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     

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.     

RNA interference of cofilin in Chinese hamster ovary cells improves recombinant protein productivity

RNA interference (RNAi) has been recently applied to improve the yield and quality of recombinant proteins produced in Chinese hamster ovary (CHO) cells, the most commonly used mammalian cell line for production of complex biopharmaceuticals. Proteomic profiling of CHO cells undergoing gene amplification identified cofilin, a key regulatory protein of actin cytoskeletal dynamics, as a cellular target for genetic engineering studies. Transient reduction of cofilin by small interfering RNA (siRNA) enhanced specific productivity in recombinant CHO cells by up to 80%. CHO cell lines expressing cofilin-specific short hairpin RNA (shRNA) vectors showed up to a 65% increase in specific productivity. These results suggest that modulation of cofilin, and its regulatory pathways, may be a new approach to enhance recombinant protein productivity in CHO cells.     

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442. © 2010 Wiley Periodicals, Inc.     

Cover Picture: Biotechnology Journal 9/2015

The cover of this regular issue of BTJ is inspired by several articles in which Chinese hamster ovary (CHO) cells were used. These articles include metabolite profiling in CHO cells, the one‐step generation of a triple knockout line using CRISPR‐Cas technology and analysis of antibody glycosylation patterns in different CHO cell lines. © asolo79 – Fotolia.com     

Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC‐MS) analytics to define the molecular loci by which two yield‐enhancing feeds improve recombinant antibody yields from a model GS‐CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing.     

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene‐of‐interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long‐term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non‐UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one‐step amplification procedure. However, non‐UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long‐term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non‐UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1014–1025, 2015     

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl‐2 family proteins such as Bcl‐2 and Bcl‐x_L potently inhibit apoptosis, no studies have examined the use of the Bcl‐2 family protein, Mcl‐1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl‐1 protein and a Mcl‐1 mutant protein that is not degraded by the proteasome in a serum‐free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl‐1 led to increased viabilities in fed‐batch culture, with cell lines expressing the Mcl‐1 mutant maintaining ～90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl‐1‐expressing cell lines were isolated that consistently showed increases in antibody production of 20–35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl‐1‐expressing cell lines, and Mcl‐1‐expressing cells exhibited 3‐fold lower caspase‐3 activation when compared with the control cell lines. Altogether, the expression of Mcl‐1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO cells for therapeutic protein production is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this cell line, stability of expression was retained for 40-50 generations after which time a 40% loss in antibody production was detected. The instability observed within the cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative cell time values and an apparent increased sensitivity to cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in cellular metabolism during LTC (alterations to extracellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning cellular phenotype changes require further detailed analysis at a molecular level.     

The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside

Recently, with the dramatic increase in demand for therapeutic antibodies, Chinese hamster ovary (CHO) cell culture systems have made significant progress in recombinant antibody production. Over the past two decades, recombinant antibody productivity has been improved by more than 100-fold. Medium optimization has been identified as an important key approach for increasing product concentrations. In this study, we evaluated the effects of deoxyuridine addition to fed-batch cultures of antibody-expressing CHO cell lines. Furthermore, we investigated the effects of combined addition of deoxyuridine, thymidine, and deoxycytidine. Our results suggest that addition of these pyrimidine nucleosides can increase CHO cell growth, with no significant change in the specific production rate. As a result of the increased cell growth, the antibody concentration was elevated and we were able to achieve more than 9 g/L during 16 days of culture. Similar effects of nucleoside addition were observed in fed-batch cultures of a Fab fragment-expressing CHO cell line, and the final Fab fragment concentration was more than 4 g/L. This nucleoside addition strategy could be a powerful platform for efficient antibody production.     

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high‐yielding Chinese hamster ovary (CHO) transient expression system. Suspension‐adapted CHO‐K1 host cells were engineered to express the gene encoding Epstein‐Barr virus (EBV) nuclear antigen‐1 (EBNA‐1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA‐1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA‐1‐only transfectants. This was confirmed with the retransfection of an EBNA‐1‐only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA‐1‐only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:132–141, 2014     

Optimized signal peptides for the development of high expressing CHO cell lines

Recombinant biotherapeutic proteins such as monoclonal antibodies are mostly produced in Chinese hamster ovary (CHO) cells and pharmaceutical companies are interested in an appropriate platform technology for the development of large‐scale production processes. A major aim of our study was therefore to improve the secretion efficiency of a recombinant biotherapeutic antibody by optimizing signal peptides. Reporter molecules such as gaussia and vargula luciferase or secreted alkaline phosphatase are frequently used to this end. In striking contrast, we used a biotherapeutic antibody that was fused to 16 different signal peptides during our study. In this way, the secretion efficiency of the recombinant antibody has been analyzed by transient expression experiments in CHO cell lines. Compared to the control signal peptide, it was not possible to achieve higher efficiencies with signal peptides derived from a variety of species or even natural immunoglobulin G signal peptides. The best results were obtained with natural signal peptides derived from human albumin and human azurocidin. These results were confirmed by fed‐batch experiments with stably transfected cell pools, in which cell‐specific productivities up to 90 pg cell^?1 day^?1 and product concentrations up to 4 g L^?1 could be determined using the albumin signal peptide. Finally, the applicability of the identified signal peptides for both different antibodies and non‐antibody products was demonstrated by transient expression experiments. In conclusion, it was found that signal peptides derived from human albumin and human azurocidin are most appropriate to generate cell lines with clearly improved production rates suitable for commercial purposes in a product‐independent manner. Biotechnol. Bioeng. 2013; 110: 1164–1173. © 2012 Wiley Periodicals, Inc.     

Unstable expression of recombinant antibody during long-term culture of CHO cells is accompanied by histone H3 hypoacetylation

The gradual loss of recombinant protein expression in CHO cell lines during prolonged subculture is a common issue, referred to as instability, which seriously affects the industrial production processes of therapeutic proteins. Loss of recombinant gene copies, due to the genetic instability of CHO cells, and epigenetic silencing of transgene sequences, are the main reported causes of production instability. To increase our understanding on the molecular mechanisms inherent to CHO cells involved in production instability, we explored the molecular features of stable and unstable antibody producing cell lines obtained without gene amplification, to exclude the genetic instability induced by the gene amplification process. The instability of recombinant antibody production during long-term culture was caused by a 48–53 % decrease in recombinant mRNA levels without significant loss of recombinant gene copies, but accompanied by a ~45 % decrease in histone H3 acetylation (H3ac). Thus, our results suggest a critical role of H3ac in the stability of recombinant protein production.