1. Introduction
In 2019, the COVID-19 virus ravaged the world, and the outbreak of the pandemic affected every aspect of people’s lives [
1]. As a great invention, vaccines are important in preventing the transmission of viruses and reducing the risk of infection [
2]. There were several kinds of vaccines that dominated during the epidemic, such as inactivated vaccines, recombinant vaccines and mRNA vaccines [
3,
4]. Inactivated vaccines, due to their high safety and mature production technology, comprised the highest number of vaccinations during the COVID-19 epidemic. Among these kinds, inactivated vaccines and recombinant vaccines both stimulate human bodies to produce antiviral antibodies by immune stimulation through the target proteins of the virus; in the case of COVID-19, this is the S proteins [
5]. Thus, the determination of the S protein is of great significance for the quality control of vaccine products. Meanwhile, the residual of host cell proteins (HCPs) is inevitably introduced in vaccine production process. These HCPs can cause unknown immune responses which may induce serious toxicity and side effects to immune recipients [
6]. Therefore, it is necessary to determine HCP residues in such vaccine products.
Generally, the detection of S proteins and HCP residues relies on enzyme-linked immunosorbent assay (ELISA)-based assays [
7]. However, there are still some limitations in the measurement. Firstly, for target protein analysis, this method can only make a general quantitation which cannot provide additional subtype information of the target proteins at the molecular level. Second, the absolute quantification of S proteins by ELISA-based methods needs to rely on the usage of target protein standards. For the quality control of vaccine production in industry, quantitative control between batches is usually conducted according to a specific batch of reference product; thus, it is difficult to achieve comparisons between different companies’ products. In addition, the ELISA-based methods cannot cover all kinds of HCPs, resulting in the underestimate of HCP content [
8,
9]. Moreover, the ELISA-based methods cannot simultaneously analyze the content of S proteins and HCPs at the same time, which increases the procedures and duration of detection.
Liquid chromatography–mass spectrometry (LC-MS) is an analytical technology with high selectivity, high sensitivity and high-throughput features [
10,
11,
12]. Since corresponding antibodies are not required, LC-MS has the advantage of rapid method development and is therefore more suitable for emergency situations such as pandemics, avoiding risks such as poor antibody specificity. The advancement of LC-MS in proteomics enables the accurate qualitative and quantitative analysis of proteins, especially in complex samples [
13,
14,
15,
16]. Based on the analysis capacity of LC-MS at the molecular level, it is very suitable for the profile of related proteins in bio-macromolecular samples such as vaccines. Due to its universal capacity, the LC-MS method can be used for global monitoring during production (e.g., virus/host cell metabolism, MOI optimization, etc.). Not only the same vaccines produced in different batches or by different companies, but also the vaccines of different pathogens and the different kinds of vaccines (inactivated, recombinant and so on) can be analyzed in one set of methods.
Herein, we developed a nano LC-MS method for the simultaneous analysis of S proteins and HCP residues of COVID-19 inactivated vaccine products and recombinant vaccine products. Based on this, the content of S proteins and HCP residue could be quantified according to the spiked standard proteins. Additionally, different batches, different companies and different kinds of vaccine products were successfully compared, which may be valuable to improving the manufacturing technique of vaccines in the future.
2. Materials and Methods
2.1. Vaccine Samples and Standard Substance
Each of three batches of COVID-19 (wild-type strain) inactivated vaccine final bulk from two companies (company A and B) were marked with AW1, AW2, AW3, BW1, BW2 and BW3, respectively. Three batches of COVID-19 (Gamma strain) inactivated vaccine final bulk from company A were marked with AG1, AG2 and AG3, respectively. Each of three batches of COVID-19 (Omicron strain) inactivated vaccine final bulk from two companies (company A and B) were marked with AO1, AO2, AO3, BO1, BO2 and BO3, respectively. Each of three batches of COVID-19 recombinant protein vaccine final bulk from company C were marked with CW1, CW2, CW3, CB1, CB2, CB3, CD1, CD2, CD3, CO1, CO2 and CO3, respectively (W represents wild-type strain, B represents Beta strain, D represents Delta strain and O represents Omicron strain). One batch of hepatitis A virus inactivated vaccine final bulk from company F was marked with F. One batch of Haemophilus infiuenzae type b conjugate vaccine final bulk from company G was marked with G. Pierce intact protein standard mix was purchased from ThermoFisher Scientific (Waltham, MA, USA).
2.2. Reagents and Materials
Dithiothreitol (DTT) and 2-iodoacetamide (IAA) were bought from Sigma-Aldrich (St. Louis, MO, USA). Ammonium bicarbonate was bought from Acros Organics (Fair Lawn, NJ, USA). RapiGest SF and trypsin were purchased from Waters Corporation (Milford, MA, USA) and Promega (Madison, WI, USA), respectively. Formic acid (LC-MS grade) and acetonitrile (LC-MS grade) were respectively bought from ThermoFisher Scientific (Waltham, MA, USA) and Merck (Darmstadt, Germany). A 2019-nCoV Spike protein ELISA kit was bought from Sino Biological (Beijing, China). Ultrapure water (18.2 MΩ·cm) was purified through a Millipore Milli Q water purification system and was used in all experiments.
2.3. Instruments
An Easy-nLC 1200 (ThermoFisher Scientific, Waltham, MA, USA), Orbitrap Eclipse mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) and centrifuge (ThermoFisher Scientific, Waltham, MA, USA) were used in this study.
2.4. Enzymolysis of Vaccine Samples and Standard Substance
First, 10 µL of Pierce intact protein standard mix solution (200 μL of 50 mM NH4HCO3 buffer was used to dissolve one bottle of Pierce intact protein standard mix) and 110 µL of RapiGest SF (1 mg/mL, dissolved in 50 mM NH4HCO3 buffer) were added to a 100 µL vaccine sample. After being gently mixed, the mixture was kept at 60 °C for 15 min. Then, 10 µL of DTT (0.5 M, dissolved in 50 mM NH4HCO3 buffer) was added to the above mixture and incubated at 60 °C for another 60 min. After cooling down to room temperature, 10 µL of IAA (1 M, dissolved in 50 mM NH4HCO3 buffer) was added and kept in the dark for 30 min at room temperature. After that, all of the mixture was transferred to a 3K ultrafiltration device and centrifuged for 10 min under 21,000× g of centrifugal force. Then, 100 μL of NH4HCO3 buffer (50 mM) was added to run a new round of the ultrafiltration process. This process was performed twice. Subsequently, 100 μL of NH4HCO3 buffer (50 mM) and 10 μL of trypsin (0.2 mg/mL, dissolved in 50 mM NH4HCO3 buffer) were added to the upper chamber of the above ultrafiltration device and gently mixed. After enzymolysis at 37 °C overnight, 3 μL of formic acid was added, followed by centrifugation under 21,000× g of centrifugal force for 10 min. Finally, the filtrate was collected for LC-MS analysis.
For control experiment, initially, 10 µL of Pierce intact protein standard mix solution (200 μL of 50 mM NH4HCO3 buffer was used to dissolve one bottle of Pierce intact protein standard mix) was mixed with 110 µL of RapiGest SF (1 mg/mL, dissolved in 50 mM NH4HCO3 buffer). The following process was the same as for the vaccine samples.
2.5. Chromatography
An Acclaim PepMap
TM 100 C18 (75 μm × 20 mm, 2 µm) (ThermoFisher Scientific, Waltham, MA, USA) was used as a trap column and an Acclaim PepMap
TM 100 C18 (75 μm × 250 mm, 2 µm) (ThermoFisher Scientific, Waltham, MA, USA) was used as an analytical column. For mobile phases, 0.1% formic acid in water was used as phase A and 0.1% formic acid in 80% acetonitrile as phase B. The flow rate was 0.3 μL/min. The volume of sample injection was 1 μL. The details of the eluent gradient are shown in
Table 1.
2.6. Mass Spectrometry
Nanospray source (NSI) was used. The mode was positive ion; capillary voltage was 2.0 kV; capillary temperature was 300 °C; scan range (m/z) was 350–1800; and orbitrap resolution was 120,000 (MS), 30,000 (MS/MS).
2.7. LC-MS Data Analysis
Database searching was performed on Proteome Discoverer. Label free quantification was performed with the match-between-runs function. Raw protein abundances were normalized using the added six-protein standard. Differential regulated proteins were defined as more than two folds of change of abundance and a p-value less than 0.05 (student’s t-test).
4. Conclusions
In this work, we developed a nano LC-MS method for the simultaneous analysis of S proteins and HCP residues of COVID-19 inactivated vaccine products and recombinant vaccine products. By using this novel method, thousands of proteins, both known and unknown, can be qualitatively and quantitatively analyzed with only one injection. Based on this, the proteomes of 15 batches of COVID-19 inactivated vaccine samples from two companies and 12 batches of COVID-19 recombinant protein vaccine samples from one company were successfully analyzed, which provided a significant amount of valuable information. Samples in different batches or from different companies can be systematically contrasted in this way, offering powerful supplements for existing quality standards. In the future, this method can also be used for global monitoring during production (e.g., virus/host cell metabolism, MOI optimization, etc.), which may provide more supporting information for process improvement. This strategy paves the way for profiling proteomics in complex samples and provides a novel perspective on the quality evaluation of bio-macromolecular drugs.