Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1.

Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled L-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.

Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering.

The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.

Negative interference with antibody-dependent cellular cytotoxicity mediated by rituximab from its interactions with human serum proteins.

Although interactions of small molecular drugs with serum proteins have been widely studied from pharmacokinetic and pharmacodynamic perspectives, there have been few reports on the effects of serum components on therapeutic antibody functions. This study reports the effect of abundant serum proteins on antibody-dependent cellular cytotoxicity (ADCC) mediated by rituximab and Fcγ receptor III (FcγRIII). Human serum albumin (HSA) and the Fab fragment from the pooled serum polyclonal IgG were found to compromise ADCC as non-competitive inhibitors. Our nuclear magnetic resonance data provided direct evidence for the interactions of HSA with both the Fab and Fc regions of rituximab and also with the extracellular region of FcγRIII (sFcγRIII). The degree of involvement in the interaction decreased in the order of rituximab-Fab > rituximab-Fc > sFcγRIII, suggesting preferential binding of HSA to net positively charged proteins. Although much less pronounced than the effect of HSA, polyclonal IgG-Fab specifically interacted with rituximab-Fc. The NMR data also showed that the serum protein interactions cover the Fc surface extensively, suggesting that they can act as pan-inhibitors against various Fc receptor-mediated functions and pharmacokinetics. Our findings highlight the importance of considering serum-protein interactions in the design and application of antibody-based drugs with increased efficacy and safety.

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells.

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.

Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells.

Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.

Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein.

Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms.

Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system.

Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147 kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.

Characterization of Chinese hamster ovary cells with disparate chromosome numbers: Reduction of the amount of mRNA relative to total protein.

Chromosomes in Chinese hamster ovary (CHO) cells are labile. We have shown that high-chromosome-number CHO cells have greater potential to become robust producers of recombinant proteins. One explanation being the increase in transgene integration sites. However, high-chromosome-number cell clones produce more IgG3 following culture of single-cell clones, even under conditions that yield the same number of integrations as cells with normal chromosome numbers. Here, we characterized high-chromosome-number cells by transcriptome analysis. RNA standards were used to normalize transcriptomes of cells that had different chromosome numbers. Our results demonstrate that the mRNA ratio of ß-actin and many other genes in high-chromosome-number cells to that in normal-chromosome-number cells per cell (normalized to RNA standards) was smaller than the equivalent genomic size and cell volume ratios. Many genes encoding membrane proteins are more highly expressed in high-chromosome-number cells, probably due to differences in cell size caused by the increase in chromosomes. In addition, genes related to histone modification and lipid metabolism are differentially expressed. The reduced transcript level required per protein produced in total and the different intracellular signal transductions might be key factors for antibody production.

Effect of the disulfide isomerase PDIa4 on the antibody production of Chinese hamster ovary cells.

Therapeutic monoclonal antibodies recognize and bind specific molecules on the surface of target cells, stimulating the immune system, which can attack these targeted cells. These antibodies are produced by mammalian cells, including Chinese hamster ovary (CHO) cells, because the formation of antibodies requires complicated posttranslational modifications, including peptidyl-prolyl cis/trans isomerization, disulfide bond formation, and glycosylation. Currently, it is thought that the efficient production of secretory proteins is limited by posttranslational processes. The ER is the biosynthesis site of all secreted and membrane proteins. The accumulation of unfolded proteins in the ER causes the ER stress response. During the ER stress state, various molecular chaperones are expressed to prevent proteins from the aggregate formation. The molecular chaperone involved in ER stress likely plays an essential role in the production of secretory proteins. The purpose of this study was to improve the production of monoclonal antibodies by cells. We elucidated the function of ER chaperones in the production of a monoclonal antibody. First, we quantitatively measured the mRNA expression levels of protein disulfide-isomerase family members. In CHO HcD6 cells treated with tunicamycin, the expression level of pdia4 was significantly increased. Second, we investigated the relationship between PDIa4 and antibody productivity in pdia4-knockdown cells. Both a decrease in the amount of secreted antibody and the accumulation of immature antibodies inside the cells were observed. Recombinant PDIa4 was able to refold the antibodies and Fabs. These results indicate that PDIa4 affects the production of monoclonal antibodies by catalyzing disulfide bond formation in these antibodies in CHO cells.

Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling.

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.

Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats.

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.

Secretory leakage of IgG1 aggregates from recombinant Chinese hamster ovary cells.

Aggregation of therapeutic antibodies is one of the most important issues to be resolved in manufacturing processes because of reduced efficacy and immunogenicity. Despite aggregation studies in vitro, little is known about the aggregation mechanism in cell culture processes. In this study, we investigated the process of aggregate formation of IgG1 antibodies during the culture of Chinese hamster ovary (CHO) cells to determine how aggregation occurs. A recombinant CHO cell line was cultivated in a bioreactor, and purified IgG1 from daily culture supernatants was analyzed by size exclusion chromatography. We found a linear correlation between the peak plots of IgG1 by-products, dimeric and aggregated IgG1, and integrated viable cell density, indicating that these by-products were secreted from CHO cells at a constant secretion rate. In addition, aggregate formation was not reproduced in pseudo-culture experiments, and the solution structures of intracellular and extracellular IgG1 aggregates were similar. These results support the concept of secretory leakage of IgG1 by-products. Secreted aggregates appeared to be in an alternatively folded state, which can pass through the protein quality control system in mammalian cells.

Analysis of intracellular IgG secretion in Chinese hamster ovary cells to improve IgG production.

The production of biopharmaceutical immunoglobulin G (IgG) using cultured mammalian cells, especially Chinese hamster ovary (CHO) cells is well established and has been markedly improved through the modification of cells and cell culture engineering technologies. The establishment of high-production cell lines remains a challenge. The intracellular secretion of IgG has been investigated to identify and solve the rate-limiting steps in antibody production. However, strategies that regulate the expression of proteins that are related to antibody secretory pathway have not consistently improved their production. In this study, key features and limitations of the antibody secretion process in recombinant CHO cells were analyzed to develop more efficient approaches for establishing high-production cells. By chase assay with protein translation inhibitors, IgG secretion reached a plateau when at least 20% of IgG remained in the cells. The secretion kinetics and retention ratio of IgG varied between IgG subclasses (two types of IgG1 and an IgG3 subclass). Immunofluorescent microscopy and size exclusion chromatography showed that the remaining intracellular IgG localized mainly within the endoplasmic reticulum (ER) and less with the cis-Golgi network, despite the formation of fully assembled IgG. These results show that remaining intracellular IgG is a target for enhancing antibody secretion, even in high-production CHO cells.

Secretion analysis of intracellular "difficult-to-express" immunoglobulin G (IgG) in Chinese hamster ovary (CHO) cells.

The Chinese hamster ovary (CHO) cell line is the most widely used host cell for therapeutic antibody production. Although its productivity has been improved by various strategies to satisfy the growing global demand, some difficult-to-express (DTE) antibodies remain at low secretion levels. To improve the production of various therapeutic antibodies, it is necessary to determine possible rate-limiting steps in DTE antibody secretion in comparison with other high IgG producers. Here, we analyzed the protein secretion process in CHO cells producing the DTE immunoglobulin G (IgG) infliximab. The results from chase assays using a translation inhibitor revealed that infliximab secretion could be nearly completed within 2 h, at which time the cells still retained about 40% of heavy chains and 65% of light chains. Using fluorescent microscopy, we observed that these IgG chains remained in the endoplasmic reticulum and Golgi apparatus. The cells inefficiently form fully assembled heterodimer IgG by making LC aggregates, which may be the most serious bottleneck in the production of DTE infliximab compared with other IgG high producers. Our study could contribute to establish the common strategy for constructing DTE high-producer cells on the basis of rate-limiting step analysis.

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III.

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

Mechanism of induced folding: Both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants.

A considerable number of functional proteins are unstructured under physiological condition. These "intrinsically disordered" proteins exhibit induced folding when they bind their targets. The induced folding comprises two elementary processes: folding and binding. Two mechanisms are possible for the induced folding: either folding before binding or binding before folding. We found that these two mechanisms can be distinguished by the target-concentration dependence of folding kinetics. We also created two types of mutants of staphylococcal nuclease showing the different inhibitor-concentration dependence of induced folding kinetics. One mutant obeys the scheme of binding before folding, while the other the folding before binding. This is the first experimental evidence demonstrating that both mechanisms are realized for a single protein. Binding before folding is possible, when the protein lacks essential nonlocal interaction to stabilize the native conformation. The results cast light on the protein folding mechanism involved in the intrinsically disordered proteins.

Construction of Anti-HER2 Recombinants as Targeting Modules for a Drug-delivery System Against HER2-positive Cells.

BACKGROUND/AIM: Recombinant antibodies have been investigated and used in applications such as targeting modules of drug-delivery systems (DDS) against cancers. This study aimed to prepare recombinant antibodies against HER2, containing sortase A (SrtA) recognition sequence, that are applicable as targeting modules in DDS after linkage with the drug-carrier containing oligoglycine-acceptor peptide by SrtA transpeptidation. MATERIALS AND METHODS: The recombinant trastuzumab fragment antibodies (scFvs and Fab) with the SrtA-recognition motif (LPXTG) at their C-terminal were constructed and expressed in Escherichia coli and Chinese hamster ovary (CHO) cells, respectively. The reactivity of the purified recombinant antibodies towards HER2-expressing cells was also evaluated via immunofluorescent staining. RESULTS: Fab demonstrated higher yield and purity and better reactivity towards HER2-expressing cells (HCT-15 and HeLa) when compared to scFvs. CONCLUSION: The CHO expression system possesses superior yield and purity when compared to the E. coli expression system with respect to the preparation of recombinant antibodies applicable in targeting modules for DDS (DDS-TM). Moreover, a Fab variant prepared in this study demonstrated the potential to be a DDS-TM against HER2-expressing cancer cells.

Enhanced IgG1 production by overexpression of nuclear factor kappa B inhibitor zeta (NFKBIZ) in Chinese hamster ovary cells.

Several engineering strategies have been employed to improve the production of therapeutic recombinant proteins in Chinese hamster ovary (CHO) cell lines. We have focused on unfolded protein response-based engineering and reported that ATF4 overexpression increases protein production. In this study, transcriptome analysis of ATF4-overexpressed CHO cells was performed using high-coverage expression profiling, to search for another key factor contributing to recombinant protein production. We observed the upregulated expression of transcription factor, nuclear factor (NF)-kappa-B inhibitor zeta (NFKBIZ or Iκbζ), in ATF4-overexpressed cells. A total of 1917 bp of CHO NFKBIZ cDNA was cloned, and two stable cell lines overexpressing NFKBIZ were constructed. We investigated the effects of NFKBIZ on IgG1 production in CHO cells. Although the two stable cell lines, NFKBIZ-A and -B, had the opposite phenotypes in cell growth, the specific IgG1 production rate of both cell lines was enhanced by 1.2-1.4-fold. In the NFKBIZ-A cell line, the synergistic effect between enhanced viable cell density and improved specific IgG1 production rate brought about a large increase in the final IgG1 titer. Luciferase-based NF-κB signaling assay results suggest that altered p50/p50 signaling seems to be due to the opposite phenotypes in cell growth. No difference was observed in the translational levels and intracellular assembly states of IgG1 between mock and two NFKBIZ cell lines, indicating that the secretion machinery of correctly folded IgG1 was enhanced in NFKBIZ-overexpressing cell lines.

Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis.

Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production.