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

McLeod, Jane; O'Callaghan, Peter M.; Pybus, Leon P.; Wilkinson, Stephen J.; Root, Tracy; Racher, Andrew J.; James, David C.

doi:10.1002/bit.23146

Cited by 20 publications

(17 citation statements)

References 45 publications

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“…The LC polypeptide amounts showed no correlation with the final titer or qP, although the LC mRNA content showed a positive correlation with the final titer ( R = 0.73 on day 3). Overall, these results are consistent with previous reports that the IC HC polypeptide and mRNA contents show a stronger correlation with mAb productivity than the LC content, indicating that HC can be a limiting factor for the final titer . Although the correlation with LC was lower, there was some correlation between LC transcript and the final titer; this is not unexpected as LC polypeptide is necessary to drive HC constant domain 1 (CH1) folding, mAb assembly, and secretion .…”

Section: Resultssupporting

confidence: 91%

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Pekle

Smith²,

Rosignoli³

et al. 2019

Biotechnology Journal

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Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high‐producing clonal cell lines. During cell line development, cell cloning using fluorescence‐activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single‐cell level to identify cell attributes predictive of productivity. IFC assays are developed to quantify the organelle content and recombinant heavy‐chain (HC) and light‐chain (LC) polypeptide as well as messenger RNA (mRNA) amounts in single cells. The assays are then validated against orthogonal standard flow cytometry, western blot, and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) methods. The authors describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single‐cell level, allowing the isolation of such cells during the cloning process. From the analysis, HC polypeptide and mRNA are found to be predictive of productivity early in the culture; however, specific organelle content did not show any correlation with productivity.

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Section: Resultssupporting

confidence: 91%

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Pekle

Smith²,

Rosignoli³

et al. 2019

Biotechnology Journal

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“…Although there was only a small variation in the expression level of IgG genes among the sub-clones, a wide range of specific productivities was observed among them. A weak positive relationship between productivity and the transcript level can be seen in heavy chain, but not in the light chain (evidence also shown in McLeod et al (2011)). The lack of a strong direct correlation between the transgene transcript level and the product titer has also been observed before (Fann et al, 2000;Flickinger et al, 1992;Leno et al, 1992).…”

Section: Gene Sets Changed Duringmentioning

confidence: 79%

Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells

Vishwanathan

Lê

Jacob

et al. 2013

Biotech & Bioengineering

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Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.

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“…The synthetic promoters presented in this study enable precise control of recombinant gene expression in CHO host cells over a broad dynamic range. For easy to express proteins, where transcription rates have been shown to exert a high level of control over production (McLeod et al, ; O'Callaghan et al, ), they could be utilized to maximize recombinant gene transcription levels (e.g., by using synthetic promoter 2/01). For difficult to express proteins (e.g., bispecific antibodies, fusion proteins), where maximizing transcription is unlikely to be beneficial, they could be utilized to provide optimized protein‐specific transcription activity kinetically coordinated with polypeptide‐specific folding and assembly rates.…”

Section: Discussionmentioning

confidence: 99%

Synthetic promoters for CHO cell engineering

Brown

Sweeney

Mainwaring

et al. 2014

Biotech & Bioengineering

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We describe for the first time the creation of a library of 140 synthetic promoters specifically designed to regulate the expression of recombinant genes in CHO cells. Initially, 10 common viral promoter sequences known to be active in CHO cells were analyzed using bioinformatic sequence analysis programs to determine the identity and relative abundance of transcription factor regulatory elements (TFREs; or transcription factor binding sites) they contained. Based on this, 28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter element and secreted alkaline phosphatase (SEAP) reporter gene. After evaluation of the relative activity of TFREs by transient expression in CHO-S cells, we constructed a first generation library of 96 synthetic promoters derived from random ligation of six active TFREs inserted into the same reporter construct backbone. Comparison of the sequence and relative activity of first generation promoters revealed that individual TFRE blocks were either relatively abundant in active promoters (NFκB, E-box), equally distributed across promoters of varying activity (C/EBPα, GC-box) or relatively abundant in low activity promoters (E4F1, CRE). These data were utilized to create a second generation of 44 synthetic promoters based on random ligation of a fixed ratio of 4 TFREs (NFκB 5: E-box 3: C/EBPα 1: GC-box 1). Comparison of the sequence and relative activity of second generation promoters revealed that the most active promoters contained relatively high numbers of both NFκB and E-box TFREs in approximately equal proportion, with a correspondingly low number of GC-box and C/EBPα blocks. The most active second generation promoters achieved approximately twice the activity of a control construct harboring the human cytomegalovirus (CMV) promoter. Lastly, we evaluated the function of a subset of synthetic promoters exhibiting a broad range of activity in different CHO cell host cell lines (CHO-S, CHO-K1, and CHO-DG44) and across extended fed-batch transient expression in CHO-S cells. In general, the different synthetic promoters both maintained their relative activity and the most active promoters consistently and significantly exceeded the activity of the CMV control promoter. For advanced cell engineering strategies our synthetic promoter libraries offer precise control of recombinant transcriptional activity in CHO cells spanning over two orders of magnitude.

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

Cited by 20 publications

References 45 publications

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells

Synthetic promoters for CHO cell engineering

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