Characterization of metalloprotease and serine protease activities in batch CHO cell cultures: control of human recombinant IFN-γ proteolysis by addition of iron citrate

Clincke, Marie-Françoise; Guédon, Emmanuel; Yen, Frances T; Ogier, Virginie; Goergen, Jean‐Louis

doi:10.1186/1753-6561-5-s8-p115

Cited by 14 publications

(13 citation statements)

References 3 publications

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“…Of the 219 proteins identified in the eight sample iTRAQ, 100 were common to the six sample iTRAQ; all 219 proteins identified to a three or more unique peptide confidence in the eight sample analysis were present in the six sample comparison. Interestingly, proteins identified and retained include proteases such as cathepsins and serine proteases, such molecules having previously been shown to result in degradation of mAbs after purification , and thioredoxin reductase, which has been identified as playing a major role in the reduction of intact mAb in cell culture supernatants .…”

Section: Resultsmentioning

confidence: 99%

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO‐S cell line

Chiverton

Evans

Pandhal

et al. 2016

Biotechnology Journal

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There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO‐S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.

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Section: Resultsmentioning

confidence: 99%

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO‐S cell line

Chiverton

Evans

Pandhal

et al. 2016

Biotechnology Journal

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“…Furthermore, a higher concentration of Ctsd that cleaves the Fc-moiety was observed in DUKX-Fc as compared to in DG-Fc ( Supplementary Tables SVI and SVII). MMP-3, -9, -10, -12, and -19 have also been shown to cleave the recombinant proteins (Clincke et al, 2011;Nelissen et al, 2003;Rodriguez et al, 2010;Sandberg et al, 2006). Serine and cysteine proteases cleave IgG1, and vascular endothelial growth factor receptor 1 (D1-D3)-Fc fusion protein (Chakrabarti et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Proteomic analysis of host cell protein dynamics in the supernatant of Fc‐fusion protein‐producing CHO DG44 and DUKX‐B11 cell lines in batch and fed‐batch cultures

Park

Jin²,

et al. 2017

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells are the most widely used host cell lines for the commercial production of therapeutic proteins including Fc-fusion proteins. During the culture of recombinant CHO (rCHO) cells, host cell proteins (HCPs), secreted from viable cells and released from dead cells, accumulate extracellularly, potentially impairing product quality. In this study, the HCPs that accumulated extracellularly in batch and fed-batch cultures of Fc-fusion protein-producing rCHO cell lines (DG-Fc and DUKX-Fc) were identified and quantified using nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by gene ontology and functional analysis. When the proteome database of Cricetulus griseus was used as a reference to identify the HCPs, more HCPs were identified for DG-Fc (1632 HCPs in batch culture and 1733 HCPs in fed-batch culture) than for DUKX-Fc (1114 HCPs in batch culture and 1002 HCPs in fed-batch culture). Clustering analysis of HCPs, which were classified into four clusters according to their concentration profiles during culture, showed that the concentration profiles of HCPs affecting the quality of Fc-fusion proteins correlated with changes in Fc-fusion protein quality. Taken together, the dataset of HCPs obtained in this study using the two different rCHO cell lines provides insights into the determination of appropriate target proteins to be removed during the culture and purification steps so as to ensure good Fc-fusion protein quality. Biotechnol. Bioeng. 2017;114: 2267-2278. © 2017 Wiley Periodicals, Inc.

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“…The strategy we have developed provides a practical method for the large‐scale production of unclipped, glycan restricted, Env proteins to enable the testing of clade B HIV vaccine concepts including monomeric and trimeric envelope proteins, as well as guided immunization strategies. Further studies may also elucidate whether a C1s‐deficient CHO cell line or other CHO protease‐deficient CHO cell line is beneficial for the expression of other recombinant proteins found to be proteolyzed in CHO cells including but not limited to monoclonal antibodies, human Factor VIII, or IFN‐γ (Bee et al, ; Clincke, Guedon, Yen, Ogier, & Goergen, ; Dorai et al, ; Kaufman, Wasley, & Dorner, ).…”

Section: Discussionmentioning

confidence: 99%

“…Previously, groups have partially characterized CHO proteases involved in proteolytic degradation and have stated that the CHO protease of interest is a metalloprotease or serine protease on the basis of screening assays with protease inhibitors and limited amino acid sequence from mass spectroscopy experiments. At the time, they were not able to identify the protease because of the lack of an annotated CHO genome (Clincke et al, ; Dorai et al, ; Du et al, ; Sandberg et al, ). With the publication of the CHO genome in 2011 (Xu et al, ) and common MS/MS techniques, we have determined the identity of the protease and created an engineered knockout cell line to eliminate proteolysis of recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells

Byrne

et al. 2019

Biotech & Bioengineering

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Proteolysis associated with recombinant protein expression in Chinese HamsterOvary (CHO) cells has hindered the development of biologics including HIV vaccines.When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1 s (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s −/− MGAT1 − CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs).The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation.Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases. K E Y W O R D S

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Characterization of metalloprotease and serine protease activities in batch CHO cell cultures: control of human recombinant IFN-γ proteolysis by addition of iron citrate

Cited by 14 publications

References 3 publications

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO‐S cell line

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO‐S cell line

Proteomic analysis of host cell protein dynamics in the supernatant of Fc‐fusion protein‐producing CHO DG44 and DUKX‐B11 cell lines in batch and fed‐batch cultures

Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells

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