Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level
Abstract: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)… Show more
“…For the Fab C samples (Figure S8a,b), there was no correlation between the copies of gDNA and copies of LC or HC mRNA copies (p = 0.5457 and 0.2277, respectively). The lack of correlation between gDNA and mRNA copy numbers again suggests that random integration is a major Bottlenecks at the mRNA level have previously been reported for other molecules (Godfrey et al, 2017;Jiang et al, 2006;Lee et al, 2009;Mason et al, 2012;Mead et al, 2009;Pekle et al, 2019). For IgG1, posttranscriptional processes must limit product yields as reported in the literature for other recombinant targets (Chusainow et al, 2009;Godfrey et al, 2017;Hussain et al, 2017;Ley et al, 2015;Mason et al, 2012;Mead et al, 2009Mead et al, , 2015Mohan et al, 2008;O'callaghan et al, 2010;Reisinger et al, 2008).…”
Section: Assessment Of Secreted and Intracellular Igg And Fab Proteinmentioning
Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small-and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.
“…For the Fab C samples (Figure S8a,b), there was no correlation between the copies of gDNA and copies of LC or HC mRNA copies (p = 0.5457 and 0.2277, respectively). The lack of correlation between gDNA and mRNA copy numbers again suggests that random integration is a major Bottlenecks at the mRNA level have previously been reported for other molecules (Godfrey et al, 2017;Jiang et al, 2006;Lee et al, 2009;Mason et al, 2012;Mead et al, 2009;Pekle et al, 2019). For IgG1, posttranscriptional processes must limit product yields as reported in the literature for other recombinant targets (Chusainow et al, 2009;Godfrey et al, 2017;Hussain et al, 2017;Ley et al, 2015;Mason et al, 2012;Mead et al, 2009Mead et al, , 2015Mohan et al, 2008;O'callaghan et al, 2010;Reisinger et al, 2008).…”
Section: Assessment Of Secreted and Intracellular Igg And Fab Proteinmentioning
Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small-and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.
“…Generally, these methods eliminate in situ spatial information during sample preparation and capture "bulk" information of all cells within a given sample. Therefore, achieving single-cell information using the following methods is still challenging, particularly in proteomics due to the inability to amplify proteins [179][180][181][182][183][184][185] . Details on the type of MS and RNA-seq approaches that could be coupled with the methods in this section are reviewed in [186][187][188] .…”
Section: Sequencing-based Methods In Spatial Transcriptomics and Proteomicsmentioning
confidence: 99%
“…Another major consideration is that the approach requires cells to be in suspension, and dissociation of adherent cells or tissues may cause aberrant localization of molecules. Whilst performed less frequently than protein analysis, RNA transcripts can be visualized using IFC ( 162 , 163 ). Fig.…”
Section: Imaging the Spatial Transcriptome And Proteomementioning
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“…Chinese hamster ovary (CHO) cells are the expression system most widely used to produce complex human-like recombinant proteins such as monoclonal antibodies (mAbs) ( Walsh, 2018 ). As such, a kaleidoscope of processes and technologies required for the generation and purification of recombinant material from CHO cells has been explored to improve productivity, ensure consistent product quality and reduce process timelines ( Vito et al, 2020 ; Povey et al, 2014 ; Pekle et al, 2019 ; Shukla et al, 2018 ; Budge et al, 2021 ). Yet there remains a need and desire to further improve such expression systems, particularly for so termed difficult to express (DTE) proteins ( Roobol et al, 2020 ).…”
Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.
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