The development of vaccines, from research and development to early preclinical studies, to commercial production, testing, and batch release, involves extensive analysis and testing to ensure the effectiveness and safety of vaccine products. The biological analysis in vaccine development faces challenges in terms of sample matrix, analyte types, and workflow efficiency. For example, vaccine samples used for analysis vary greatly, and the range of analytes is wide, including polysaccharides, proteins, and peptides, all of which must be detected in different matrices. In addition, the new generation of vaccines involves an increasing number of antigens, such as the HPV (human papillomavirus) vaccine, which has multiple types including bivalent, quadrivalent, and nonavalent, greatly increasing the complexity of testing. Immunoassay is commonly used to determine the titer, purity, and efficacy of vaccines, as well as their immunogenicity in animals and humans. ELISA immunoassay based on porous plates has become one of the gold standard techniques in vaccine bioanalysis, including quantitative antigen epitopes, evaluation of impurity levels, and monitoring of titer and immunogenicity in vaccine development. However, the increasingly urgent project schedule has prompted researchers to seek improvements in vaccine immunoassay performance, including:

Use smaller amounts of precious reagents such as vaccine candidate molecules and samples from small experimental animals

The detection method can conveniently process samples of serum and plasma, aluminum containing adjuvants, and oil emulsion adjuvants

Reduce hands-on operation time

Reduce the development time of detection methods

Gyrolab ® The system provides a solution for these needs and offers many additional benefits in vaccine development. The open platform of Gyrolab supports rapid detection and development of various analytes, sample types, and formulations, and provides ready to use reagent kits for various applications. Gyrolab at Bioaffy ™ Nano upgraded immune detection in microfluidic CD minimizes interference from multiple matrices and has low sample and reagent consumption, which can meet the needs of the latest generation of vaccines and small experimental animal experiments; High throughput and automation modes enable parallel analysis of multiple analytes, greatly reducing hands-on time and providing data quickly to meet challenging project timelines and faster decision-making. The Gyrolab software workstation complies with the requirements of 21 CFR Part 11, and its developed testing methods and data meet regulatory environmental requirements, and can be smoothly transferred between pharmaceutical factories and CROs.

　　This article introduces several application cases of using the Gyrolab system to improve vaccine development performance and efficiency, in order to illustrate the advantages of the Gyrolab system in practical use.

Case 1:Detect antigen-specific antibodies in serum within 1.5 hours

Detecting the quantity and quality of antibodies produced by memory B cells (MBC) in vaccinated subjects is key to understanding how to induce lifelong protective antibodies. The relative frequency of human peripheral blood antigen-specific memory B cells is generally determined by serial limiting dilution assay (sLDA) and ELISA analysis of cell culture supernatant. However, this method is time-consuming, labor-intensive, and requires a large amount of experimental materials. Therefore, Novartis' vaccine and diagnostic development researchers have transferred ELISA immunoassay to the Gyrolab system to increase production. The goal of this study is to combine Neisseria antigen with three adjuvants and inoculate mice, and determine the IgG subtypes produced by mouse memory B cells through immune detection. Researchers tested two forms of detection:

　　Direct Assay,

Used to evaluate: ① the ability of biotinylated antigens to bind to the streptomycin affinity column in Gyrolab Bioaffy microfluidic CD and capture specific IgG; ② Sensitivity of detecting antigen-specific antibodies in cell culture supernatant; ③ Evaluate the affinity of antibodies to specific antigens through binding curves in the Gyrolab Viewer.

　　Reverse Assay,

Used to evaluate the ability of Alexa fluorescently labeled antigen detection to bind specific IgG to CD affinity columns.

Direct Assay

Figure 1. Gyrolab detection and ELISA identified identical positive and negative samples in the supernatant of B cell culture of mice inoculated with Neisseria gonorrhoeae. The Y-axis represents the antibody titers detected by Gyrolab, and positive samples identified by ELISA are marked with green boxes.

Figure 2. The gyro lab direct detection method can simultaneously detect antibodies belonging to different IgG subtypes in a few microliters of sample.

Compared with ELISA, antigen-specific antibody detection on the Gyrolab system has many advantages, as only a few microliters of sample are needed to analyze the quality of antibody reactions, i.e. the relative number of IgG subtypes.

Case 2:Reagent screening - Comparison of vaccine batches for release testing

The biological analysis of multivalent vaccines is becoming increasingly complex, requiring the development of a large number of detection methods. Because multivalent vaccines contain multiple antigens, release testing methods must be developed for each antigen component to ensure dosage and efficacy, so the number of tests for each product must be multiplied by the number of antigens. Merck's vaccine analysis and development team has developed a highly flexible Gyrolab detection method that can minimize the development of multivalent vaccine detection methods and reagent consumption (such as labeled detection antibodies). This method is based on biotinylated goat anti mouse IgG immobilized on a streptavidin column of Gyrolab Bioaffy CD. After adding analyte specific capture antibodies (such as mouse alpha PS mAb) to the sample, a second analyte specific antibody (such as rabbit alpha PS pAb) is used to detect the captured analyte, and finally Alexa Fluor is used ® 647 labeled donkey anti rabbit antibodies were subjected to fluorescence detection (Figure 3). This 5-layer "double sandwich" detection form does not require labeling of reagents specific to the analyte, and analyte specific reagents can be easily replaced, so it only requires minimal development to be applied to the detection of multivalent polysaccharides and multivalent protein vaccines, as well as batch differences in anti serum.

Figure 3. 5-layer "double sandwich" Gyrolab detection form. PS: Polysaccharide analyte

The Gyrolab assay was used to compare two batches of anti serum for three antigen tests. By using the Gyrolab Viewer software module, the binding reaction of each data point on the affinity column in CD microchannels can be visualized, making the data comparable to more time-consuming efficacy curves (Figure 4).

Figure 4. Panel A: Comparing batches of anti serum against antigens A, B, and C using fluorescence intensity data (left) and reaction curves (right). Panel B: The Gyrolab Viewer binding intensity curve image of antigen B shows that the fluorescence intensity on the column of the new batch is lower, and the peak tailing is increased, indicating that the antibody affinity of this batch is lower than that of the old batch.

Although the two batches showed similar performance against antigen A, the old batch performed better against antigen B, with a larger peak area in the Gyrolab Viewer combined with the spectrum, and a higher signal-to-noise ratio during titration. The new batch shows a clear fluorescent tail on the column, indicating a lower affinity for the antigen. For antigen C, both batches of serum showed obvious tailing, with the old batch showing more peak tailing. During titration, the background of the old batch is relatively high and the signal-to-noise ratio is low. Therefore, the visualization of Gyrolab Viewer combined with spectra, reaction curves, and signal-to-noise ratio provides valuable insights into understanding serum batches.

The Merck team has concluded that the Gyrolab system provides valuable statistical and graphical data that, when evaluated together, is consistent with detection data based on porous plates, as well as additional benefits such as fast turnaround time, high-throughput, and the ability to visualize binding strength.

Case 3:The dynamic range of antigen titer quantification has been expanded to 4 logs

Vaccine development companies need to expand the dynamic range of immunoassays to measure glycoprotein antigens adsorbed on aluminum adjuvants. The researchers transferred ELISA to the Gyrolab system, expanding the dynamic range from 5-300 mIU/mL to~0.6-40000 mIU/mL. Gyrolab detection simultaneously extended the upper and lower limits of quantification.

Figure 5. Gyrolab provides a powerful detection method that greatly expands the dynamic range when measuring antigen titers.

Case 4:Vaccine efficacy trials supporting formula development

Evaluating the consistency of efficacy between batches is an important component of vaccine quality control. Efficacy testing can be used for stability studies, validation and optimization of fermentation and purification processes, comparison of new and old facilities, and evaluation of the effectiveness of formula or adjuvant changes.

Merck compared the performance of the Gyrolab system and Luminex assay in mouse efficacy tests. This study involves in vivo immunity in mice by diluting normalized antigen samples to a concentration range of 6-12 doses. The effective dose (ED50) is determined based on the maximum dose converted by 50% of mouse serum in each treatment group. Obtaining an ED50 value requires up to 220 mouse serum samples, so this tedious task requires a large number of small samples. Gyrolab detection has been proven to be reliable and robust, with sensitivity and reproducibility (CV<10%) comparable to Luminex assay (R2=0.97), but with shorter operating time and faster turnaround time.

Figure 6. Gyrolab detection has high repeatability, CV<10%

Figure 7. The results obtained from the immune detection of Gyrolab and Luminex are comparable