Abstract
Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.
Highlights
The majority of recombinant glycoprotein therapeutics are manufactured in CHO (Chinese Hamster Ovary) cells due to their high productivity (1–10 grams per liter), genetic stability, and ability to be grown in large-scale suspension culture [1,2,3]
We studied the expression of another recombinant protein, human Factor VIII, known to be susceptible to proteolysis in CHO cells to explore the substrate specificity of Component 1s (C1s) and how it differs from thrombin, another enzyme able to cleave the GPGRAF consensus sequence
CHO cells are widely used for the production of most protein therapeutics, limited proteolysis, or clipping, is a common problem in large scale production of recombinant proteins
Summary
The majority of recombinant glycoprotein therapeutics are manufactured in CHO (Chinese Hamster Ovary) cells due to their high productivity (1–10 grams per liter), genetic stability, and ability to be grown in large-scale suspension culture [1,2,3]. Many recombinant proteins including monoclonal antibodies, antibody fusion proteins, and IFN-γ are partially degraded or “clipped” by endogenous CHO cell proteases during the cell culture or recovery process [4,5,6,7,8,9] This is the case for glycoprotein 120 (gp120), the monomeric subunit of the HIV-1 envelope protein (Env), used in many of the HIV vaccines tested to date in human vaccine efficacy trials [10,11,12,13]. HIV isolates are classified into different genetic clades based on impartial sequence analysis [20,21] These include clades C and CFRF01_AE viruses, prevalent in Africa and Asia respectively, and clade B viruses in North America, Europe, the Caribbean and Australia. Env proteins proteolyzed in this manner are difficult to manufacture and purify in quantities required for immunization of populations at high risk for infection [24,26]
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