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Evaluation of Cellular Mechanisms Involved in Recombinant Antibody Expression in Transiently Transfected Chinese Hamster Ovary Cells

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Evaluation of Cellular Mechanisms Involved in Recombinant Antibody Expression in Transiently Transfected Chinese Hamster Ovary Cells Evaluation of Cellular Mechanisms Involved in Recombinant Antibody Expression in Transiently Transfected Chinese Hamster Ovary Cells The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Masterjohn, Elizabeth. 2016. Evaluation of Cellular Mechanisms Involved in Recombinant Antibody Expression in Transiently Transfected Chinese Hamster Ovary Cells. Master's thesis, Harvard Extension School. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33797315 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Evaluation of Cellular Mechanisms Involved in Recombinant Antibody Expression in Transiently Transfected Chinese Hamster Ovary Cells Elizabeth Masterjohn A Thesis in the Field of Biotechnology for the Degree of Master of Liberal Arts in Extension Studies Harvard University May 2016 Abstract Transient recombinant antibody production in Chinese hamster ovary (CHO) cells is often used to screen large panels of candidates for potential therapeutic use. However, a significant proportion of antibodies express at a level too low for adequate in vitro characterization. This study compares the pathways, including the unfolded protein response (UPR), involved in the transient expression of both low and high expressing antibody clones. Twenty historically low expressing antibodies from two different hybridoma campaigns were re-evaluated for antibody productivity using transient co- transfection of heavy and light chain plasmids in CHO 3E7 cells. Five antibodies had significantly improved antibody production while one antibody had moderately improved antibody production when expressed in CHO 3E7 cells compared to their respective historical data. Seven individual antibody chains were affected at the transcriptional level with minimal or no detectable levels of mRNA. These seven chains were used in different combinations for ten of the antibodies screened. For those ten antibodies, the lack of detectable mRNA, determined by Northern blot analysis, correlated with low levels of intracellular and secreted antibody. The low levels of mRNA could potentially be caused by enhanced degradation due to the physical characteristics of these sequences (Cooper, G. M., 2000). One variant had low levels of light chain mRNA but had secreted titers of antibody similar to its corresponding control and was the only antibody that possessed these features. The remaining four variants had adequate levels of mRNA and intracellular antibody, determined by Western blot analysis, but minimal amounts of secreted antibody, potentially implicating UPR induction. Antibodies 5L+19H (low expression) and 5L+6H (high expression) along with mock and untransfected cells were analyzed for UPR gene regulation using the Affimetrix CHO Gene array. Several observations were made. First, the transfection process alone had the most impact on differential gene expression affecting 1146 genes out of 29,700 genes assayed. Second, the UPR related gene CHOP was noted to be upregulated for both antibody transfections compared to mock, and HERPUD1 was shown to be upregulated for the low expressing antibody compared to mock. This suggests UPR induction had begun for both experimental transfections, however, the specific pathway or pathways that have been activated cannot be determined with the limited number of genes that were found to be upregulated. Additionally, when the two experimental transfections were compared to each other we found no significant difference in differential gene expression of UPR related genes. This implies that 72 hours post-transfection, the level of UPR induction was similar for an antibody that has low levels of secretion and one that has high levels of secretion. Finally, four key UPR associated genes were found to be downregulated in mock transfected cells compared to untransfected cells, (CHOP, GADD34, ERDJ4 and XBP1). Acknowledgements William Brondyk, PhD: Thesis Director, Sanofi Genzyme Dr. Brondyk is the Vice President of Research Biologics at Sanofi Genzyme and provided guidance and support for this project and critical review of the manuscript. Stephen Denkin, PhD: Research Advisor, Harvard University Dr. Denkin is the director of the Biotechnology ALM program, and provided expertise in meeting all program requirements. Kimberly Bishop Kimberly is an Associate Scientific Director in Research Biologics at Sanofi Genzyme who provided critical content and editorial review of the manuscript. Timothy Connors Timothy is a Senior Scientist in Research Biologics at Sanofi Genzyme who provided archived plasmids and sequence information. Christine DeMaria & Jin Zhang Christine is the Associate Scientific Director of the Therapeutic Protein Expression group and Jin is a Staff Scientist I in the same group. Christine and Jin provided protocols, guidance and expertise for Northern blot analysis. v Yuriy Alekseyev, Ph.D. Dr. Alekseyev is a Research Associate Professor of Pathology and Laboratory Medicine and is the Director of the Microarray and Sequencing Resource Center at Boston University and ran the CHO Array as well as provided support and guidance for array analysis. Todd Kitterman Todd is a Senior Principal Systems Engineer at Raytheon who provided critical editorial review of the manuscript. The 3E7 CHO/EBNA cells and pTT5 expression vector were licensed from the National Research Council Canada (NRC). The CHO Gene micro-array was performed at the Boston University Microarray facility. All remaining research was performed at Sanofi Genzyme, 49 New York Avenue, Framingham, MA. vi Table of Contents Acknowledgements………………………………………………………………….…..v List of Tables……………………………………………………………………….…....x List of Figures…………...…………………………………………………….…….…..xi I. Introduction………………………………………………………………....…….…..1 Classical antibody generation overview…………………………………......…..1 Limitations of cellular expression………………………………...……….…….3 UPR regulation………………………………………………………..…...…….3 Predictions of limitations in a transient system…………………………….....…4 Limitations of cellular expression systems in a transient environment………….4 Implications of understanding limitations in a transient system………...………5 II. Materials and Methods…………………………………………………………...…..7 Expression vector…………………………………………………………......…7 Cell line and cell culture………………………………………………….......….8 Transient expression……………………………………………....…..…8 Quantitation of secreted antibody…………………………………………….…9 vii Western blot analysis………………………………………………………...…10 Northern blot analysis…………………………………………………………..10 RNA isolation……………………………………………………..…….11 Gel electrophoresis…………………………………………………...…11 RNA transfer……………………………………………………....……12 DNA probe generation………………………………………………….12 Hybridization………………………………………………………..….13 Cricetulus griseus (Chinese hamster) gene array……………………………....14 Microarray experimental design………………………………………..14 Microarray………………………………………………………………15 Normalization and quality assessment………………………………….16 Student’s t test………………………………………………………….16 Microarray analysis………………………………………………....…..17 III. Results…………………………………………………………………………...…18 Determination of target antibody panel………………………………………...18 Inhibition at the RNA level………………………………………………….….23 Intracellular antibody expression…………………………………………….…25 Investigation of UPR induction………………………………………..……….29 viii IV. Discussion…………………………………………….…………………….…….…35 Experimental review……………………..………………………………..….…35 Limitations in transcription…………………………………………………..….37 Intracellular protein synthesis……………………………………………….…..39 UPR regulation in a transient system…………………………………….…..….40 V. Conclusions……………………………………………………………………….….45 Appendix 1…………………..……………………………………………………….….47 Northern blots…………………………………………………………….……..47 Appendix 2…………………………………………………………………………...….48 Western blots……………………………………………………………………48 Appendix 3………………………………………………………………………..…….49 Microarray Statistical Analysis……………………………………………..…..49 Bibliography……………………………………………………………………….……53 ix List of Tables Table 1. Mock transfection versus untransfected cells………………………………....49 Table 2. Low expression (5L+19H) versus mock transfected cells………………….....50 Table 3. High expression (5L+6H) versus mock transfected cells…………………..…51 Table 4. Low expression (5L+19H) versus high expression (5L+6H)………………....52 x List of Figures Figure 1. Antibody campaign flow chart……………………………………………….19 Figure 2. Transient antibody expression titers……………………………………….....20 Figure 3. Organizational chart of antibody expression titers………………………..….22 Figure 4. Northern blot and relative quantitated mRNA………………………...……..47 Figure 5. Western blot of intracellular protein………………………………………….48 Figure 6. Western blot of conditioned media………………………………………..….31 Figure 7. Volcano plots of differential gene regulation………………………………....33 Figure 8. Fold change of UPR specific genes in mock versus untransfected cells…......36 Figure 9. Fold change of differentially expressed genes……………………….…….…37 Figure 10. UPR Pathway of differentially expressed genes……………...………….….45 xi Chapter I Introduction The development of humanized monoclonal antibodies for therapeutic use has notably improved over the last few decades, however significant hurdles still remain. Many humanized antibodies are initially evaluated in transient mammalian expression systems which sometimes fail to produce these recombinant
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