Officer (COO), Kashiv BioSciences, LLC, New Jersey, USA

INTRODUCTION

Mammalian cell cultures such as Chinese hamster ovary (CHO) cells are widely used for the commercial production of therapeutic biomolecules like monoclonal antibodies and proteins¹. Several process parameters like pH, temperature, pO2, CO2, air flow, feeding, agitation rate etc. are essential to maintain cells in a culture environment². Temperature and pH being one of the critical parameters requires optimization for enhanced cell growth, viability, productivity for the generation of therapeutic biomolecules. The optimization of fed-batch culture for the production of monoclonal antibodies with desired quantity with desired quality is a challenging process and usually it depends on particular biotherapeutic molecule as well³. Omalizumab is a recombinant DNA-derived humanized IgGlK monoclonal antibody with a molecular weight of approximately 149 kD that selectively binds to human immunoglobulin (IgE). Omalizumab inhibits the interaction of IgE with high affinity IgE receptor (FceRI)⁴. Acidic species or acidic variants produced during the production process are defined as the antibody variants that elute earlier than the main peak during CEX or later than the main peak during AEX analysis. Sialic acid or deamidation has been commonly reported to contribute to the formation of acidic species. These charge variants substantially affect the in vitro and in vivo properties of antibodies⁵. Galactose a unit for the glycosylation chain reaction by linking it next to a N-acetylglucosamine sugar via galactosyltransferase⁶. Galactosylation is known to affect the process of complement dependent (CDC), as the highest modes of action (MOA) of monoclonal antibodies. Furthermore, the low amount of terminal galactosylation required to increase serum half-life of antibody ^[7,8]. The present study follows the production of omalizumab antibody at temperature 36.5° C ± 0.5 and maintaining pH 6.8± 0.1 during production phase to get enhanced titer and desired characteristics likely reduced galactosylation and/or reduced acidic variants in omalizumab antibody. 

MATERIAL AND METHODS

The monoclonal antibody cell bank vial was thawed in Shake flask 125 (SF125) (corning make) into the seed media supplemented with growth nutrient and cell counts were checked. The incubation was continued for 3-4 days during which cells reach the optimum cell density for subculture. cells were subsequently expanded to Shake flask 250 (SF250) and Shake flask 500 (SF500) to generate sufficient inoculum for 5L glass bioreactors (working volume 3500 mL) and Shake flask 125 (SF125), Shake flask 500 (SF500) and Shake flask 3000 (SF3000) to N-1 50 L (seed bioreactor) to generate sufficient inoculum for 200L bioreactors (working volume 185 L) and 500L bioreactor (working volume 360 L). The production culture starts with production media supplemented with growth nutrient. Additions are made to the bioreactor over the 12-day run including scheduled feeding of Feed 1 and Feed 2, with glucose solution and antifoam added as needed. Production fed-batch bioreactor run was executed at 5L, 200L and 500L bioreactor (Sartorius make) using process parameters as mentioned in Table 1-3. The overall batch cycle was for 12 days and was harvested depending upon the culture viability.

Table 1: Process parameters for 5L bioreactor

Table 2: Process parameters for 200L bioreactor

Table 3: Process parameters for 500L bioreactor

RESULT AND DISCUSSION

The study was performed for the production of omalizumab without modulating the temperature with setpoint 36.5^oC±0.5 and pH 6.8± 0.1 to obtain increased omalizumab titer with reduced percentage of acidic variants and reduced percentage of total galactosylation in omalizumab. 

In 5L bioreactor, the fed-batch process parameters were kept at 36.5^oC temperature and pH set point of 7.1 ± 0.3 day 0-3, 6.8 ± 0.1 day 3 till harvest. Along with it control process with 37.5^oC and pH 6.7 in 5L bioreactor fed-batch run was conducted. Harvest was performed in both the batches by centrifugation of cells at followed by 0.2 um filtration. NPEL generated from day 12 harvests and samples were analyzed for titer by protein A HPLC, %Acidic charge variants by CEX-HPLC method and N-Glycosylation (% Total galactosylation) was checked by HILIC-UPLC method. Following observations were made pertaining to Titer and product quality as below.

1. Titer was increased by 135.6% (Batch 1), 128.2% (Batch 2), 162.8% (Batch 3).
2. Total % Galactosylation was decreased by 16.1 % (Batch 1), 31.8% (Batch 2), 15.7% (Batch 3).
3. %Acidic charge variants were decreased by 29.1% (Batch 1), 30.6% (Batch 2), 28.8% (Batch 3).

2) Process run at 36.5 ^oC and pH 6.8± 0.1 vs Process with 35.5 °C and pH 6.8

The process with 35.5°C temperature and pH set point of 7.1 ± 0.3 day 0-3, 6.8 day 3 – day 12 (harvest) in 5L bioreactor fed-batch run was conducted. Harvest is performed by centrifugation of cells at followed by 0.2 um filtration. NPEL generated from day 12 harvest and samples were analyzed for titer by Protein A HPLC, %Acidic charge variants by CEX-HPLC method and N-Glycosylation (% Total galactosylation) was checked by HILIC-UPLC method. Following observations are made pertaining to Titer and product quality shown below when compared with 36.5 °C and pH 6.8± 0.1.

1. Titer was increased by 64.23.%.
2. Total % Galactosylation was decreased by 2.38 %.

3) 200L bioreactor fed-batch process run at 36.5 ^oC and pH 6.8± 0.1  

The process with 36.5°C temperature and pH set point of 7.1 ± 0.3 day 0-3, 6.8 ± 0.1 day 3-harvest in 200L bioreactor fed-batch run was conducted. Harvest was performed by two-stage depth filtration followed by 0.2 um filtration. NPEL generated from Day 12 harvest and sample ware analyzed for titer by protein A HPLC, %Acidic charge variants by CEX-HPLC method and N-Glycosylation (% Total galactosylation) was checked by HILIC-UPLC method. Following observations were made pertaining to Titer and product quality shown below when compared with control process results at 37.5°C and pH 6.7.

1. Titer was increased by 105.75%.
2. Total Galactosylation was decreased by 41.98 %.
3. %Acidic charge variants were decreased by 11.9%.

4) 500L bioreactor fed-batch process run at 36.5 ^oC and pH 6.8± 0.1  

The process with 36.5°C temperature and pH set point of 7.1 ± 0.3 day 0-2, 7.0 ± 0.3 day 2-3, 6.8 ± 0.1 day 3-harvest in 500L bioreactor fed-batch run was conducted. Harvest was performed by two-stage depth filtration followed by 0.2 um filtration. NPEL generated from Day 12 harvest and sample ware analyzed for Titer by protein A HPLC, %Acidic charge variants by CEX-HPLC method and N-Glycosylation (% Total galactosylation) was checked by HILIC-UPLC method. Following observations were made pertaining to Titer and product quality shown below when compared with control process results at 37.5°C and pH 6.7.

1. Titer was increased by 150.75%.
2. Total % Galactosylation was decreased by 40.59%.
3. %Acidic charge variants were decreased by 6.91 %.

CONCLUSION

The mammalian cell culture process is very complex and significantly affects the quality and quantity of protein. The selection of pH, temperature, duration, media selection, and other parameters make the cell culture complex and scientists need to optimize culture conditions to obtain the desired product. In the present study, we investigated the effect of specific temperature and pH on the quality and quantity of Omalizumab which is produced at a large scale through a fed-batch cell culture process. It is found that a specific temperature of 36.5° C ± 0.5 and pH 6.8± 0.1 maintained during fed-batch cell culture is most suitable for the production of omalizumab as it provides high titer with reduced undesired product such as reduced galactosylation and reduced acidic variant of Omalizumab. Further, this study avoids the requirement of temperature shift during cell culture, and thereby cell culture can be performed for long duration without shifting the temperature.   

Acknowledgments

We are thankful to Kashiv Team for their valuable support and guidance during this work.

Conflict of interests

Authors declare that there is no conflict of interest.

REFERENCES

1. Sharker SM, Rahman A. A Review on the Current Methods of Chinese Hamster Ovary (CHO) Cells Cultivation for the Production of Therapeutic Protein. Curr Drug Discov Technol. 2021; 18(3):354-364. https://doi.org/10.2174/1570163817666200312102137 PMid:32164511

2. https://www.bioreactors.net/bioreactor-operational-requirements (accessed 4 July 2023)

3. Radhakrishnan D et al. Strategies to enhance productivity and modify product quality in therapeutic proteins. Curr. Opin. Chem. Eng. (2018).  https://doi.org/10.1016/j.coche.2018.09.005

4. Busse W, Corren J, Lanier BQ, McAlary M, Fowler-Taylor A, Cioppa GD, van As A, Gupta N. Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma. J Allergy Clin Immunol. (2001) Aug; 108(2):184-90. https://doi.org/10.1067/mai.2001.117880 PMid:11496232

5. Du Y, Walsh A, Ehrick R, Xu W, May K, Liu H. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs. (2012) Sep-Oct; 4(5):578-85. https://doi.org/10.4161/mabs.21328 PMid:22820257 PMCid:PMC3499298

6. McDonald AG, Hayes JM, Bezak T, Głuchowska SA, Cosgrave EF, Struwe WB, Stroop CJ, Kok H, van de Laar T, Rudd PM, Tipton KF, Davey GP. Galactosyltransferase 4 is a major control point for glycan branching in N-linked glycosylation. J Cell Sci. 2014; 127(Pt 23):5014-26. https://doi.org/10.1242/jcs.151878 PMid:25271059 PMCid:PMC4248093

7. Hodoniczky J, Zheng YZ, James DC. Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog. (2005) Nov-Dec; 21(6):1644-52.  https://doi.org/10.1021/bp050228w PMid:16321047

8. Szabo M, Filep C, Nagy M, Sarkozy D, Szigeti M, Sperling E, Csanky E, Guttman A, N-glycosylation structure - function characterization of Omalizumab, an anti-asthma biotherapeutic product. J. Pharm. Biomed. Anal., 209 (2022), Article 114483,  https://doi.org/10.1016/j.jpba.2021.114483 PMid:34864596