Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens

Abstract

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

“…The biological basis for false-positives is multifactorial, but the influence of the production platform- and process-related peculiarities or impurities on performance of a diagnostic protein are factors that are often underestimated. While the viral NP is almost exclusively being produced in bacteria [ 48 , 49 ], we expressed the spike receptor binding domain in HEK cells, CHO cells, insect cells and plants [ 4 , [50] , [51] , [52] ]. To find out which of these systems leads to the highest quality and manufacturability of the RBD diagnostic antigen of potentially high demand, we evaluated these production platforms and pre-validated the proteins based on diagnostic performance with a large set of pre-COVID-19 and COVID-19 sera using the Luminex platform.…”

“…Pre-COVID-19 cohort, Meduni Wien Biobank : The pre-COVID-19 cohort covered a total of 1126 samples from healthy, non-SARS-CoV-2-infected individuals collected before 2020 to guarantee seronegativity. Banked human samples including sera from voluntary donors ( n = 265, median age 38 [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] years, 59.0% females), samples from a large population-based cohort aged 8–80 years, representing a cross-section of the Austrian population ( N = 494, collected 2012–2016 from November to March to increase the likelihood of infection with other respiratory viruses, median age 43 [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] ,…”

A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays

“…The biological basis for false-positives is multifactorial, but the influence of the production platform and process-related peculiarities or impurities on protein performance are factors that are often underestimated. While the viral NP is almost always produced in bacteria (12,13), we expressed the spike receptor binding domain in HEK cells, CHO cells, insect cells and plants (4,(14)(15)(16). To find out which of these systems leads to the highest quality and manufacturability of the RBD diagnostic antigen of potentially high demand, we evaluated these production platforms and pre-validated the proteins based on diagnostic performance with a large set of pre-COVID-19 and COVID-19 sera using the Luminex platform.…”

Site-specific steric control of SARS-CoV-2 spike glycosylation

“…In order to define the variability in trimeric recombinant S protein glycosylation and compare recombinant and viral derived S protein we obtained preparations of recombinant S protein from a range of laboratories. These include the Amsterdam Medical Centre (Amsterdam), Harvard Medical School (Harvard), Switzerland, The Wellcome centre for Human genetics (Oxford) and Biological Sciences/University of Texas at Austin (Southampton/Texas) 1,29,[53][54][55][56] . Whilst there are minor differences between the constructs used to produce the S protein the overall design is similar.…”

“…For CHO-based production of a trimeric Spike protein the construct Spike_ΔCter_ΔFurin_2P_T4_His was used 53 . Briefly, based on the CHO-vector pXLG-6 (ExcellGene SA), containing a puromycin resistance marker and optimized expression elements, the variant spike sequence was inserted, containing a scambled furin cleavage site sequence, a two-prolin sequence for blocking the protein in the prefusion form, a 3' T4-trimerization DNA, followed by a hexahistidin tag sequence.…”