(T0930-11-64) Evaluation of CQA Outcomes in Cell Line Changes in Upstream Biomanufacturing

Purpose: Therapeutic antibodies are primarily produced in mammalian cell lines such as murine myeloma (NS0) and Chinese hamster ovary (CHO). The selection of the expression system depends on productivity, product quality, and manufacturer preferences and is often influenced by historical experiences. Significant efforts have been made to genetically engineer host cells to enhance product quality and robustness, with relative glycosylation typically control being central to quality control due to its impact on antibody clearance and bioactivity. Precise glycosylation control is challenging due to clonal variations, media, and culture conditions. Choosing the final production clone is critical, particularly before Phase 3 clinical trials, as changing cell lines later requires demonstrating product comparability and may necessitate additional clinical studies. Ensuring consistent product quality is a challenge during the scale-up from development to commercial manufacturing. Previously, we cultured the NISTCHO cell line under tightly controlled bioreactor conditions to explore the impact of cell-substrate changes. In this study, we examined the critical quality attributes of two non-originator NS0 expression cell lines (NISTNS0-c59 and NISTNS0-c66) producing nNISTmAb-FDA, and one CHO expression cell line (NISTCHO) producing cNISTmAb-FDA. The model mAbs express similar quality attributes to the NIST RM 8671.

Methods: Vials of NISTNS0-c59, NISTNS0-c66, and NISTCHO were thawed and propagated using a protocol adapted by NIST from the Sigma-Aldrich CHOZN® Platform Technical Bulletin. Ambr250 bioreactors were used to cultivate the cells in triplicate with identical process parameters. Fifteen-milliliter culture volumes were sampled on days 4, 7, and 9 for at-line analysis using Spin Trap ProA columns. Purified samples were aliquoted and stored at -80°C for critical quality attribute assessments, including glycan assays, charge variant heterogeneity, CE-SDS, and protein aggregation. The released N-glycan assay was performed using an Agilent IPC glycan kit and analyzed by Waters Xevo Q-TOF. Charge variant and CE-SDS assays were conducted using LabChip GXII. Protein aggregation was analyzed using size exclusion chromatography.

Results: Results showed that although all three cell lines were cultivated under the same process parameters, the glycan distribution of the non-originator NISTmAb differed slightly from the NIST RM 8671, particularly between cNISTmAb-FDA and NIST RM 8671. Preliminary charge variant results indicated that nNISTmAbs had slightly higher acidic peaks than cNISTmAb and NIST RM 8671.

Conclusion: We hypothesize that the glycan differences between cell lines are due to media composition or genetic differences in the cell lines. To further investigate, we have adapted the NISTCHO cell line to hybridoma media to study critical quality attributes under identical chemical and physical bioreactor processes.

References: 1. Li F, Vijayasankaran N, Shen AY, Kiss R, Amanullah A. Cell culture processes for monoclonal antibody production. MAbs. 2010 Sep-Oct;2(5):466-79. doi: 10.4161/mabs.2.5.12720.
2. Yandrofski K, Mouchahoir T, De Leoz ML, Duewer D, Hudgens JW, Anderson KW, Arbogast L, Delaglio F, Brinson RG, Marino JP, Phinney K, Tarlov M, Schiel JE. Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics. Front Mol Biosci. 2022 May 5;9:876780. doi: 10.3389/fmolb.2022.876780.
3. Erica Berilla, Casey Kohnhorst. Exploration of non-originator NISTmAb generated from NS0 clones and cNISTmAb generated from NISTCHO, Poster, 2023 FDA Science Forum.

Acknowledgements: This project was supported in part by an appointment to the Research Fellowship Program at the Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA.
This abstract reflects the views of the author and should not be construed to represent FDA’s views or policies.
Certain commercial equipment, instruments, or materials are identified in this presentation to foster understanding. Such identification does not imply recommendation or endorsement by FDA, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
The authors declare no competing financial interest.