Generation of reference CHO cell lines and insights into media additive use for influencing mAb production and quality
Date
2023
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Chinese hamster ovary (CHO) cell lines with favorable attributes are used to commercially biomanufacture hundreds of therapeutic proteins and antibodies. Companies heavily invest in private Research and Development sectors, enabling the latest innovations of cell line development and custom media/feed blends to ensure reliable and highly productive cell platforms for mAb production. In comparison, academic researchers often face limitations in acquiring high producing cell lines, as they lack the resources and proprietary knowledge to generate these types of cells. This disparity is a challenge to the comparability between academic and industrial discoveries, as drastic variability can exist between low and high producers. As industrial CHO cell lines reach better performance characteristics, this uncertainty in the comparability of studies conducted using low producing cell lines continues to grow. To circumvent this issue, it is imperative that academic researchers have access to a high producing reference CHO cell line which will expedite and broaden understanding of a specific cell line through a communal approach of research while increasing the translation of academic break throughs to industry. ☐ To address these issues, we establish two monoclonal antibodies (mAbs) expressing, CHO “reference cell lines” from different lineages as part of a university- industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) funded project. During the development of these reference cell lines, we characterize key growth and mAb production attributes while confirming key performance outcomes and technology transferability across two academic laboratories. As part of these studies, we generate fed-batch cultivation data from shake flask and scaled-down bioreactor processes with data presented confirming titers over 2 g/L in commercial medias. Furthermore, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. ☐ Next, we describe a developed cell line development (CLD) platform approach for creating clones with varying productivities using the reference cell line. We then describe a method for purifying the mAb using protein A chromatography, followed by a reliable, consistent glycosylation analysis using mass spectrometry. The proposed workflow can be applied for a robust CLD process optimization to generate robust clones, enhance product expression, and improve product quality attributes. Lastly, using our developed reference cell line clone platform, we evaluate the potential titer enhancing and glycosylation impact of the histone deacetylase inhibitor, sodium butyrate, as a productivity enhancer in relationship to mAb production levels. ☐ Finally, we performed an in-depth factorial design study using the reference cell line to evaluate the influence of glycan modifying media additives such as 2-F- peracetyl fucose (2FP), galactose, and N-acetylmannosamine (manNAc) supplemented at various stages of cell growth. The resulting growth, production, and glycan distribution data help improve our understanding of media supplementation as a strategic tool for achieving desirable quality glycan profiles while also enabling faster process development times. ☐ Collectively, this work provides a universal, industrially relevant CHO culture platform, consisting of two “reference cell lines” to accelerate biomanufacturing innovation amongst the academic community. Furthermore, this work serves as a foundational study on methods and strategies for improving mAb production and influencing glycan profiles for the academic community to build upon for these universal reference cell lines.
Description
Keywords
Chinese hamster ovary, Glycosylation, Sodium butyrate, Cell lines, Monoclonal antibodies