TGA发布Pfizer与Moderna COVID-19 mRNA疫苗质量保证与控制检测结果

Samples

Batch-release testing for Comirnaty® and Spikevax™ mRNA COVID-19 vaccines in this report occurred between 9 February 2021 to 26 July 2024 (however, batch release of these products is ongoing). Samples from different batches and formulations of Comirnaty® (Pfizer Australia Pty Ltd) and Spikevax™ multidose vials and pre-filled syringes (Moderna Australia Pty Ltd) were used in this study. Samples were stored in accordance with their approved storage conditions.

Test procedures

Table 2 provides a list of analytical test methods used to test mRNA vaccines as a part of the TGA batch-release^19-22. Researchers seeking to perform the same assays should request the complete manufacturer's testing protocols directly from each manufacturer due to the commercial confidentiality of some of the testing protocols.

* This is not a test performed for release of the batch. Testing was performed on a subset of batches after their release.

RNA identity - RT-qPCR

To confirm that the correct RNA species is present in the samples, both Comirnaty® and Spikevax™ vaccines were tested by RT-qPCR amplification using sequence-specific primers and probes. RNA was extracted from samples using the QiAMP RNA Viral Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reactions were performed using the QuantStudio 3 RT-PCR System (Thermo Fisher Scientific, Massachusetts, U.S.), with a commercially available one-step RT-PCR kit. For each assay, nuclease-free water was used as a negative control, and a drug substance (DS) reference material was used as a positive PCR control. Negative extraction controls were prepared using nuclease-free water subjected to the RNA extraction process. Where applicable, positive extraction controls were prepared using a reference batch of vaccine drug product (DP). All samples and controls were tested using three technical replicates with product-specific thermocycling conditions, including a reverse transcription step followed by multiple cycles of melting and annealing. The mRNA sequence identity was confirmed by sample amplification in all test replicates, with cycle threshold (C_T) values below a product-specific cut-off. Assay suitability was determined by control performance: all positive control material replicates were required to exhibit C_T values below the cut-off, and all negative control replicates were required to exhibit either no amplification or C_T values greater than the cut-off value. A sample was accepted if all three replicates produced the consistent results.

RNA identity/ratio - RPIP-HPLC - Spikevax™

This assay confirms the identities and relative content of the two mRNA species present in the bivalent vaccine formulation. To confirm the identity and ratio of RNA in Spikevax™ bivalent products, samples were tested by RPIP-HPLC using the Waters Acquity H-Class ultra-performance liquid chromatography (UPLC) system (Waters, USA). RNA samples were denatured in a deformulation buffer to disrupt secondary RNA structures using a commercially available kit (Zymo Research, USA). RNA was purified by spin-column with a wash buffer, RNase-free water and centrifugation at 15,000 x G for 1 minute. For digestion, prepared RNA samples and template-guided RNase H digestion mixture were incubated at 65°C for 30 minutes followed by the addition of pre-prepared quench solution and DIEAA solution according to the manufacturer’s instructions. Resulting RNA fragments were separated by RPIP-HPLC using the manufacturer’s recommended gradient conditions with a flow rate of 0.45 mL/min. The separated RNA fragments were detected in-line by UV absorbance at 260 nm and peaks corresponding to known RNA standards were used to determine RNA identities. The RNA ratio for samples was determined by comparing the sample RNA % peak area against the % peak area of RNA product standard constants. Acceptance criteria were set as % interference ≤ 5%, % relative standard deviation (RSD) of reference standard RNA peak area ≤ 5%, % RSD of reference standard RNA peak retention time ≤ 5%, % recovery of each bracketing standard peak area as compared to the average peak area of reference standard RNA peak of the reference standard 90 – 110%.

Lipid content - HPLC-CAD

This assay confirms the correct type and amount of each of the four lipids that comprise the LNP.

For Comirnaty®, identification and quantitation of ALC-0159, cholesterol, ALC-0315 and DSPC were achieved using the Waters Acquity H-Class UPLC system (Waters, USA) with a charged aerosol detector (CAD) (Thermo Fisher Scientific, Massachusetts, U.S). The mobile phase consisted of a combination of aqueous (e.g., water or buffered solutions) and organic solvents to minimise background noise and ensure compatibility with the CAD. Standard stock preparations were made using an alcohol solution and the lipid reference standards, with controls prepared using a dilution of the stock solution based on the manufacturer’s instructions. DP samples were diluted in methanol to ensure lipid concentrations met the ranges specified in the standard curves specified by the manufacturer. Samples were analysed using HPLC-CAD. Sample acceptance criteria were set as % RSD ≤ 10 as well as % relative retention time (RRT) for each lipid according to the acceptance specified by the manufacturer.

For Spikevax™, identification and quantitation of PEG 2000 DMG, cholesterol, SM-102 and DSPC was achieved using the Waters Acquity H-Class UPLC system (Waters, USA) with a CAD detector (Thermo Fisher Scientific, Massachusetts, U.S). The mobile phase consisted of a combination of aqueous (e.g., water or buffered solutions) and organic solvents to minimise background noise and ensure compatibility with the CAD. Standard stock preparations were made using an alcohol solution and the lipid reference standards, with controls prepared using a dilution of the stock solution based on the manufacturer’s instructions. DP samples were diluted in ethanol to ensure lipid concentrations met the ranges specified in the standard curves specified by the manufacturer. Samples were analysed using HPLC-CAD. Sample acceptance criteria were set as % RSD ≤ 10 as well as % RRT for each lipid according to the acceptance criteria specified by the manufacturer.

mRNA content and encapsulation efficiency

For Comirnaty® samples, to confirm the correct content of mRNA is present in each dose, quantitation of total and % encapsulated RNA in LNP was achieved using RiboGreen fluorescence (Thermo Fisher Scientific, Massachusetts, U.S.) with the SpectraMax M5e spectrophotometer system (Molecular Devices, USA). A standard curve was prepared with DS mRNA based on the manufacturer’s instructions using qualified RNA DS reference material. DP samples consisting of encapsulated RNA were compared against a DS standard curve to measure the amount of free RNA (without non-ionic surfactant) and total RNA (with non-ionic surfactant added to release the encapsulated RNA from the LNPs). RiboGreen was added to bind to RNA and generate a fluorescence signal that was measured to determine the amount of free RNA and total RNA. The amount of encapsulated RNA was calculated by subtracting the free RNA content from the total RNA, and the encapsulation efficiency was calculated as the ratio between encapsulated and total RNA, expressed as a percentage. Assay acceptance criteria included a minimum goodness-of-fit for each standard curve and a maximum variability in fluorescence measurements for the replicates of each standard curve dilution.

For Spikevax™ samples, the amount of total mRNA present in the vaccine dose was determined by AEX-HPLC. Free (unencapsulated) mRNA content was measured by a spectrophotometric assay and used to determine the encapsulation efficiency of Spikevax™ vaccine samples in accordance with the manufacturer’s instructions. Encapsulated mRNA was calculated as the difference between total mRNA as measured by AEX-HPLC (described below) and free mRNA content. Encapsulation efficiency was calculated as the ratio between the encapsulated and total mRNA content, expressed as a percentage. The assay involved the complexation of a dye with free mRNA in vaccine test samples and measurement of the associated change of absorbance. Vaccine test samples were prepared according to the manufacturer’s instructions to a constant concentration and mixed with stock solution before absorbance measurement with the M5e spectrophotometer. Baseline absorbance was determined using the stock solution mixed with vaccine formulation buffer without mRNA. The shift in absorbance is proportional to the free RNA content, which was calculated by applying an empirically determined constant relating to absorbance changes and RNA concentration, as provided by the manufacturer.

RNA integrity - capillary gel electrophoresis

RNA integrity of Comirnaty® vaccines was assessed using capillary gel electrophoresis (5300 Fragment Analyzer System, Agilent, California, U.S). According to the manufacturer’s specifications, samples and reference materials were prepared by incubation with a non-ionic surfactant and alcohol solution to release RNA from LNPs. The released RNA was denatured at 70°C for 2 minutes prior to separation by capillary gel electrophoresis. The analysis was performed using the commercially available Standard Sensitivity (SS) RNA kit (DNF-471-1000, Agilent, California, USA). Separation of RNA according to size was achieved by the application of an 8 kV voltage through the capillary array. The inclusion of a fluorescent intercalating dye in the running gel allowed for detection of RNA fragments as they passed before a LED light source linked to a charge-coupled device (CCD) detector. The migration of RNA through each capillary was normalised using an internal marker, and fragment sizes were determined using an RNA ladder. Samples were tested in technical triplicates, with a blank as a negative control and a reference material DP as a positive control. RNA integrity was reported as the % area of the main peak compared to total RNA present in the sample. The system suitability and assay acceptance criteria involved assessing the specificity and range of the RNA ladder and positive control.

RNA purity - RPIP-HPLC

For Spikevax™, the purity of mRNA in monovalent and multivalent final drug product formulations was determined by RPIP-HPLC with UV detection at 260 nm. The method separates mRNA size variants based on their length by gradient elution using ion-pairing agents in the mobile phases using Waters Acquity H-Class UPLC system (Waters, USA). The purity is reported as the relative area % of the main peak, which represents full-length mRNA.

RNA content - AEX-HPLC

For Spikevax™, quantitation of total RNA content for final drug product samples was achieved using AEX- HPLC with UV detection at 260 nm. Agilent 1260 Infinity II HPLC system (Agilent Technologies, USA) was used. The results were reported as the concentration of total mRNA present in the formulations.

LNP size and polydispersity - DLS

Analysis of hydrodynamic particle size and the polydispersity index PDI of Comirnaty® and Spikevax™ vaccines was achieved using DLS with a Malvern Zetasizer Pro Blue (Malvern Panalytical, UK). NIST Traceable Nanosphere Particle Size Standards (Thermo Fisher Scientific, USA) were used as reference standards to ensure accurate particle size measurement. All work was performed in a biosafety cabinet to ensure a dust-free environment. Mean hydrodynamic diameter and PDI were calculated by the instrument software. Reference standards for Comirnaty® and Spikevax™ samples were diluted to the appropriate concentrations before measurement. For both products, system suitability, assay, and sample acceptance criteria were determined by the accuracy and precision of the measurements for the standards and/or samples.

Endotoxin - LAL

The test for bacterial endotoxins was conducted in accordance with the methods specified in the European Pharmacopoeia 2.6.14 and United States Pharmacopoeia <85>.

In vitro expression

A cell-based in vitro expression (IVE) assay was used to confirm cell transfection by the LNPs, and translation of the mRNA sequence into the spike protein. HEK239T (Human embryonic kidney 239T cells) (ATCC, Washington, DC, USA), were incubated with Comirnaty® and Spikevax™ products according to Pfizer’s testing method. For each assay, nuclease-free water and a DP reference material were used as negative and positive controls, respectively. Post incubation, cells were harvested and stained with a cell viability dye, followed by a fixation step. To detect the spike protein, the fixed cells were incubated with an antibody specific to the SARS‑CoV‑2 spike protein, followed by incubation with a secondary antibody conjugated to phycoerythrin. Immunofluorescent cells were detected on a BD FACSCanto II (Becton Dickinson, New Jersey, USA). Assay acceptance was determined by the number of analysed cells, the percentage of viable cells, the precision of the technical replicates, and the results from the controls. Sample acceptance was determined by the percentage of single, viable fluorescent cells.

Monitoring for residual DNA content

Testing commenced in 2023 using samples from the two most recently supplied COVID-19 vaccine strains (bivalent original +BA.4-5 and monovalent XBB1.5) of both Comirnaty® and Spikevax™ products. Testing was conducted in accordance with the standard test procedures for residual deoxyribonucleic acid (DNA) quantitation as described in the European Pharmacopoeia 2.6.35.

Statistical analysis

The mean, along with the standard deviation and percent coefficient of variation [(standard deviation/mean) x 100] was used as the primary method of analysis to evaluate variability within the test results and assess batch-to-batch consistency.