Jane McLeod

Cell line-specific control of recombinant monoclonal antibody production by CHO cells.

In this study we compare the cellular control of recombinant human IgG4 monoclonal antibody (Mab) synthesis in different CHO cell lines. Based on comprehensive empirical analyses of mRNA and polypeptide synthetic intermediates we constructed cell line‐specific mathematical models of recombinant Mab manufacture in seven GS‐CHO cell lines varying in specific production rate (qMab) over 350‐fold. This comparative analysis revealed that control of qMab involved both genetic construct and cell line‐specific factors. With respect to the former, all cell lines exhibited excess production of light chain (LC) mRNA and polypeptide relative to heavy chain (HC) mediated by more rapid LC transcription and enhanced LC mRNA stability. Downstream of this, cell lines differed markedly in their relative rates of recombinant mRNA translation, Mab assembly and secretion although HC mRNA abundance and the rate of HC translation generally exerted most control over qMab—the latter being directly proportional to qMab. This study shows that (i) cell lines capable of high qMab exceed a threshold functional competency in all synthetic processes, (ii) the majority of cells in parental and transfected cell populations are functionally limited and (iii) cell engineering strategies to increase Mab production should be cell line specific. Biotechnol. Bioeng. 2010;106: 938–951.

Impact of gene vector design on the control of recombinant monoclonal antibody production by chinese hamster ovary cells

In this study, we systematically compare two vector design strategies for recombinant monoclonal antibody (Mab) synthesis by Chinese hamster ovary (CHO) cells; a dual open reading frame (ORF) expression vector utilizing separate cytomegalovirus (CMV) promoters to drive heavy chain (HC) and light chain (LC) expression independently, and a single ORF vector design employing a single CMV promoter to drive HC and LC polypeptide expression joined by a foot and mouth disease virus F2A polypeptide self‐cleaving linker sequence. Initial analysis of stable transfectants showed that transfectants utilizing the single ORF vector designs exhibited significantly reduced Mab production. We employed an empirical modeling strategy to quantitatively describe the cellular constraints on recombinant Mab synthesis in all stable transfectants. In all transfectants, an intracellular molar excess of LC polypeptide over HC polypeptide was observed. For CHO cells transfected with the single ORF vectors, model‐predicted, and empirical intracellular intermediate levels could only be reconciled by inclusion of nascent HC polypeptide degradation. Whilst a local sensitivity analysis showed that qMab of all transfectants was primarily constrained by recombinant mRNA translation rate, our data indicated that all single ORF transfectants exhibited a reduced level of recombinant gene transcription and that Mab folding and assembly reactions generically exerted greater control over qMab. We infer that the productivity of single ORF transfectants is limited by ER processing/degradation “capacity” which sets a limit on transcriptional input. We conclude that gene vector design for oligomeric recombinant proteins should be based on an understanding of protein‐specific synthetic kinetics rather than polypeptide stoichiometry.

An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer.

In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG4 monoclonal antibody production by GS‐CHO cells throughout a state‐of‐the‐art industrial fed‐batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy‐intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell‐specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole‐process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell—where control of productivity is inherently protein product and cell line specific. Biotechnol. Bioeng. 2011;108:2193–2204.

Interdisciplinary working in public health research: a proposed good practice checklist

Background Guidance on how different disciplines from the natural, behavioural and social sciences can collaborate to resolve complex public health problems is lacking. This article presents a checklist to support researchers and principle investigators to develop and implement interdisciplinary collaborations. Methods Fourteen individuals, representing 10 disciplines, participated in in-depth interviews to explore the strengths and challenges of working together on an interdisciplinary project to identify the determinants of substance use and gambling disorders, and to make recommendations for future interdisciplinary teams. Data were analysed thematically and a checklist was derived from insights offered by participants during interview and discussion among the authors on the implications of findings. Results Participants identified 18 scientific, interactional and structural strengths and challenges of interdisciplinary research. These findings were used to develop an 18-item BASICS checklist to support future interdisciplinary collaborations. The five domains of the checklist are: (i) Blueprint, (ii) Attitudes, (iii) Staffing, (iv) Interactions and (v) Core Science. Conclusion Interdisciplinary work has the potential to advance public health science but the numerous challenges should not be underestimated. Use of a checklist, such as BASICS, when planning and managing projects may help future collaborations to avoid some of the common pitfalls of interdisciplinary research.