Immunoassays help in sharpening a potent healthcare tool
Vaccines are key in the prevention of infectious disease and as immunotherapies for other conditions including certain cancers and Alzheimer’s disease. The development of vaccines has generally been a slow and painful process, taking 10–15 years and with a high attrition rate compared to standard drugs. The recent record-breaking speed of vaccine development aimed at fighting the ongoing COVID-19 pandemic represents a paradigm shift, but the basic demands on vaccine R&D and bioprocess remain the same — vaccines must be shown to be safe, pure, potent, and effective for regulatory approval.
Immunoassays deliver critical data to support vaccine R&D and process development. The need to meet ever more pressing deadlines means that fast, efficient and flexible immunoassay platforms such as Gyrolab® system are at a premium. Gyrolab system has become established in the majority of biopharmaceutical companies and contract research organizations (CRO) worldwide, including a broadening application in vaccine development and manufacturing
Conventional moves towards next generation
Conventional vaccines reduce the risk of illness by inducing a protective immune response by exposing the recipient to antigens presented in a number of ways:
- Inactivated, but previously virulent, micro-organisms
- Live, attenuated microorganisms (mostly viral, some bacterial) that also include pathogens of other animals that cause mild or no disease in the recipient — heterologous vaccines
- Protein-based vaccines e.g. toxoids, subunits, and protein-polysaccharide conjugates
Limitations in these approaches have led to the development of a number of next-generation vaccine strategies that offer the promise of more effective, rational and rapid vaccine development, including:
- DNA vaccines involving the insertion and expression of viral or bacterial DNA in human or animal cells, triggering immune system recognition
- RNA (mRNA) vaccines comprising viral RNA packaged within a carrier such as lipid nanoparticles
- Antigen-presenting cells (APC)
- Viral vectors that deliver synthetic genetic material from the pathogen into the recipient’s cell and instruct it to make a viral protein that stimulates an immune response
- Cancer vaccines based on peptide neoantigens
These new approaches offer a number of significant advantages, particularly when rapid development is needed in the case of a pandemic such as COVID-19. For example, six COVID-19 vaccine candidates based on adenovirus vectors are in clinical trials, of which one has shown high efficacy in Phase III trials. Added to that, two RNA-based vaccines are also showing high efficacy. Such developments only 10 months after the SARS-CoV-2 genome was published in January 2020 are astonishing and demonstrate how next-generation technology could revolutionize protection against infectious disease.
Meeting the need for speed and efficiency in vaccine bioanalysis
The rapidity of COVID-19 vaccine development, together with the threat of future pandemics, may well represent a fundamental change in the way vaccines are developed. This need for speed and efficiency can be expected to increase the demand for ever more effective bioanalytical tools, including platforms to run the immunoassays that play such a fundamental role in supplying reliable data for vaccine R&D. These tools also underpin the efforts to streamline process development, which includes a move toward data-driven quality by design (QbD), real process understanding, in-process testing using process analytical technologies (PAT), feedback control, and continuous improvement over the life cycle of each product.
High-performance immunoassays in vaccine development and bioprocess
Vaccine development involves a large number of tests to support vaccine R&D, from early, pre-clinical studies to final production, testing, and release of batches. Bioanalysis during vaccine development presents many challenges in assay diversity and complexity, sample matrices, analyte types, and demands on workflow efficiency. Immunoassays are used to characterize vaccine titer, vector titer, purity, affinity, and potency, as well as immunogenic response in both animals and humans.
Samples for analysis vary greatly, with a wide range of analytes that include polysaccharides, proteins and peptides that must be measured in diverse matrices. Added to that, the new generation of vaccines involves increasing numbers of antigens.
Plate-based immunoassays have become the gold standard for many aspects of vaccine bioanalysis, including quantifying antigen epitopes, assessing impurity levels, and vaccine potency assays such as vaccine titer and immunogenicity. The search for improved immunoassay performance includes:
- The use of smaller amounts of precious reagents, such as vaccine candidate molecules, and samples from small test animals
- Assays that can readily handle serum and plasma samples, formulations with aluminum particles, and emulsions
- Reduction of hands-on time
- Reduction of assay development time
Examples of Gyrolab immunoassays in vaccine R&D and manufacturing
Gyrolab system can be used in a wide range of applications within vaccine R&D and manufacturing.
Rapid detection of antigen-specific antibodies in serum
Measuring the quantity and quality of antibodies in vaccinated subjects is key to understanding how protective lifelong antibody responses can be induced. To increase throughput, a team at Novartis Vaccines and Diagnostics transferred a laborious ELISA to Gyrolab system to enable rapid analysis of IgG subclasses to characterize memory B cell responses in mice vaccinated with Neisseria antigen together with three adjuvants.
Immunogenicity responses in a preclinical mouse study detecting IgG antibody subtypes simultaneously in a single run were analyzed with Gyrolab immunoassays to screen responses from different formulations.
Gaining insights into antibody affinity
The efficiency of vaccine development is greatly assisted by precise and accurate data that characterizes binding affinity between epitopes and elicited antibodies. Affinity data generated using Gyrolab systems can, for example, be used to test sera prepared using different immunization schemes based on cutoff criteria.
Affinity data (a) can be used to test sera prepared using different immunization schemes based on cutoff criteria (orange lines, b).
Figure 4a redrawn based on the original figure.
Reagent screening and comparison of vaccine lots for release testing
The increasing complexity of multivalent vaccines requires the development of large numbers of assays, including assays to measure active vaccine analyte levels prior to release. This need stimulated the Vaccines Analytical Development group at Merck, USA to develop a highly flexible Gyrolab assay that would minimize assay development and consumption of precious reagents such as labeled detection antibodies.
Comparison of two antiserum lots using fluorescence intensity data (top left panel), and response curves (top right panel), for antigen B. The old lot performs much better, with a greater peak area in Gyrolab Viewer, and higher signal/background and slope when titrated. The new lot shows significant on-column fluorescence tailing (bottom panels), which suggests a low-affinity interaction with the antigen.
Serology testing for SARS-CoV-2 antibodies
Serology assays that detect antibodies in serum deliver important data that support the development of effective vaccines and therapeutics. To help in the development of vaccines against SARS-CoV-2, Gyros Protein Technologies has released a three-step bridging Gyrolab antibody immunoassay designed to measure antibodies against SARS-CoV-2 virus.
Antigen titer – broadening the analytical range
A vaccine tester needed to extend the dynamic range of the immunoassay being used to measure a glycoprotein antigen adsorbed on aluminum gel. Transferring the ELISA to Gyrolab system extended the dynamic range from 5–300 mIU/mL to ~0.6–40 000 mIU/mL. Gyrolab systems are now being used by the customer to measure a number of antigens in bioprocess.
Gyrolab assays to measure antigen titer frequently have an analytical range that is far broader than ELISA.
Gyrolab assays also enable the rapid generation of titer data with high precision, combined with reliable robustness, and automation that increases reproducibility and reduces hands-on time.
Robust measurement of HCP impurities throughout a vaccine bioprocess workflow
Removal of host cell protein (HCP) impurities is a major challenge in the production of biopharmaceuticals, including vaccines based on, for example, recombinant subunits.
GSK Vaccines used Gyrolab assays to quantify HCPs during the purification of polyclonal antibodies from HEK 293 cells in in-process samples with a robustness that surpassed ELISA. These ELISAs suffered from a distinct dilution dependence for samples from later steps in the purification process, had a narrower analytical range, and consumed more sample/antibody reagent.
Gyrolab assays proved to be more robust than ELISA and were able to measure HCP levels in samples from all steps in the purification process.
These results highlight the value of automating and miniaturizing impurity analysis with Gyrolab systems, which can also shorten turn-around time and deliver reproducible high-quality data, with fewer manual operations. Find out more about how Gyrolab technology can help in the process-related impurity analysis of biotherapeutics.
Potency testing to support formulation
Assessing batch-to-batch consistency in potency is an important component of vaccine QC. This testing can be used in stability studies, validation and optimization of fermentation and purification, comparing new facilities, and evaluating the effect of changes in formulation or adjuvant.
To this end, Merck compared the performance of Gyrolab system with a Luminex assay for mouse potency testing. The Gyrolab assay proved to be reliable and robust, delivering sensitivity and reproducibility (CV <10%) comparable with a Luminex assay (R2 = 0.97) but with less hands-on time and a shorter turnaround time.
Gyrolab and Luminex immunoresponse assays gave comparable results.
Determining vector particle concentration (titer)
Viral-vectored vaccines are live viruses that are genetically engineered to express one or more heterologous antigens. Determining the vector particle concentration is an important step in developing and manufacturing such vaccines. Vector production is an expensive process that results in a small volume of highly valuable product — for example 200 L of bioreactor product concentrated down to 20 mL. This means that analytical methods that require only a small volume of sample are at a premium.
Gyrolab systems in combination with Gyrolab AAVX Titer Kit or Gyrolab p24 Kit quickly deliver high quality titer data for a range of AAV serotypes, or lentivirus (p24), respectively, using smaller sample volumes and with better overall performance compared to ELISA.