Case study on human α1‐antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

Hansen, Henning Gram; Kildegaard, Helene Faustrup; Lee, Gyun Min; Kol, Stefan

doi:10.1002/biot.201600409

Cited by 7 publications

(5 citation statements)

References 29 publications

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“…4D). This is in accordance with previous work, which showed that A1AT activity is not linked to its N-glycosylation and CHO WT produced rhA1AT has similar activity to plA1AT [10,38]. Furthermore, differences of CHO-S WT-and 10x KO-derived rhA1AT were made visible using IEF gel analysis, where rhA1AT from the two clones revealed similar patterns to plA1AT (Fig.…”

Section: Discussionsupporting

confidence: 92%

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Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

Amann

Hansen

Kol

et al. 2019

Metabolic Engineering

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Abbreviations:A1AT, alpha-1-antitrypsin; AATD, alpha-1-antitrypsin deficiency; AUC, area under curve; B3gnt2, UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2; Cas9, CRISPR-associated protein 9; C1INH, C1 esterase inhibitor; CHO, Chinese hamster ovary; CRISPR, clustered regularly interspaced short palindromic repeats; FACS, fluorescence-activated cell sorting; FITC, Fluorescein isothiocyanate; Fut8, alpha-(1,6)-fucosyltransferase; Glul, glutamate-ammonia ligase; HAE, hereditary angioedema; HM, high-mannose; indel, insertion or deletion; IVC, integral of viable cells; KO, knock-out; mAb, monoclonal antibody; Mgat4A, mannosyl (alpha-1,3-)-glycoprotein beta-1,4-Nacetylglucosaminyltransferase isozyme A; Mgat4B, mannosyl (alpha-1,3-)glycoprotein beta-1,4-N-acetylglucosaminyltransferase isozyme B; Mgat5, mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase; MSX, methionine sulfoximine; sgRNA, single guide RNA; SNA, sambucus nigra agglutinin; Sppl3, signal peptide peptidase like 3; St3gal3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; St3gal4, ST3 beta-galactoside alpha-2,3-sialyltransferase 4; St3gal6, ST3 beta-galactoside alpha-2,3-sialyltransferase 6; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; VCD, viable cell density; WT, wild type Abstract Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency.Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.

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Section: Discussionsupporting

confidence: 92%

“…HEPES buffer and 1% Antibiotic-Antimycotic (Gibco, Waltham, MA) per well as described previously [38]. For cell sorting, fluorescent-positive cell populations were gated based on non-transfected WT CHO-S cells.…”

Section: Single Cell Cloning Of Genome Edited Cells Using Facsmentioning

confidence: 99%

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

Amann

Hansen

Kol

et al. 2019

Metabolic Engineering

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“…Etanercept titers were determined in supernatants by bio‐layer interferometry using protein A biosensors and an Octet RED96 system (FortéBio, Pall, Menlo Park, CA) as previously described, with an increased shaking speed of 1000 rpm. The absolute titers of etanercept were calculated using a calibration curve generated from a dilution series of Enbrel (Pfizer, New York City, NY; Lot R51698) and absolute quantification was validated by Coomassie‐stained SDS‐PAGE gels as previously described (data not shown) …”

Section: Methodsmentioning

confidence: 99%

Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept

Pristovšek

Hansen

Sergeeva

et al. 2018

Biotechnology Journal

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The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.

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“…The production of HIV Env vaccines has mainly employed ELISA for the quantification of vaccine products secreted onto the cultivation media. While ELISA is inexpensive and relatively easy to implement in any production setting, the waiting times for ELISA incubations and washes can be long and the variability of the measurement can often be too wide for the method to be considered accurate (H. G. Hansen et al, 2016). Another method for the detection and quantitation of protein products in crude supernatants is the western blot, which also depends on the recognition of the protein analyte by a primary antibody.…”

Section: Discussionmentioning

confidence: 99%

A reverse phase HPLC method for the quantification of HIV gp145 glycoprotein levels in cell culture supernatants

Feliciano

lez

Akamine

et al. 2020

Preprint

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A reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly in culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent media, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 x 50 mm, 3.5 μm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 x 12.5 mm, 5μm), using 0.1% TFA and 2% n-propanol as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile as mobile phase B. The column temperature was 80ºC, the flow rate 1 ml/min and the absorbance monitored at 280 nm. The procedures and capabilities of the method were evaluated against the present criteria for linearity, limit of detection (LOD), accuracy, precision, and robustness of the International Conference on Harmonization (ICH) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The methods showed good linearity (R2 = 0.9996), a lower LOD of 2.4 μg/ml, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

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Case study on human α1‐antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

Cited by 7 publications

References 29 publications

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept

A reverse phase HPLC method for the quantification of HIV gp145 glycoprotein levels in cell culture supernatants

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