Characterization of a SARS-CoV-2 spike protein reference material

Abstract

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography–tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet–visible spectrophotometry (UV–Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L−1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL−1 (k = 2), respectively.

Heterologous DNA-prime/protein-boost immunization with a monomeric SARS-CoV-2 spike antigen redundantizes the trimeric receptor-binding domain structure to induce neutralizing antibodies in old mice

“…In our system, we determined higher molecular weights for both the trimeric S2-P ΔTM/GCN4-IZ antigen and the monomeric S6-P ΔTM antigen that differ from the calculated values, i.e., 670 kDa instead of 540 kDa for the trimer and 370 kDa instead of 180 kDa for the monomer. This was not unexpected, since their migration behavior during SEC could be affected among others by their glycosylation state possibly leading to an overestimation of the molecular weight up to 100% ( 54 ), their conformation, and from the molecular weight standard proteins ( 49 , 50 ). Furthermore, the level of glycosylation might vary depending on the antigen itself, the cells used for production, and the level and/or time of expression.…”

Production and Characterization of a SARS-CoV-2 Nucleocapsid Protein Reference Material

“…Symbols w x denote the mass fraction of X in the solution ( X = A, A*, or B), m x ( xy ) is the mass of solution x to prepare the blend xy , and r x is the measured isotope amount ratio of x . Validation of this technique has been demonstrated in interlaboratory comparisons − and has facilitated the development of multiple protein reference materials. ,, …”

“…Reference materials support measurement accuracy and enhanced reliability, and thus can be valuable tools to assess comparability between test kits. We recently characterized a spike protein reference material for use in various applications including lateral flow assays. With regard to nucleocapsid, production of recombinant protein has been reported using a variety of methods in different cell types, including bacterial, insect, and mammalian cells. − Such diverse approaches generate products with variable characteristics, including differences in post-translational modification and oligomeric status, which will likely impact their performance in different assays.…”

“…Reference materials support measurement accuracy and enhanced reliability, and thus can be valuable tools to assess comparability between test kits. We recently characterized a spike protein reference material 11 for use in various applications including lateral flow assays. With regard to nucleocapsid, production of recombinant protein has been reported using a variety of methods in different cell types, including bacterial, insect, and mammalian cells.…”

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

“…In response to the COVID-19 pandemic, an important focus of our research has been the production of recombinant SARS-CoV-2 spike proteins in CHO cells. These proteins, specifically soluble, trimeric, prefusion-stabilized spike ectodomain constructs (Stuible et al, 2021), have been investigated as candidate vaccine antigens and also as reagents for serosurveillance studies (Akache et al, 2021(Akache et al, , 2022Chisanga et al, 2022;Colwill et al, 2022;Rudi et al, 2022;Stark et al, 2022;Stocks et al, 2021). With the frequent emergence of new variants, we have had the opportunity to generate dozens of spike-expressing CHO pools over the last 2 years, which has allowed us to appreciate better the performance and robustness of this expression platform.…”