Cathepsin L Causes Proteolytic Cleavage of Chinese‐Hamster‐Ovary Cell Expressed Proteins During Processing and Storage: Identification, Characterization, and Mitigation

Luo, Haibin; Liu, Tie; Cao, Mingyan; Hunter, Alan K.; Pabst, Timothy M.; Du, Jiali; Field, Raymond; Li, Yuling; Wang, William K.

doi:10.1002/btpr.2732

Cited by 33 publications

(22 citation statements)

References 27 publications

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“…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. Similar to our work, other groups in recent years have also utilized the CHO genome to identify CHO proteases involved in the degradation of biologics (Cathepsin D, Cathepsin L, matriptase, etc…) or to do large‐scale proteomics studies (Bee et al, ; Laux et al, ; Luo et al, ; Park et al, ; Valente, Lenhoff, & Lee, ). Identifying the myriad of proteases or host cell proteins in CHO cells is necessary because of the continuing use of the CHO cell line for the expression of therapeutic proteins.…”

Section: Discussionmentioning

confidence: 58%

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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Section: Discussionmentioning

confidence: 58%

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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“…Fragmentation of drug Luo et al, 2019;Migani et al, 2017;Vanderlaan et al, 2018 Cathepsin (D,E) a Aspartyl protease with activity in both acidic and neutral pH Drug quality Beatson et al, 2011;Vanderlaan et al, 2018 Note: A hyperlink to the BioPhorum website (https://www.biophorum.com/host-cell-proteins/) is provided which directs the user to a live database that will continue to be updated with high-risk HCPs. Expanded list of references are provided in the Biophorum website.…”

Section: Drug Qualitymentioning

confidence: 99%

“High‐risk” host cell proteins (HCPs): A multi‐company collaborative view

Jones

Palackal

Wang

et al. 2021

Biotech & Bioengineering

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Host cell proteins (HCPs) are process-related impurities that may copurify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation. Some have been shown to be difficult to remove by purification. Why should the biopharmaceutical industry worry about these high-risk HCPs? What approach could be taken to understand the origin of its copurification and address these *Marisa Jones and Nisha Palackal should be considered joint first authors About Biophorum Development Group (BPDG): Since its inception in 2004, BioPhorum has become a trusted environment in which senior leaders of the biopharmaceutical industry come together to share and discuss openly the emerging trends and challenges facing their industry.BioPhorum currently comprises more than 3800 active participants in seven "phorums" covering cell and gene therapy, drug substance, development, fill-finish, a technology roadmap, information technology, and supply partners. The Host Cell Protein (HCP) Workstream is part of the Development Group (BPDG). This article is a composite view of opinions shared by the whole of the BPDG-HCP Workstream and should not be attributed to the individual positions of the participating companies.

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“…CHO cells are widely used for production of recombinant glycoprotein therapeutics due to their high productivity (1-10 g per liter), genetic stability and ability to grow in large-scale suspension culture [199][200][201] in serum-free medium. However, many recombinant proteins, including monoclonal antibodies, antibody fusion proteins and IFN-γ, are partially degraded or proteolyzed by endogenous CHO cell proteases during the cell culture or recovery process [202][203][204][205][206][207]. Novel approaches to solve proteolysis and enhance glycosylation for production of HIV envelope proteins as vaccine candidates are being explored [208].…”

Section: Mammalian Cellsmentioning

confidence: 99%

Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies

Cid¹,

Bolı́var

2021

Biomolecules

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To date, vaccination has become one of the most effective strategies to control and reduce infectious diseases, preventing millions of deaths worldwide. The earliest vaccines were developed as live-attenuated or inactivated pathogens, and, although they still represent the most extended human vaccine types, they also face some issues, such as the potential to revert to a pathogenic form of live-attenuated formulations or the weaker immune response associated with inactivated vaccines. Advances in genetic engineering have enabled improvements in vaccine design and strategies, such as recombinant subunit vaccines, have emerged, expanding the number of diseases that can be prevented. Moreover, antigen display systems such as VLPs or those designed by nanotechnology have improved the efficacy of subunit vaccines. Platforms for the production of recombinant vaccines have also evolved from the first hosts, Escherichia coli and Saccharomyces cerevisiae, to insect or mammalian cells. Traditional bacterial and yeast systems have been improved by engineering and new systems based on plants or insect larvae have emerged as alternative, low-cost platforms. Vaccine development is still time-consuming and costly, and alternative systems that can offer cost-effective and faster processes are demanding to address infectious diseases that still do not have a treatment and to face possible future pandemics.

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Cathepsin L Causes Proteolytic Cleavage of Chinese‐Hamster‐Ovary Cell Expressed Proteins During Processing and Storage: Identification, Characterization, and Mitigation

Cited by 33 publications

References 27 publications

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

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

“High‐risk” host cell proteins (HCPs): A multi‐company collaborative view

Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies

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