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. 2022 Dec 28;663:115034. doi: 10.1016/j.ab.2022.115034

Fingerprinting trimeric SARS-CoV-2 RBD by capillary isoelectric focusing with whole-column imaging detection

Jialiang Du a,1, Gang Wu a,1, Quanyao Chen a,b,1, Chuanfei Yu a, Gangling Xu a, Anhui Liu b, Lan Wang a,∗
PMCID: PMC9794521  PMID: 36586502

Abstract

Because the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is the immunodominant antigen, the S protein and its receptor-binding domain (RBD) are both targets currently to be genetically engineered for designing the broad-spectrum vaccine. In theory, the expressed protein exists as a set of variants that are roughly the same but slightly different, which depends on the protein expression system. The variants can be phenotypically manifested as charge heterogeneity. Here, we attempted to depict the charge heterogeneity of the trimeric SARS-CoV-2 RBD by using capillary isoelectric focusing with whole-column imaging detection (cIEF-WCID). In its nature form, the electropherogram fingerprints of the trimeric RBD were presented under optimized experimental conditions. The peaks of matrix buffers can be fully distinguishable from peaks of trimeric RBD. The isoelectric point (pI) was determined to be within a range of 6.67–9.54 covering the theoretical pI of 9.02. The fingerprints of three batches of trimeric RBDs are completely the same, with the intra-batch and batch-to-batch relative standard deviations (RSDs) of both pI values and area percentage of each peak no more than 1.0%, indicating that the production process is stable and this method can be used to surveillance the batch-to-batch consistency. The fingerprint remained unchanged after incubating at 37 °C for 7 d and oxidizing by 0.015% H2O2. In addition, the fingerprint was destroyed when adjusting the pH value to higher than 10.0 but still stable when the pH was lower than 4.0. In summary, the cIEF-WCID fingerprint can be used for the identification, batch-to-batch consistency evaluation, and stability study of the trimeric SARS-CoV-2 RBD, as part of a quality control strategy during the potential vaccine production.

Keywords: cIEF-WCID, Isoelectric point, SARS-CoV-2, Spike, Receptor-binding domain

Graphical abstract

Image 1

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The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged as a huge threat to human beings early at the turn of the 21st century [1]. Scientists identified that spike (S) protein played as the immunodominant antigen of the virus in a very short period, especially in its native conformation [2,3]. The S protein is a transmembrane glycoprotein with a molecular weight of approximately 150 kDa, forming a prominent homotrimer on the viral particle surface [4]. S protein consists of two functional subunits cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage point), which remain non-covalently bound in the prefusion conformation [5,6]. The S2 subunit is also composed of multiple domains, and its function is mainly to mediate the fusion of virus and host cells. The distal S1 subunit is structurally divided into four distinct domains: N-terminal domain (NTD), receptor-binding domain (RBD), C-terminal domain 1 (CTD1), and C-terminal domain 2 (CTD2) [7] & Shang et al., 2020). RBD is mainly responsible for binding with the receptor, angiotensin-converting enzyme 2 (ACE2), on the surface of host cells, thereby mediating virus infection of host cells [8]. Due to the great selective pressure, the mutation sites mainly exist in the amino acid sequence of S protein, especially the RBD region, which may increase the affinity of the virus to the ACE2 receptor and weaken the neutralizing antibody effect, thereby enhancing the virulence and infectivity of the virus, accelerating the escape of the virus, and reducing the protective effect of the vaccine [9]. Therefore, S protein and RBD are both targets to be genetically engineered for designing the broad-spectrum vaccine against multiple circulating strains of SARS-CoV-2 [30]; [10,11].

Generally, the recombinant S protein and RBD subunit vaccines were manufactured in a cell-based system that supports protein expression [[12], [13], [14], [15]]. The recombinant proteins will show heterogeneity in different expression systems and under different production process conditions, contributed by the post-translational modifications (PTMs) [16,17]. The isoelectric point (pI) is a powerful tool to determine the presence of protein isoforms [18]. Furthermore, it is noted that the isoelectric point is one of the prime keys for understanding various biochemical properties of protein sequences [19,20]. Krebs et al. investigated the pIs of several commercial RBD, S1, S1/S2, and hACE2 proteins [21]. However, these proteins are all His-tagged and/or Fc-tagged, which may influence the peak shapes and uncover the actual pI values of untagged proteins. In this study, we depicted the electropherogram fingerprint of the CHO-expressed trimeric RBDs in its natural formation by using capillary isoelectric focusing with whole-column imaging detection (cIEF-WCID) to provide a tool for batch-to-batch consistency surveillance of the potential SARS-CoV-2 vaccine based on the trimeric RBD subunit.

1. Materials and methods

1.1. Information on the trimeric SARS-CoV-2 RBD

The trimeric SARS-CoV-2 RBD was produced in the Chinese Hamster Ovary (CHO) cell expression system [22,23]. It consists of three protein repeats with 219 amino acids, and the theoretical pI is 9.02. The trimeric RBD solution and matrix buffer (containing histidine and sodium chloride) were stored at 4 °C. Three batches of trimeric RBD solutions were used in this study.

1.2. Sample preparation

The samples were prepared by using the desalting and concentrating process. Briefly, 500 μl sample solution was centrifuged at 12,000 rpm for 10 min in the 10 kDa ultrafiltration tube to a final volume of approx. 50 μl. This concentrated solution will be used to improve the detection of charge variants. The above-concentrated solution was centrifuged again at 12,000 rpm for 10 min in the 10 kDa ultrafiltration tube by pre-adding ultra-pure water to remove the residual salt ingredients. The desalting solution will be used to reduce unwanted interferences.

Three batches of the trimeric SARS-CoV-2 RBDs were used to evaluate the batch-to-batch consistency, reflecting assay stability. The coefficient of variation (CV) of both pI values and area percentage (%Area) were calculated to describe the degree of deviation of the test results. The stability of the samples under different experimental conditions was also investigated, including 1) incubation for 1, 3, and 7 d at 37 °C; 2) pH value from 3.8 to 10.0; and 3) under oxidizing conditions with H2O2 concentrations of 0.003%, 0.009%, and 0.015%.

1.3. The assay conditions

A mixed amphoteric electrolyte was used for separating the charge variants. Briefly, 35 μl 1% methylcellulose solution was mixed with 3 μl of Pharmalytes (pH 3–10) (GE Healthcare) and 1 μl of Pharmalytes (pH 8–10.5) (GE Healthcare), a certain volume of prepared sample solution, 10 μl 200 mM IDA (iminodiacetic acid), 5 μl of Arginine and 1 μl of each pI markers (4.05 and 10.45) (Protein Simple), and ultra-pure water in a 1.5 ml centrifuge tube to make a final volume of 200 μl.

1.4. cIEF-WCID detection

cIEF-WCID was done using the Maurice analyzer (ProteinSimple; USA). It was equipped with a 50 mm × 100 μm internal diameter × 200 μm outer diameter silica capillary column. On the inside the capillary was coated with fluorocarbon. The pre-treated samples were applied to the column as described above. After the column was filled with samples (detected by WCID), apply 1.5 kV DC voltage to pre-focus for 1 min, followed by 3 kV DC voltage for 7 min to achieve IEF separation. After this period, the focus is completed and the electropherogram with fluorescence signal is recorded. Exposure time was optimized as 3 s. After focusing, the 3 kV DC voltage was turned off, and the sample solution in the IEF column was flushed into the waste bottle. Between runs, the column was washed with 0.5% methylcellulose for 1 min.

2. Results & discussion

2.1. Varieties of charge variants were determined in the trimeric SARS-CoV-2 RBDs

This study used cIEF-WCID to analyze the of charge heterogeneity of the trimeric RBDs in their native form. First, the assay conditions were modified including ampholytes, pI markers, and concentration. To truly reflect the charge heterogeneity and the proximity to the theoretical isoelectric point, a mix of the wide range of ampholytes (3-10) and the narrow range of ampholytes (8–10.5) at the ratio of 1:3 was used As shown in Fig. 1 , the large interval of pIs was determined lying in the range from 6.67 to 9.54 (20 peaks). The same phenomenon has also been reported for the unique S proteins from six continents [10]. The theoretical pI values of the trimeric RBDs were calculated as 9.02 by inputting the FASTA protein sequence into the ProtParam tool from the Bioinformatics Resource Portal ExPASy, and thus approximately near the upper edge of the experimentally determined range 6.67–9.54. The above data indicated that the charge heterogeneity is more complex than expected. A major disadvantage is that calculated IEP values do not consider that some amino acids are buried when the protein folds and do not consider any post-translational modifications [24]. Due to the post-translational modifications such as glycosylation, and the presence of a lipid membrane, it is imperative that enveloped viruses have a measured IEP and do not use the calculated value from the spike protein amino acid sequence [24].

Fig. 1.

Fig. 1

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Electropherogram fingerprints of the trimeric RBDs in a mix of the wide range of ampholytes (3-10) and the narrow range of ampholytes (8–10.5) at the ratio of 1:3, corresponding with the pI markers 4.05 and 10.45. The peaks numbered 1–20 represent 20 different charge variants. The fluorescence in the y-axis was used as signal values for better visualization.

In a previous study, the total molecular weight of the trimeric RBDs (approx. 88 kDa) detected by MALDI-TOF MS was larger than the theoretical value (approx.74 kDa) [25]. The difference was partly related to protein glycosylation modification verified by the UPLC-MS analysis. Besides glycosylation modification, other PTMs will also result in charge heterogeneity [26]. Thus, in our next study, the variants will be further analyzed using cIEF-MS [27] to clarify the reasons for the formation of variants and explore the correlation between variants and immunogenicity.

The charge heterogeneity of the trimeric RBD used in this study is also different with previous reported poly-His-tagged RBD proteins [21]. It indicates that His-tag proteins changed the charge heterogeneity of recombinant RBD protein by both in addition of additional His-tag and the resulting alterations in protein structure. The recently published report also concluded that the poly-His-tagged SARS-CoV-2 RBD proteins significantly impair protein immunogenicity against the virus [28].

2.2. Desalting treatment was unnecessary to remove interference from the formulation buffer

Desalting treatment was also used to improve the peak-to-peak resolution and reduce the potential interference of salt components on assay results. Through treatment, the original formulation buffer was replaced by purified water. Results showed that the peak profile of the target trimeric RBD does not change significantly before and after desalting treatment (Fig. 2 ), indicating that the formulation buffer has no significant effect on the fingerprints of the trimeric RBD. Considering that the desalting treatment will be required in the subsequent mass spectrometry analysis, the desalting treatment will be considered here.

Fig. 2.

Fig. 2

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Electropherogram fingerprints of the trimeric RBDs before (Blue) and after (Green) desalting treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.3. Protein concentration should be high enough when determining the pIs of a protein with a variety of charge variants

Compared to other recombinant proteins with a single pI, such as monoclonal antibodies, the trimeric RBD loading must reach the level of mg/ml (i.e., μg per injection) for obtaining electropherograms with good separation and suitable sample-to-noise ratio (S/N) value. In this study, the S/N value is higher, and the peak shape is better visualized when it is concentrated up to 1 mg/ml (the loading amount reaches 20 μg per injection) (Fig. 3 ), which is slightly higher than the previous report 17.85 μg/injection [21]. As mentioned above, the trimeric RBD comprises up to 20 variants. Thus the concentration of individual variants will be one-tenth of the total protein concentration on average. Thus the protein concentration should be considered to avoid unrealistic fingerprints when the studied protein is rich in charge variants.

Fig. 3.

Fig. 3

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Electropherogram fingerprints of the trimeric RBDs before (Green) and after (Blue) concentrated. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.4. The cIEF-WCID fingerprints can reflect batch-to-batch consistency

The demonstration of batch-to-batch consistency is indispensable for the quality control of protein production [29]. For evaluating the reproducibility of the assay and the batch-to-batch consistency, three runs were performed for each batch of the trimeric RBDs. The pI values and %Area were collected and analyzed. Totally 20 peaks were detected per assay (Fig. 4 ), which can be artificially divided into three groups, including <7.0 (group 1), 7.0–9.0 (group 2), and >9.0 (group 3). The intra-batch and inter-batch repetition data showed no more than 1.0% relative standard deviations (RSDs) for each pI point (Table 1 ). In addition, the RSDs of %Area of each peak in groups 1 and 2 were below 20%, but the RSDs of %Area of each peak in group 3 varied unexpectedly. Apart from this, the distribution characteristics of the pIs of the charge variants can still be used as a measure for batch-to-batch consistency and the stability of the production process.

Fig. 4.

Fig. 4

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Electropherogram fingerprints of trimeric RBDs in triple batches (a: batch #1; b: batch #2; c: batch #3; d: batch #1, #2 & #3).

Table 1.

Intra-batch and batch-to-batch consistency (theoretical pI: 9.02, n = 3).

2.4.

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2.5. The SARS-CoV-2 trimeric RBD is stable for 7 d at 37 °C

The trimeric RBD folds independently of other parts of the spike protein, and the folding is robust in different folding environments [30]. The thermal stability of trimeric RBD was detected by differential scanning calorimetry (DSC) with a melting temperature (Tm) value was 47.2°C, indicating good thermal stability [22]. To explore changes in the charge heterogeneity of this vaccine in an accelerated stability test, the matrix buffer and three batches of trimeric RBDs were incubated at 37 °C for 1, 3, and 7 d to evaluate the thermo-stability. As shown in Fig. 5 , the fingerprints of the trimeric RBDs experienced no visible changes even being incubated at 37 °C for 7 d. Moreover, the pI values and %Area of each variant (Table 2 ) also did not shift unacceptably after incubation for 7 d, indicating that this trimeric RBD stayed stable for at least 7 d even when exposed to an ambient temperature of 37 °C, which is very helpful for its use in high-temperature areas or countries.

Fig. 5.

Fig. 5

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Electropherogram fingerprints of the trimeric RBDs after incubation at 37 °C for 1 d (Green), 3 d (Grey) and 7 d (Pink) compared with the untreated one (Blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 2.

pI values and %Area of the trimeric RBDs before and after incubation at 37 °C.

Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
pI Mean 5.85 6.21 6.63 6.75 7.38 7.86 8.15 8.32 8.51 8.64 8.76 8.86 8.95 9.08 9.19 9.27 9.36 9.44 9.49 9.59
RSD% 0.02 0.08 0.05 0.04 0.09 0.12 0.07 0.09 0.10 0.09 0.08 0.08 0.10 0.09 0.07 0.07 0.07 0.05 0.06 0.07
%Area Mean 0.54 1.50 2.20 1.68 6.27 8.56 9.58 8.99 8.22 7.19 5.78 4.89 4.41 3.94 5.21 4.47 5.58 4.96 3.91 2.13
RSD% 14.99 17.22 4.35 10.74 5.27 5.58 2.06 0.74 2.75 3.54 3.71 2.00 4.18 2.76 4.42 5.94 6.61 7.27 7.84 3.17
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2.6. Oxidation does not affect the charge heterogeneity of trimeric RBDs

As proteins react rapidly with many oxidants, they are highly susceptible to oxidative damage, which can generate a broad spectrum of post-translational modifications [31]. Here, H2O2 was used as an oxidant for evaluating the effect of oxidation on the charge heterogeneity of trimeric RBD [32]. It is reported that H2O2 (in the final concentration of 0.003%) can completely oxidize 50 μg monoclonal antibody with a molecular weight of 150 kDa [33]. In this study, H2O2 in the final concentration of 0.003%, 0.009%, and 0.015% were used to oxidize the trimeric RBD in excess. As shown in Fig. 6 and Table 2, even spiked with 0.015% H2O2, the fingerprints, pI values, and %Area (Table 3 ) remain unchanged. It indicates that oxidation with H2O2 does not affect the charge heterogeneity of trimeric RBD, which is completely different from oxidation in monoclonal antibodies.

Fig. 6.

Fig. 6

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Electropherogram fingerprints of the trimeric RBDs after oxidation by using 0.003% H2O2 (Green), 0.009% H2O2 (Blue), and 0.015% H2O2 (Grey) compared with the untreated one (Red). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 3.

pI values and %Area of the trimeric RBDs before and after spiked with H2O2.

Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
pI Ave 5.92 6.24 6.67 6.78 7.34 7.86 8.15 8.32 8.50 8.63 8.76 8.85 8.94 9.08 9.19 9.27 9.35 9.41 9.46 9.55
RSD% 0.26 0.17 0.19 0.22 0.12 0.15 0.13 0.09 0.07 0.10 0.09 0.07 0.09 0.07 0.07 0.08 0.09 0.08 0.07 0.08
%Area Ave 0.29 1.16 2.40 1.37 5.60 7.53 9.54 9.71 9.86 8.78 7.67 6.83 5.72 4.70 4.50 3.61 3.84 3.09 2.57 1.26
RSD% 11.77 9.39 11.00 7.68 1.91 2.01 0.45 1.42 4.10 4.20 1.83 2.07 1.31 3.56 3.38 4.48 2.99 0.87 2.81 3.97
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2.7. pH value

Previous studies indicated that pH value is involved in viral infection and immune escape [[34], [35], [36]]. The SARS-CoV-2 spike employs mobile RBDs to engage the human ACE2 receptor and to facilitate virus entry [34], which may be partially pH-regulated that leading to an increased population of up conformation at physiological pH and a gradual decrease of open conformations on either side of neutral pH [35]. On the other hand, the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibodies due to the low-pH-endosomal pathways [34], indicating that a monoclonal antibody with pan-neutralizing activities to both up and down forms of RBD is needed [36]. In this study, we explored the effect of pH on the stability of the trimeric RBD using changes in the composition of charge variants as indicators. As expected, the formation of charge variants is still unchanged even under extremely acidic conditions (approx.3.5) but completely changed under basic conditions (approx. 10.0) (see Fig. 7 ). This may be the possible reason explaining the variation of %Area among three batches in the pH > 9.0 region (see 3.4).

Fig. 7.

Fig. 7

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Electropherogram fingerprints of the trimeric RBDs after changing the pH value to 3.5 and 10.0 by adding 5 M HCl (Green) and 5 M NaOH (Blue) compared with the untreated one under pH 6.5 (Grey). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3. Conclusion remarks

In our previous study, the cIEF-WCID was applied to finger-printing the HuNoV VLPs in the degeneration forms of monomer VP1 proteins [37]. Unlike HuNoV VLPs, the trimeric SARS-CoV-2 RBDs are not formed by polymerizing three different RBD proteins but folding a triple tandemly expressed protein product [22] & Li et al., 2021). Herein, the charge heterogeneity of the trimeric RBD was assayed and evaluated in the native formation using cIEF-WCID. The applicability of this method for the specific identification, the evaluation of the batch-to-batch consistency of the protein production and the consistency of production process, and the stability study was also confirmed. However, it would be even better for the development of an RBD-based vaccine with broad-spectrum activity against multiple SARS-CoV-2 variants [[12], [13], [14], [15]] if cIEF-MS technology [27] could be used to analyze how exactly these charge variants are formed, and whether/how these charge variants affect the immunogenicity of the candidate trimeric SARS-CoV-2 RBD vaccine.

Ethical approval

This article does not contain any studies with human participants or animals performed by any authors.

Funding

This study was financially supported by the National Key R&D Program of China (No. 2021YFF0600804), sponsored by the Ministry of Science and Technology of the People's Republic of China.

CRediT authorship contribution statement

Jialiang Du: Conceptualization, Methodology, Data curation, Writing – original draft, Writing – review & editing. Gang Wu: Conceptualization, Methodology, Data curation, Writing – original draft, Writing – review & editing. Quanyao Chen: Conceptualization, Methodology, Data curation, Writing – original draft, Writing – review & editing. Chuanfei Yu: Data curation, Writing – review & editing, Funding acquisition. Gangling Xu: Data curation, Writing – review & editing, Funding acquisition. Anhui Liu: Data curation, Writing – review & editing. Lan Wang: Methodology, Investigation, Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Data availability

Data will be made available on request.

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