Multiplex secretome engineering enhances recombinant protein production and purity

AbstractHost cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

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Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling

Journal of Chromatography B ◽

10.1016/j.jchromb.2018.02.032 ◽

2018 ◽

Vol 1083 ◽

pp. 44-56 ◽

Cited By ~ 9

Author(s):

Matic Grom ◽

Mirijam Kozorog ◽

Simon Caserman ◽

Andrej Pohar ◽

Blaž Likozar

Keyword(s):

Cell Culture ◽

Monoclonal Antibody ◽

Affinity Chromatography ◽

Chinese Hamster Ovary ◽

Protein A ◽

Chinese Hamster ◽

Cho Cell ◽

Equilibrium Modeling ◽

Cho Cell Culture

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Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

International Journal of Molecular Sciences ◽

10.3390/ijms20010161 ◽

2019 ◽

Vol 20 (1) ◽

pp. 161 ◽

Cited By ~ 8

Author(s):

Tuhidul Islam ◽

Amith D. Naik ◽

Yasuhiro Hashimoto ◽

Stefano Menegatti ◽

Ruben G. Carbonell

Keyword(s):

Binding Capacity ◽

Protein A ◽

Chinese Hamster ◽

High Capacity ◽

Alkaline Solutions ◽

Peptide Ligand ◽

Dynamic Binding ◽

Ligand Density ◽

Host Cell Proteins ◽

Igg Binding

This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.

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Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform

BioTechniques ◽

10.2144/btn-2020-0074 ◽

2020 ◽

Vol 69 (3) ◽

pp. 186-192

Author(s):

Kathleen Van Manen-Brush ◽

Jacob Zeitler ◽

John R White ◽

Paul Younge ◽

Samantha Willis ◽

...

Keyword(s):

Host Cell ◽

Chinese Hamster Ovary ◽

Expression System ◽

Chinese Hamster ◽

Cell Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Host Cell Proteins ◽

Common Technique ◽

Automated Instrument ◽

Cell Expression

Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.

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Surfactant Protein A2 (SP-A2) Variants Expressed in CHO Cells Stimulate Phagocytosis of Pseudomonas aeruginosa More than Do SP-A1 Variants

Infection and Immunity ◽

10.1128/iai.01341-06 ◽

2007 ◽

Vol 75 (3) ◽

pp. 1403-1412 ◽

Cited By ~ 51

Author(s):

Anatoly N. Mikerov ◽

Guirong Wang ◽

Todd M. Umstead ◽

Mario Zacharatos ◽

Neal J. Thomas ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Posttranslational Modifications ◽

Insect Cell ◽

Mammalian Cells ◽

Protein A ◽

Chinese Hamster ◽

Surfactant Protein ◽

Cho Cell ◽

Cell Association ◽

First Time

ABSTRACT Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. Two functional genes, SP-A1 and SP-A2, encode human SP-A. As we showed before, baculovirus-mediated insect cell-expressed SP-A2 enhances the association of P. aeruginosa with rat alveolar macrophages (rAMs) more than does SP-A1. However, true phagocytosis (internalization) was not shown, and insect cell derived proteins lack or are defective in certain mammalian posttranslational modifications that may be important for SP-A1 and SP-A2 activity and specificity. Here we used SP-A1 (6A2, 6A4) and SP-A2 (1A0, 1A1) allele variants expressed by CHO (Chinese hamster ovary) mammalian cells to study their effect on association and/or internalization of P. aeruginosa by rAMs and/or human AMs (hAMs) and to study if phagocytosis can be modulated differentially and/or more effectively by CHO cell-expressed SP-A variants than by insect-cell expressed SP-A variants. For cell association and internalization assessments, light microscopy and fluorescence-activated cell sorter analyses were used, respectively. We found the following for the first time. (i) SP-A2 variants enhanced phagocytosis (cell association and/or internalization) of P. aeruginosa more than SP-A1 variants did, and the cell association correlated with internalization. (ii) Differences in the activities of SP-A variants were observed in the following order: 1A1>1A0>6A2>6A4. (iii) rAMs, although more active than hAMs, are an appropriate model, as SP-A2 variants exhibited activity higher than that seen for SP-A1 variants with either rAMs or hAMs. (iv) CHO cell-expressed SP-A was considerably more active than insect cell-expressed variants. We conclude that SP-A2 variants stimulate phagocytosis of P. aeruginosa more effectively than SP-A1 variants and that posttranslational modifications positively influence the phagocytic activity of SP-A.

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Investigating the influence of physiologically relevant hydrostatic pressure on CHO cell batch culture

Scientific Reports ◽

10.1038/s41598-020-80576-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Menglin Shang ◽

Taehong Kwon ◽

Jean-Francois P. Hamel ◽

Chwee Teck Lim ◽

Bee Luan Khoo ◽

...

Keyword(s):

Hydrostatic Pressure ◽

Cell Growth ◽

Cho Cells ◽

Large Scale ◽

Chinese Hamster ◽

Perfusion Culture ◽

Mammalian Host ◽

Manufacturing Cost ◽

Cho Cell ◽

Significant Difference

AbstractChinese hamster ovary (CHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins, such as monoclonal antibodies. Enhancement of productivity of these CHO cells is one of the top priorities in the biopharmaceutical industry to reduce manufacturing cost. Although there are many different methods (e.g. temperature, pH, feed) to improve protein production in CHO cells, the role of physiologically relevant hydrostatic pressure in CHO cell culture has not been reported yet. In this study, four different hydrostatic pressures (0, 30, 60, and 90 mmHg) were applied to batch CHO cells, and their cell growth/metabolism and IgG1 production were examined. Our results indicate that hydrostatic pressure can increase the maximum cell concentration by up to 50%. Moreover, overall IgG1 concentration on Day 5 showed that 30 mmHg pressure can increase IgG1 production by 26%. The percentage of non-disulphide-linked antibody aggregates had no significant change under pressure. Besides, no significant difference was observed between 30 mmHg and no pressure conditions in terms of cell clumping formation. All these findings are important for the optimization of fed-batch or perfusion culture for directing cell growth and improving antibody production.

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Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells

10.1101/318865 ◽

2018 ◽

Author(s):

Ganglong Yang ◽

Yingwei Hu ◽

Shisheng Sun ◽

Chuanzi Ouyang ◽

Weiming Yang ◽

...

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Large Scale ◽

Chinese Hamster Ovary Cells ◽

Expression System ◽

Chinese Hamster ◽

Reversed Phase ◽

Cho Cell ◽

Cell Lysate ◽

Therapeutic Glycoproteins

AbstractThe Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization of the glycosylation profiles is critical to understand the important role of glycosylation on therapeutic glycoproteins from CHO cells. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10,338 N-linked glycosite-containing IGPs were identified, representing 1,162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to accelerate the production of recombinant therapeutic glycoproteins.

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Characterization of a human surfactant protein A1 (SP-A1) gene-specific antibody; SP-A1 content variation among individuals of varying age and pulmonary health

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00249.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. L1052-L1063 ◽

Cited By ~ 43

Author(s):

Hephzibah Rani S. Tagaram ◽

Guirong Wang ◽

Todd M. Umstead ◽

Anatoly N. Mikerov ◽

Neal J. Thomas ◽

...

Keyword(s):

Specific Antibody ◽

Protein A ◽

Age Groups ◽

Chinese Hamster ◽

Lavage Fluid ◽

Surfactant Protein ◽

Cho Cell ◽

Significant Difference ◽

Lung Health ◽

Culture Positive

The human surfactant protein A (SP-A) locus consists of two functional genes (SP-A1, SP-A2) with gene-specific products exhibiting qualitative and quantitative differences. The aim here was twofold: 1) generate SP-A1 gene-specific antibody, and 2) use this to assess gene-specific SP-A content in the bronchoalveolar lavage fluid (BALF). An SP-A1-specific polyclonal antibody (hSP-A1_Ab68-88_Col) was raised in chicken, and its specificity was determined by immunoblot and ELISA using mammalian Chinese hamster ovary (CHO) cell-expressed SP-A1 and SP-A2 variants and by immunofluorescence with stably transfected CHO cell lines expressing SP-A1 or SP-A2 variants. SP-A1 content was evaluated according to age and lung status. A gradual decrease ( P < 0.05) in SP-A1/SP-A ratio was observed in healthy subjects (HS) with increased age, although no significant change was observed in total SP-A content among age groups. Total SP-A and SP-A1 content differed significantly between alveolar proteinosis (AP) patients and HS, with no significant difference observed in SP-A1/SP-A ratio between AP and HS. The cystic fibrosis (CF) ratio was significantly higher compared with AP, HS, and noncystic fibrosis (NCF), even though SP-A1 and total SP-A were decreased in CF compared with most of the other groups. The ratio was higher in culture-positive vs. culture-negative samples from CF and NCF ( P = 0.031). A trend of an increased ratio was observed in culture-positive CF (0.590 ± 0.10) compared with culture-positive NCF (0.368 ± 0.085). In summary, we developed and characterized an SP-A1 gene-specific antibody and used it to identify gene-specific SP-A content in BALFs as a function of age and lung health.

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Determination of Corynebacterium diphtheriae toxigenicity by a colorimetric tissue culture assay

Journal of Clinical Microbiology ◽

10.1128/jcm.7.1.91-96.1978 ◽

1978 ◽

Vol 7 (1) ◽

pp. 91-96

Author(s):

J R Murphy ◽

P Bacha ◽

M Teng

Keyword(s):

Growth Inhibition ◽

Diphtheria Toxin ◽

Large Scale ◽

Chinese Hamster ◽

Corynebacterium Diphtheriae ◽

Biologically Active ◽

Assay Method ◽

Phenol Red ◽

Cho Cell ◽

Ph Indicator

Chinese hamster ovary (CHO) cell cultures in microtiter wells are sensitive to growth inhibition and killing by picogram quantities of diphtheria toxin. In the absence of biologically active toxin, the CHO cell culture produces sufficient acidic metabolites to change the phenol red pH indicator from pink to yellow within 56 h. In the presence of 10 pg of toxin per well, growth inhibition can be observed microscopically within 24 h. Diphtheria toxin can be qualitatively assayed from culture supernatants of Corynebacterium diphtheriae or from beta-phage agar plaque plugs. The colorimetric CHO cell assay method for determining toxigenicity allows for the large-scale screening of either diphtheria toxigenicity or antitoxin titration of sera.

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