Prolonged endoplasmic reticulum (ER) stress reduces protein synthesis and induces apoptosis in mammalian cells. When dimethyl sulfoxide (DMSO), a specific monoclonal antibody productivity ...(qmAb)‐enhancing reagent, is added to recombinant Chinese hamster ovary (rCHO) cell cultures (GSR cell line), it induces ER stress and apoptosis in a dose‐dependent manner. To determine an effective ER stress inhibitor, three ER stress inhibitors (BiP inducer X BIX, tauroursodeoxycholic acid, and carbazole) are examined and BIX shows the best production performance. Coaddition of BIX (50 μm) with DMSO extends the culture longevity and enhances qmAb. As a result, the maximum mAb concentration is significantly increased with improved galactosylation. Coaddition of BIX significantly increases the expression level of binding immunoglobulin protein (BiP) followed by increased expression of chaperones (calnexin and GRP94) and galactosyltransferase. Furthermore, the expression levels of CHOP, a well‐known ER stress marker, and cleaved caspase‐3 are significantly reduced, suggesting that BIX addition reduces ER stress‐induced cell death by relieving ER stress. The beneficial effect of BIX on mAb production is also demonstrated with another qmAb‐enhancing reagent (sodium butyrate) and a different rCHO cell line (CS13‐1.00). Taken together, BIX is an effective ER stress inhibitor that can be used to increase mAb production in rCHO cells.
Endoplasmic reticulum (ER) stress is induced in Chinese hamster ovary (CHO) cell culture, especially with the addition of productivity‐enhancing chemicals. BiP inducer X (BIX) is an effective ER stress inhibitor for use in recombinant CHO cell cultures for improved monoclonal antibody (mAb) production.
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.
Ethanol extract (FRE) and water extract (FRW) of Ficus racemosa (family: Moraceae) were subjected to free radical scavenging both by steady state and time resolved methods such as nanosecond pulse ...radiolysis and stopped-flow spectrophotometric analyses. FRE exhibited significantly higher steady state antioxidant activity than FRW. FRE exhibited concentration dependent DPPH, ABTS −, hydroxyl radical and superoxide radical scavenging and inhibition of lipid peroxidation with IC50 comparable with tested standard compounds. In vitro radioprotective potential of FRE was studied using micronucleus assay in irradiated Chinese hamster lung fibroblast cells (V79). Pretreatment with different doses of FRE 1h prior to 2 Gy γ-radiation resulted in a significant (P < 0.001) decrease in the percentage of micronucleated binuclear V79 cells. Maximum radioprotection was observed at 20 μg/ml of FRE. The radioprotection was found to be significant (P < 0.01) when cells were treated with optimum dose of FRE (20 μg/ml) 1 h prior to 0.5, 1, 2, 3 and 4 Gy γ-irradiation compared to the respective radiation controls. The cytokinesis-block proliferative index indicated that FRE does not alter radiation induced cell cycle delay. Based on all these results we conclude that the ethanol extract of F. racemosa acts as a potent antioxidant and a probable radioprotector.
Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the ...targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the
DHFR
expression cassette and transgene landing pad into a CHO genomic hot spot,
C12orf35
locus, of
DHFR
-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the
DHFR
and
EGFP
copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (
q
mAb
) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a
q
mAb
of 9.1–11.0 pg/cell/day. Fluorescence
in situ
hybridization analysis showed colocalization of
DHFR
and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.
Chinese hamster ovary (CHO) cells represent the predominant platform in biopharmaceutical industry for the production of recombinant biotherapeutic proteins, especially glycoproteins. These ...glycoproteins include oligosaccharide or glycan attachments that represent one of the principal components dictating product quality. Especially important are the N-glycan attachments present on many recombinant glycoproteins of commercial interest. Furthermore, altering the glycan composition can be used to modulate the production quality of a recombinant biotherapeutic from CHO and other mammalian hosts. This review first describes the glycosylation network in mammalian cells and compares the glycosylation patterns between CHO and human cells. Next genetic strategies used in CHO cells to modulate the sialylation patterns through overexpression of sialyltransfereases and other glycosyltransferases are summarized. In addition, other approaches to alter sialylation including manipulation of sialic acid biosynthetic pathways and inhibition of sialidases are described. Finally, this review also covers other strategies such as the glycosylation site insertion and manipulation of glycan heterogeneity to produce desired glycoforms for diverse biotechnology applications.
Continuous manufacturing opens up new operation windows with improved product quality in contrast to documented lot deviations in batch or fed-batch operations. A more sophisticated process control ...strategy is needed to adjust operation parameters and keep product quality constant during long-term operations. In the present study, the applicability of a combination of spectroscopic methods was evaluated to enable Advanced Process Control (APC) in continuous manufacturing by Process Analytical Technology (PAT). In upstream processing (USP) and aqueous two-phase extraction (ATPE), Raman-, Fourier-transformed infrared (FTIR), fluorescence- and ultraviolet/visible- (UV/Vis) spectroscopy have been successfully applied for titer and purity prediction. Raman spectroscopy was the most versatile and robust method in USP, ATPE, and precipitation and is therefore recommended as primary PAT. In later process stages, the combination of UV/Vis and fluorescence spectroscopy was able to overcome difficulties in titer and purity prediction induced by overlapping side component spectra. Based on the developed spectroscopic predictions, dynamic control of unit operations was demonstrated in sophisticated simulation studies. A PAT development workflow for holistic process development was proposed.
Protein A chromatography is an effective capture step to separate Fc‐containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary ...among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture‐related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus‐like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell‐Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product‐specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb‐induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal.
Protein A chromatography is an effective capture step to separate Fc‐containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture‐related impurities.
Improving the rate of recombinant protein production in Chinese hamster ovary (CHO) cells is an important consideration in controlling the cost of biopharmaceuticals. We present the first predictive ...model of productivity in CHO bioprocess culture based on gene expression profiles. The dataset used to construct the model consisted of transcriptomic data from 70 stationary phase, temperature-shifted CHO production cell line samples, for which the cell-specific productivity had been determined. These samples were utilised to investigate gene expression over a range of high to low monoclonal antibody and fc-fusion-producing CHO cell lines. We utilised a supervised regression algorithm, partial least squares (PLS) incorporating jackknife gene selection, to produce a model of cell-specific productivity (Qp) capable of predicting Qp to within 4.44
pg/cell/day root mean squared error in cross model validation (RMSE
CMV). The final model, consisting of 287 genes, was capable of accurately predicting Qp in a further panel of 10 additional samples which were incorporated as an independent validation. Several of the genes constituting the model are linked with biological processes relevant to protein metabolism.
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