The material properties of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its proteins are discussed. We review the viral structure, size, rigidity, lipophilicity, isoelectric point, buoyant density and centrifugation conditions, stability against pH, temperature, UV light, gamma radiation, and susceptibility to various chemical agents including solvents and detergents. Possible inactivation, downstream, and formulation conditions are given including suitable buffers and some first ideas for quality-control methods. This information supports vaccine development and discussion with competent authorities during vaccine approval and is certainly related to drug-targeting strategies and hygienics. Several instructive tables are given, including the pI and grand average of hydropathicity (GRAVY) of SARS-CoV-1 and-2 proteins in comparison. SARS-CoV-1 and SARS-CoV-2 are similar in many regards, so information can often be derived. Both are unusually stable, but sensitive at their lipophilic membranes. However, since seemingly small differences can have strong effects, for example, on immunologically relevant epitope settings, unevaluated knowledge transfer from SARS-CoV-1 to SARS-CoV-2 cannot be advised. Published knowledge regarding downstream processes, formulations and quality assuring methods is, as yet, limited. However, standard approaches employed for other viruses and vaccines seem to be feasible including virus inactivation, centrifugation conditions, and the use of adjuvants.

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor‐binding domain (RBD) with His‐ and Fc‐tag, the S1 subunit with His‐tag, the S1/S2 subunits with His‐tag and the human angiotensin‐converting enzyme 2 (hACE2) with His‐tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24–9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36–9.88 (8.91) and for the S1/His it is 7.30–8.37 (7.80). The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS‐CoV‐2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.

SDS gel electrophoresis is a commonly used approach for monitoring purity and apparent molecular mass (Mr) of proteins, especially in the field of quality control of biopharmaceutical proteins. The technological installation of CE-SDS as the replacement of the slab gel technique (SDS-PAGE) is still in progress, leading to a continuous improvement of CE-SDS instruments. Various CE-SDS instruments, namely Maurice (CE-SDS/CE-SDS PLUS) and Wes by ProteinSimple as well as the microchip gel electrophoresis system LabChip® GXII Touch TM HT by PerkinElmer were tested for precision and repeatability compared to SDS-PAGE (Bio-Rad). For assessing these quality control parameters, standard model proteins with minor post-translational modifications were used. Overall, it can be concluded that the CE-SDS-based methods are similar to SDS-PAGE with respect to these parameters. Quality characteristics of test systems gain more significance by testing proteins that do not behave like model proteins. Therefore, glycosylated proteins were analyzed to comparatively investigate the influence of glycosylation on Mr determination in the different instruments. In some cases, high deviations were found both among the methods and with regard to reference values. This article provides possible explanations for these findings.

The development of capillary electrophoresis, especially CE‐SDS devices, has led CE‐SDS to become an established tool in a wide range of applications in the analysis of biopharmaceuticals and is increasingly replacing its method of origin, SDS‐PAGE. The goal of this study was to evaluate the comparability of molecular weight (MW) determination especially by CE‐SDS and SDS‐PAGE. For ensuring comparability, model proteins that have little or no posttranslational modifications and an IgG antibody were used. Only a minor influence of sample preparation conditions, including sample buffer, temperature conditions, and different reducing agents on the MW determination were found. In contrast, the selection of the MW marker plays a decisive role in determining the accurate apparent MW of a protein. When using different MW markers, the deviation in MW determination can exceed 10%. Interestingly, CE‐SDS and 10% SDS‐PAGE hardly differ in their trueness of MW determination. The trueness in relation to the reference MW for each protein was calculated. Although the trueness values for the model proteins considered range between 1.00 and 1.11 using CE‐SDS, they range between 0.93 and 1.03 on SDS‐PAGE, depending on the experimental conditions chosen.

Two imaged capillary isoelectric focusing methods were developed to provide insight into the quality and stability of messenger ribonucleic acid (mRNA) vaccines, specifically, mRNA encapsulated in lipid nanoparticles (LNPs). A variety of stressed and lipid composition‐modified samples were measured and detected by their UV absorption. The results were supported by the data of an encapsulation assay and particle sizing. One method, using 9 M urea as an additive, shows two broad and jagged peaks in which the peak shape offers detailed information. The summed peak area of both peaks showed RSDs from 2% to 8% when one batch was measured in triplicate and apparently depends on the size of the LNPs. In the second method, a combination of 5.5 M urea and 2 M N‐ethylurea was used. This method is characterized by a high repeatability of the isoelectric point (pI, <0.5%). The repeatable peak area (RSD of 2%–7%) correlates linearly with the mRNA content, which also applies to the first method, and added stress is evident by the change in pI and peak area. Furthermore, experiments with the addition of a fluorescent dye were performed (fluorescence detection), which tremendously increased the sensitivity of the methods. Both methods can be used to characterize the stability of mRNA‐loaded LNPs, for example, when investigating various storage times at different temperatures and freeze–thaw cycles, as well as the ability of the methods to distinguish lipid compositions and measure batch‐to‐batch variability.