Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine–guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6‐thioguanine (6‐TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6‐TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb‐A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb‐B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity. Biotechnol. Bioeng. 2010;107: 163–171. © 2010 Wiley Periodicals, Inc.     

A CHO strain producing high-level human IL-6 with the 3' deletion construct.

A Chinese Hamster Ovary (CHO) strain producing high-level human interleukin 6 (IL-6) was obtained, by introduction of the IL-6 cDNA from which the 3' AU-rich region was deleted. The IL-6 mRNA of this strain was stable. The productivity was more than 15 times higher than the previously obtained clone, which has intact IL-6 cDNA in the same expression vector. This strain produced IL-6 at a high level of 30 micrograms ml-1 in batch culture, or a continuous 20 micrograms per 10(6) cells per day in microcarrier cultivation.     

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     

Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.     

Sustaining an efficient and effective CHO cell line development platform by incorporation of 24‐deep well plate screening and multivariate analysis

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24‐deep well plate (24‐DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24‐DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24‐DWP achieved the highest productivity improvement upon scaling‐up to shake flasks. Product quality of the protein purified from shake flasks and 24‐DWP were also compared. The 24‐DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N‐glycan species, and increase the high‐mannose N‐glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled‐up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24‐DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175–186, 2018     

Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic

The Biogen upstream platform is capable of delivering equivalent quality material throughout the cell line generation process. This allows us to rapidly deliver high‐quality biopharmaceuticals to patients with unmet medical needs. The drive to reduce time‐to‐market led the cell engineering group to develop an expression system that can enable this strategy. We have developed a clonal Chinese Hamster Ovary (CHO) host cell line that can routinely produce consistent antibody material at high titers throughout the cell line generation process. This host line enables faster delivery of early phase material through use of the highly productive stable pool or a mixture of high performance clones. Due to unique characteristics of this cell line, the product quality of material from early cell populations is very comparable to material from the final clones. This lends itself to a “fast‐to‐tox” strategy whereby toxicology studies can be performed with representative material from an earlier cell population, thus accelerating the clinical timelines. Our new clonal host offers robust and consistent performance that enables a highly productive, flexible process and faster preclinical timelines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1468–1475, 2017     

Production of Functional Soluble Dectin-1 Glycoprotein Using an IRES-Linked Destabilized-Dihydrofolate Reductase Expression Vector

Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to β-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since β-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of Saccharomyces cerevisiae. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.     

Polycation nanostructured lipid carrier, a novel nonviral vector constructed with triolein for efficient gene delivery

A novel nonviral gene transfer vector was developed by modifying nanostructured lipid carrier (NLC) with cetylated polyethylenimine (PEI). Polycation nanostructured lipid carrier (PNLC) was prepared using the emulsion-solvent evaporation method. Its in vitro gene transfer properties were evaluated in the human lung adenocarcinoma cell line SPC-A1 and Chinese Hamster Ovary (CHO) cells. Enhanced transfection efficiency of PNLC was observed after the addition of triolein to the PNLC formulation and the transfection efficiency of the optimized PNLC was comparable to that of Lipofectamine™2000. In the presence of 10% serum the transfection efficiency of the optimal PNLC was not significantly changed in either cell line, whereas that of Lipofectamine™2000 was greatly decreased in both. Thus, PNLC is an effective nonviral gene transfer vector and the gene delivery activity of PNLC was enhanced after triolein was included into the PNLC formulation.     

Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.     

Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification

Recombinant Chinese hamster ovary (CHO) parental clones expressing a humanized antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy chain (HC) and light chain (LC) cDNA expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 representative parental clones were subjected to stepwise selection for increasing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 microM, their clonal variations in regard to antibody expression were found to be significant. Among 23 parental clones, only one clone (hu17) showed the significant increment of specific antibody productivity (q(Ab)) with increasing MTX concentration up to 0.32 microM. Compared with the parental clone (hu17), the q(Ab) of hu17 resistant at 0.32 microM MTX (hu17-0.32) was enhanced approximately 12.5-fold. To clarify the reason for the occurrence of clonal variations, Southern blot analyses of chromosomal DNAs derived from each amplified clone at 0.32 microM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it had only one 49-kb amplification unit including the LC and HC cDNAs. A fluorescent MTX competition assay showed that the resistance against MTX toxicity of the other clones without enhanced q(Ab) at 0.32 microM MTX was obtained by mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expression are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepwise selection.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Monitoring CHO cell cultures: Cell stress and early apoptosis assessment by mass spectrometry

Mammalian cells, especially CHO (Chinese Hamster Ovary), are an important host for the production of biopharmaceuticals. Early detection of cellular stress and the onset of apoptosis, ultimately leading to a reduced viability of the culture, are important with respect to process development and monitoring.     

Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media

The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed‐batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top‐down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1023–1033, 2013     

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.     

CHO细胞生长过程的建模及仿真研究

运用了一种以代谢协调和代谢调节概念为基础的生物工艺过程建模思想,以实际过程为背景建立了中国仓鼠卵巢细胞(CHO)的微载体培养生长模型,进行了仿真研究并得出了一些有意义的结论。     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

The influence of surface carbohydrates on the interaction of Fonsecaea pedrosoi with Chinese Hamster Ovary glycosylation mutant cells

In order to better understand the role played by surface glycoconjugates during cell adhesion and endocytosis by the dematiaceous fungi Fonsecaea pedrosoi, we analyzed the interaction between this microorganism and five mutants of Chinese Hamster Ovary (CHO) cells, which differ from each other in the exposition of carbohydrate residues on the cell surface. Five clones (Gat-2 parental, and the clones: Lec1, Lec2, Lec8 and ldlLec1) were tested and the adhesion and endocytic indexes were determined after 2 hours of interaction. The Lec1 and ldlLec1 clones, which present exposed mannose residues, showed the greater adhesion index (AI). On the other hand, the Lec8 clone, which exposes N-acetylglucosamine on the cell surface, presented the greater endocytic index. The role played by surface-exposed carbohydrate residues was further analyzed by addition of mannose or N-acetylglucosamine to the interaction medium and by previous incubation of the cells in the presence of the lectins Concanavalin A (ConA) and wheat germ agglutinin (WGA). The results obtained suggest that mannose residues are involved in the first step of adhesion of F. pedrosoi to the cell surface, while N-acetylglucosamine residues are involved on its ingestion process. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mitotic spindle proteomics in Chinese hamster ovary cells

Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.