Cover Picture: Biotechnology Journal 3/2014

This “regular” issue of Biotechnology Journal gathers the state‐of‐the‐art in biotechnology, including articles on CHO cells, plant biotechnology and tissue engineering. The cover image shows immunohistochemical staining of tissue sections using antibodies recognizing different target proteins (www.proteinatlas.org) and is provided by Sophia Hober et al., authors of “Antibody performance in western blot applications is context‐dependent” (http://dx.doi.org/10.1002/biot.201300341)     

Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Adaptation of dihydrofolate reductase (DHFR)‐deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time‐consuming and labor‐intensive process because nonadapted DHFR‐deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension‐adapted DHFR‐deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2–359 μM enhanced cell growth in in‐house CDM. Addition of 3.6 μM THF to in‐house CDM resulted in a more than 2.5‐fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9‐fold enhancement of maximum viable cell density. An anchorage‐ and serum‐dependent DHFR‐deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in‐house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension‐adapted DHFR‐deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR‐deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539–1546, 2016     

Apoptosis in batch cultures of Chinese Hamster Ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum‐free batch culture of CHO cells in suspension died via apoptosis—as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin‐1‐β‐Converting Enzyme (ICE) family, e.g., CPP32, Mch‐1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoro‐methyl‐ketone (z‐VAD.fmk), was unable to substantially extend the life of a serum‐free batch culture of CHO cells. In addition, z‐VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z‐VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl‐2 expression was able to significantly extend viabilities in CHO batch culture. Bcl‐2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 632–640, 1999.     

Overexpression of taurine transporter in Chinese hamster ovary cells can enhance cell viability and product yield,while promoting glutamine consumption

Transporters mediate the uptake of nutrients such as amino acids and the excretion of metabolites. The fact that transporters play crucial roles in regulating cell metabolism suggests that they might be useful targets for cell engineering to enhance the yield and/or quality of monoclonal antibody (MAb) produced by CHO cells. The taurine transporter (TAUT) is stably expressed in CHO‐DXB11 cells and is upregulated late in the culture period. We found that forcing the overexpression of TAUT delayed apoptotic cell death, extending the culture period. Thus, under fed‐batch small‐culture conditions, CHO cells that expressed pHyg‐TAUT plasmid (TAUT/CHO cells), but not those that contained the null plasmid pHyg (HYG/CHO cells), produced more MAb (P < 0.01) and less lactate (P < 0.05). In a 1‐L bioreactor, a representative high‐yield TAUT/CHO cell line (T10) showed >80% viability for more than 1 month and a 47% increase in medium MAb concentration. In T10 cells, the upregulation of TNF‐α mRNA (an apoptosis marker) and the accumulation of ammonia late in the culture period were suppressed. Moreover, if an excess of taurine was added, T10 cells efficiently consumed glutamine but not other amino acids, so T10 cells may have gained a glutamine transporter‐like function. Because a considerable amount of metabolic energy is derived from glutamine, this active glutamine consumption in T10 cells might be a reason for the improved cell viability and MAb concentration. These results demonstrate that forcing the overexpression of TAUT in CHO cells can enhance cell culture performance and increase MAb titer. Biotechnol. Bioeng. 2010;107: 998–1003. © 2010 Wiley Periodicals, Inc.     

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO‐S, CHO‐K1 8 mM glutamine, and CHO‐K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO‐S and CHO‐K1, with the diversity increasing and new variants appearing, while CHO‐K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.     

miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

In recent years, the number of complex but clinically effective biologicals such as multi‐specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult‐to‐express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next‐generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult‐to‐express proteins. In a previous study we identified miR‐143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR‐143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR‐143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen‐activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR‐143‐3p in CHO cells. Finally, siRNA‐mediated knock‐down of MAPK7 could be demonstrated to phenocopy pro‐productive effects of miR‐143. In summary, our data suggest that miR‐143 might represent a novel genetic element to enhance production of difficult‐to‐express proteins in CHO cells which may be partly mediated by down‐regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046–1058, 2017     

Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy,a web‐based target finding tool

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site‐specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome‐editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user‐friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO‐K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off‐target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high‐producing clones among a large population of low‐ and non‐productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)‐based methotrexate (MTX) selection and glutamine synthetase (GS)‐based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS‐CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L‐MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS‐knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (～2%) of bi‐allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine‐dependent growth of all GS‐knockout cell lines. Full evaluation of the GS‐knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two‐ to three‐fold through the use of GS‐knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non‐producing and low‐producing cells after 25 µM L‐MSX selection, and resulted in a six‐fold efficiency improvement in identifying similar numbers of high‐productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS‐knockout cells on recombinant protein quality is also discussed. Biotechnol. Bioeng. 2012; 109:1007–1015. © 2011 Wiley Periodicals, Inc.     

CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.     

Protein reference mapping of dihydrofolate reductase‐deficient CHO DG44 cell lines using 2‐dimensional electrophoresis

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase‐deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well‐characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2‐DE with various immobilized pH gradients (pHs 3–10, 5–8, and 3–6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver‐stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI‐TOF‐MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Acute hydrodynamic forces and apoptosis: a complex question

A second generation flow contraction device was developed and modeled which allows cells to be subjected to well‐defined hydrodynamic forces. Studies were conducted with this system on wild‐type Chinese Hamster Ovary cells (CHO‐K1) and a strain of CHO cells which expresses the human Bcl‐2? gene (CHO‐bcl‐2). In this study, the following questions were asked: (1) Does an acute hydrodynamic force induce apoptosis in wild‐type CHO and CHO‐bcl‐2 cells? (2) Does the type of culture media make a difference with respect to the induction of apoptosis or necrosis? and (3) Does culture history affect induction of apoptosis or necrosis? The results obtained with this new flow contraction device and corresponding computer simulations are consistent with previously published studies with respect to the level of energy dissipation rate (EDR) required to create significant cell lysis. Second, while detectable relative to the control in the T‐flask experiments, only a small fraction of the cells become apoptotic when exposed to a sub‐lysis level of EDR (<10⁸ W · m^?3). Third, cells cultured in suspension with serum free media do not exhibit any higher or lower sensitivity (with respect to apoptosis) to various levels of EDR when compared to control cultures grown in T‐flask and serum containing media; on the other hand, necrosis is significantly increased in experiments performed on suspended cells without serum. Fourth, the addition of the Bcl‐2 gene product might slightly reduce the occurrence of apoptosis in T‐flask culture; however, the baseline response is so low that the difference is insignificant. Biotechnol. Bioeng. 2007; 98: 772–788. © 2007 Wiley Periodicals, Inc.     

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody‐dependent cellular cytotoxicity, we disrupted α‐1, 6 fucosyltransferase (FUT8 ) gene in CHO‐S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO‐S (FUT8 ^?/?) cells was comparable with wild type CHO‐S cells. FUT8 catalyzes the transfer of a fucose residue from GDP‐fucose to N‐glycans residue. Defucosylated IgG1 antibodies produced by FUT8 ^?/? cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody‐dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.     

Use of an anti‐apoptotic CHO cell line for transient gene expression

Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti‐apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro‐apoptotic factors, Bax and Bak, in a CHO‐K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO‐K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ～3‐ to 4‐fold higher antibody titers than CHO‐K1 cells. As evidence of their anti‐apoptotic properties post‐transfection, DKO cells maintained higher viability and had reduced levels of active caspase‐3 compared to CHO‐K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post‐transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae‐ and clathrin‐mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection‐induced apoptosis and not differences in endocytosis mechanism. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1050–1058, 2013     

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11‐derived dihydrofolate reductase deficient host cell

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR‐deficient DG44, and DUXB11‐based DHFR deficient CHO. Current Genentech commercial full‐length antibody products have all been produced in the DUXB11‐derived DHFR‐deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11‐derived DHFR‐deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11‐based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14‐day fed batch cultures in shake flasks. In contrast, the DUXB11‐based host produced ～0.1 g/l for both antibodies in the same 14‐day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ～2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:980–985, 2013     

Virus susceptibility of Chinese hamster ovary (CHO) cells and detection of viral contaminations by adventitious agent testing

Biopharmaceuticals are of increasing importance in the treatment of a variety of diseases. A remaining concern associated with their production is the potential introduction of adventitious agents into their manufacturing process, which may compromise the pathogen safety of a product and potentially cause stock‐out situations for important medical supplies. To ensure the safety of biological therapeutics, regulatory guidance requires adventitious agent testing (AAT) of the bulk harvest. AAT is a deliberately promiscuous assay procedure which has been developed to indicate, ideally, the presence of any viral contaminant. One of the most important cell lines used in the production of biopharmaceuticals is Chinese hamster ovary (CHO) cells and while viral infections of CHO cells have occurred, a systematic screen of their virus susceptibility has never been published. We investigated the susceptibility of CHO cells to infection by 14 different viruses, including members of 12 families and representatives or the very species that were implicated in previously reported production cell infections. Based on our results, four different infection outcomes were distinguished, based on the possible combinations of the two factors (i) the induction, or not, of a cytopathic effect and (ii) the ability, or not, to replicate in CHO cells. Our results demonstrate that the current AAT is effective for the detection of viruses which are able to replicate in CHO cells. Due to the restricted virus susceptibility of CHO cells and the routine AAT of bulk harvests, our results provide re‐assurance for the very high safety margins of CHO cell‐derived biopharmaceuticals. Biotechnol. Bioeng. 2010;106: 598–607. © 2010 Wiley Periodicals, Inc.     

Microarray platform affords improved product analysis in mammalian cell growth studies

High throughput (HT) platforms serve as a cost‐efficient and rapid screening method for evaluating the effect of cell‐culture conditions and screening of chemicals. We report the development of a HT cell‐based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/methionine sulphoximine (MSX) CHO cell line, which produces a therapeutic monoclonal antibody, was examined using a microarray system in conjunction with a conventional shake flask platform in a non‐proprietary medium. The microarray system consists of 60‐nL spots of cells encapsulated in alginate and separated in groups via an 8‐well chamber system attached to the chip. Results show the non‐proprietary medium developed allows cell growth, production, and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base medium results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the HT microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as cell growth, metabolism, and productivity.