A CHO stable pool production platform for rapid clinical development of trimeric SARS‐CoV‐2 spike subunit vaccine antigens

Abstract

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time‐consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non‐clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP‐toxicology and early‐phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS‐CoV‐2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost‐effective clinical evaluation of potentially life‐saving vaccines.

Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development

“…Batches of SARS-CoV-2 spike protein stably produced from a stable CHO pool (named PRO1-394, PRO1-392, and PRO1-412), were generated independently from a unique frozen cell vial (Supplementary Method 3 ) 44 , 45 . Culture conditions/characteristics for each batch are described in Supplementary Table 12 .…”

Influence of variant-specific mutations, temperature and pH on conformations of a large set of SARS-CoV-2 spike trimer vaccine antigen candidates

“…Spike proteins were produced in transiently or stably transfected CHO cells as described previously ( 8 and 18 for transient and stable, respectively). CHO-DXB11 cells 40 were provided by L. A. Chasin; CHO 55E1 ™ and CHO 2353 ™ cell lines were derived from CHO-DXB11 in-house as described 21 , 41 . The identities of these cell lines were previously verified by genome sequencing (data not shown).…”

“…While different eukaryotic host cell lines have been used to produce recombinant spike trimers, including insect cells for some approved vaccines (Nuvaxovid 7 and VidPrevtyn Beta 12 ) and human HEK293 cells for many structural studies, we showed recently that CHO cells give considerably higher spike yields than HEK293 cells 8 . Combined with the ability of CHO cells, unlike insect cells, to generate proteins with human-like glycosylation 20 , we believe that CHO cells would be a preferable host for spike subunit vaccine manufacturing 21 .…”

Repressing expression of difficult‐to‐express recombinant proteins during the selection process increases productivity of CHO stable pools

“…The plasmids used for the generation of stable pools include different plasmids encoding one (pTT75, pTT81) or two (pTT109) inducible cassettes, similar to those previously described (Joubert et al, 2022(Joubert et al, , 2023Poulain et al, 2017Poulain et al, , 2019. All sequences encoding the r-proteins described in this study were codonoptimized for expression in mammalian cells and their expression is regulated by the cumate-inducible CR5 promoter composed of six CuO operator sequences from the p-cmt operon of Pseudomonas putida placed upstream of a CMV minimal promoter (Mullick et al, 2006).…”