Abstract
In this case-study, we demonstrate an approach for identifying correlations between nutrients/metabolites in the spent medium of CHO cell cultures and cell growth, mAb titre and critical quality attributes, using multivariate analyses, which can aid in selection of targets for medium and feed optimization. An extensive LC–MS-based method was used to analyse the spent medium composition. Partial least squares (PLS) model was used to identify correlations between nutrient composition and cell growth and mAb titre and orthogonal projections to latent structures (OPLS) model was used to determine the effect of the changing nutrient composition during the culture on critical quality attributes. The PLS model revealed that the initial concentrations of several amino acids as well as pyruvic acid and pyridoxine, governed the early cell growth, while the concentrations of TCA cycle intermediates and several vitamins highly influenced the stationary phase, in which mAb production was maximum. For the first time, with the help of the OPLS model, we were able to draw correlations between nutrients/metabolites during the culture and critical quality attributes, for example, optimizing the supply of certain amino acids and vitamins could reduce impurities while simultaneously increasing desirable glycoforms. The unique correlations obtained from such an exploratory analysis, utilizing conditions that are commonly adopted in early process development, present opportunities for optimizing the compositions of the growth media and the feed media for enhancing cell growth, mAb production and quality, thereby proving to be a useful preliminary step in bioprocess optimization.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahn WS, Antoniewicz MR (2012) Towards dynamic metabolic flux analysis in CHO cell cultures. Biotechnol J 7:61–74. https://doi.org/10.1002/BIOT.201100052
Altamirano C, Paredes C, Cairó JJ, Gòdia F (2000) Improvement of CHO cell culture medium formulation: Simultaneous substitution of glucose and glutamine. Biotechnol Prog 16:69–75. https://doi.org/10.1021/BP990124J
Behjousiar A, Kontoravdi C, Polizzi KM (2012) In situ monitoring of intracellular glucose and glutamine in CHO cell culture. PLoS ONE 7:e34512. https://doi.org/10.1371/JOURNAL.PONE.0034512
Esteve JM, Mompo J, Asuncion JG et al (1999) Oxidative damage to mitochondrial DNA and glutathione oxidation in apoptosis: studies in vivo and in vitro. FASEB J 13:1055–1064. https://doi.org/10.1096/fasebj.13.9.1055
Fan Y, Ley D, Andersen MR (2018) Fed-batch CHO cell culture for lab-scale antibody production. Methods Mol Biol 1674:147–161. https://doi.org/10.1007/978-1-4939-7312-5_12/COVER
Genzel Y, Ritter JB, König S et al (2005) Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells. Biotechnol Prog 21:58–69. https://doi.org/10.1021/BP049827D
Gramer MJ, Eckblad JJ, Donahue R et al (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108:1591–1602. https://doi.org/10.1002/bit.23075
Hayter PM, Curling EMA, Baines AJ et al (1992) Glucose-limited chemostat culture of chinese hamster ovary cells producing recombinant human interferon-γ. Biotechnol Bioeng 39:327–335. https://doi.org/10.1002/bit.260390311
ICH (1999) Q6B: specifications: test procedures and acceptance criteria for biotechnological/biological products step 5 note for guidance on specifications: test procedures and acceptance criteria for biotechnological/biological products
Jing Y, Borys M, Nayak S et al (2012) Identification of cell culture conditions to control protein aggregation of IgG fusion proteins expressed in Chinese hamster ovary cells. Process Biochem 47:69–75. https://doi.org/10.1016/j.procbio.2011.10.009
Kaur R, Borgayari D, Rathore AS (2017) Impact of media components on CQAs of monoclonal antibodies. Biopharm Int 30:40–46. https://doi.org/10.1038/SREP45216
Kishishita S, Katayama S, Kodaira K et al (2015) Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells. J Biosci Bioeng 120:78–84. https://doi.org/10.1016/j.jbiosc.2014.11.022
Ma N, Ellet JA, Okediadi C et al (2009) A single nutrient feed supports both chemically defined NS0 and CHO fed-batch processes: improved productivity and lactate metabolism. Biotechnol Prog 25:1353–1363. https://doi.org/10.1002/btpr.238
Rathore A (2007) Applications of multivariate data analysis in biotech processing. Biopharm Int 20:40–45
Rathore AS, Kumar Singh S, Pathak M et al (2015) Fermentanomics: relating quality attributes of a monoclonal antibody to cell culture process variables and raw materials using multivariate data analysis. Biotechnol Prog 31:1586–1599. https://doi.org/10.1002/BTPR.2155
Ritacco FV, Wu Y, Khetan A (2018) Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: history, key components, and optimization strategies. Biotechnol Prog 34:1407–1426. https://doi.org/10.1002/btpr.2706
Sellick CA, Croxford AS, Maqsood AR et al (2011) Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production. Biotechnol Bioeng 108:3025–3031. https://doi.org/10.1002/bit.23269
Sellick CA, Croxford AS, Maqsood AR et al (2015) Metabolite profiling of CHO cells: molecular reflections of bioprocessing effectiveness. Biotechnol J 10:1434–1445. https://doi.org/10.1002/biot.201400664
Strober W (2015) Trypan blue exclusion test of cell viability. Curr Protoc Immunol 111:A3.B.1-A3.B.3. https://doi.org/10.1002/0471142735.ima03bs111
Templeton N, Dean J, Reddy P, Young JD (2013) Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed-batch CHO cell culture. Biotechnol Bioeng 110:2013–2024. https://doi.org/10.1002/bit.24858
Tsang VL, Wang AX, Yusuf-Makagiansar H, Ryll T (2014) Development of a scale down cell culture model using multivariate analysis as a qualification tool. Biotechnol Prog 30:152–160. https://doi.org/10.1002/BTPR.1819
Wong DCF, Wong KTK, Goh LT et al (2005) Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89:164–177. https://doi.org/10.1002/bit.20317
Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M et al (2004) Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng 87:614–622. https://doi.org/10.1002/bit.20151
Zhang H, Lennox B (2004) Integrated condition monitoring and control of fed-batch fermentation processes. J Process Control 14:41–50. https://doi.org/10.1016/S0959-1524(03)00044-1
Zhang P, Woen S, Wang T et al (2016) Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today 21:740–765. https://doi.org/10.1016/J.DRUDIS.2016.01.006
Zhang X, Jiang R, Lin H, Xu S (2020) Feeding tricarboxylic acid cycle intermediates improves lactate consumption and antibody production in Chinese hamster ovary cell cultures. Biotechnol Prog 36:e2975. https://doi.org/10.1002/BTPR.2975
Zürcher P, Sokolov M, Brühlmann D et al (2020) Cell culture process metabolomics together with multivariate data analysis tools opens new routes for bioprocess development and glycosylation prediction. Biotechnol Prog 36:e3012. https://doi.org/10.1002/BTPR.3012
Acknowledgements
The authors are grateful to Intas Biopharmaceuticals, Ahmedabad, India, for graciously providing the CHO clone used in this study and to Shimadzu Analytical (India) Private Limited, Mumbai, India, for providing access to the Triple Quadrupole LCMS facility. The authors also thank Mr. Vinay Babu Prathap from Sartorius Stedim India Private Limited, for his inputs towards the multivariate analysis. The authors express their gratitude to the Prime Minister’s Fellowship Scheme, managed by the Confederation of Indian Industry and Science and Engineering Research Board, Govt. of India, and Himedia Laboratories Pvt. Ltd., India, for providing fellowship to M.S. The authors are thankful to Rashtriya Uchchatar Shiksha Abhiyan (RUSA) of the Ministry of Human Resource Development, Govt. of India, and the Department of Science and Technology, Govt. of India, for funding of instruments critical to the study (Sanction Order No. RUSA/Order/R&I/2016-17/274, Dt. 18/06/2016).
Author information
Authors and Affiliations
Contributions
Conceptualization and methodology: MS; formal analysis: MS, AS, VP; writing—original draft preparation: MS; writing—review and editing: VGW, PD, RJ; resources: PD, RJ; supervision: RJ.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
Research involving human participants and/or animals
No study involving human or animal subjects were conducted.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Saldanha, M., Shelar, A., Patil, V. et al. A case study: Correlation of the nutrient composition in Chinese Hamster Ovary cultures with cell growth, antibody titre and quality attributes using multivariate analyses for guiding medium and feed optimization in early upstream process development. Cytotechnology 75, 77–91 (2023). https://doi.org/10.1007/s10616-022-00561-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-022-00561-z