A quantitative real-time polymerase chain reaction (qPCR)-based method was established and validated for the detection of residual host DNA in recombinant human C1 esterase inhibitor (rhC1INH), a biopharmaceutical produced in the mammary glands of transgenic rabbits. DNA was extracted from rhC1INH samples using a sodium iodide-based extraction kit, and a TaqMan qPCR assay targeting the rabbit-specific L1Oc gene was optimized to evaluate its specificity, linearity, limit of quantification (LOQ), accuracy, precision, and robustness. The method exhibited high species specificity, with no cross-reactivity to human or other common mammalian DNA. A broad dynamic range of 0.001-100 pg/μL was achieved, with the standard curve showing excellent linearity (R[2] > 0.99) and amplification efficiency of 90-110%. The LOQ was determined to be 0.001 pg/μL, satisfying the sensitivity requirements for trace DNA detection. Spike-and-recovery assays verified favorable quantification accuracy, with recovery rates ranging from 71.83% to 97.26% at low, medium, and high concentration levels. Intra-assay and inter-assay precision were assessed, and the relative standard deviation (RSD) was no more than 15.42% at all tested concentrations, which confirmed good method repeatability. Method robustness was validated by investigating the influences of different reagent batches and lysis temperature variations, with all corresponding indicators exhibited RSD values no higher than 30%. This qPCR method features high specificity, superior sensitivity, and convenient operation, and can serve as a reliable technical means for the quality control of rhC1INH and other transgenic rabbit mammary gland-derived biopharmaceuticals.

Clesrovimab (Enflonsia™) is a long-acting prophylactic monoclonal antibody designed to protect infants from respiratory syncytial virus diseases. The potency of clesrovimab is measured by a cell-based assay using A549 cells and engineered RSV-NLP viruses. To further increase the throughput and improve the assay precision, the plate layout and data analysis models were optimized. In the updated plate layout, eleven concentrations of reference standard and five concentrations of one control sample and three test samples in duplicate were included in one 96-well plate. A Log-Log linear model using the same five concentrations of each test article was used to calculate the slope ratios for parallelism assessment. A restricted 4-parameter logistic model using logarithmic-transformed response variable was used to calculate the relative potency. The optimized cell-based assay allows more samples to be tested on one plate and has demonstrated improved precision compared to the previous version using regular 11-point 4-parameter logistic model. A comprehensive robustness study of the optimized clesrovimab cell-based assay has demonstrated fit-for-use assay performance by introducing small but deliberate variations of the selected assay parameters and reagents. The method was successfully validated and implemented for commercial release and stability testing of clesrovimab, and ultimately has been accepted by numerous global health authorities.

In October 2023, the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) presented recommendations to the World Health Organisation (WHO) Expert Committee for Biological Standardization following an audit of WHO vaccine and biotherapeutic guidelines. These recommendations aimed to promote greater and more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction and Refinement) in quality control and batch release testing. Building on the success of the WHO audit, the NC3Rs initiated a new project to accelerate wider adoption of 3Rs approaches for batch release testing by global biologics manufacturers and regulators. To facilitate this, a survey was developed and distributed to individuals who work for biologicals manufacturers, contract research organisations, national regulatory authorities and national control laboratories. This paper presents the key findings from the survey and outlines how these insights will inform the NC3Rs' ongoing work to advance the wider integration of 3Rs approaches in quality control and batch release testing of vaccines and biotherapeutics.

Licensed typhoid vaccines contain Vi polysaccharide (ViPS), a capsular antigen of Salmonella. Typhi (S.typhi), either unconjugated or conjugated to carrier proteins like tetanus toxoid (TT), diphtheria toxoid (DT), and Cross-reactive material 197 (CRM-197). Quantitation of ViPS during vaccine development, is challenging, as ViPS demonstrates limited adsorption to solid phases, resulting in poor binding and reduced sensitivity in conventional ELISA formats. We developed a novel competitive enzyme-linked immunosorbent assay (c-ELISA) using biotinylated ViPS as a detector probe for ViPS quantitation. The performance of c-ELISA with a novel Biotinylation approach was evaluated with WHO recommended orthogonal colorimetry based Hestrin assay and High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) method. Both c-ELISA and HPAEC-PAD demonstrated comparable specificity, accuracy and repeatability, however c-ELISA showed a higher sensitivity than HPAEC-PAD in the estimation of unconjugated polysaccharide (free-PS) in typhoid conjugates with a range of 90-10,000 ng/ml. Overall, the newly developed c-ELISA is a reliable and sensitive immunochemical method for the quantification of ViPS, which complements the HPAEC-PAD. Together, these tests can serve as orthogonal analytical platforms for the monitoring of the ViPS during the production of typhoid vaccines and for the quality control.

The transition toward non-animal methods for vaccine quality control has accelerated in recent years, driven by scientific advances and evolving regulatory expectations. To assess the current status of replacing animal-based methods, international stakeholders from regulatory authorities, industry, and public health institutions convened in Bangkok, Thailand from 2 to 4 December 2025 for the conference "Animal Testing Replacement for Vaccines: A One Health View - Global Outlook and Future Strategy," organized by Animal-Free Safety Assessment Collaboration and the International Alliance for Biological Standardization. This report synthesizes key discussions from the meeting, including advancements in in vitro potency, safety, and pyrogenicity testing; challenges associated with validation and global regulatory acceptance; access to critical reagents and reference standards; implementation of reliance and work-sharing models; and capacity building for national control laboratories across human and veterinary sectors. Despite recent progress in some areas, significant barriers remain, including fragmented regulatory requirements, limited access to reference standards, and inconsistent validation and implementation pathways across regions and sectors. Collectively, the outcomes underscore the need for coordinated international action to enable broader adoption of scientifically robust, non-animal testing strategies for quality control of vaccines.

Evaluating the risk of out-of-specification (OOS) occurrence before it happens is essential to prevent delays or interruptions in the supply of biological products. This study proposes a practical and quantitative evaluation approach using two indices, S1 and S2, as OOS risk screening and monitoring tools under finite-sample conditions. S1 evaluates whether the variability of data points in a dataset satisfies a low OOS risk criterion, defined as an acceptable level of inherent variability. S2 evaluates whether the observed data range remains within the specification limits with a range-proportional safety margin. Monte Carlo simulations based on normal distributions with sample sizes up to 500 demonstrated that S1 > 1 effectively identified datasets that deviated from the low OOS risk criterion, whereas S2 > 1 functioned as an early alert indicator that predicted or coincided with OOS occurrences. Regardless of the distribution type, both indices increase by definition as the spread of data points increases, suggesting their potential applicability as practical monitoring tools even for non-normal datasets. The combined use of S1 and S2 provides complementary and early warning insights into potential quality deviations and supports proactive quality risk management of biological products.

Accurate quantification of polysaccharide content is indispensable for the development and quality control of glycoconjugate vaccines. Conventional methods are often laborious, require large sample volumes, or are susceptible to interference. Ultraviolet-visible (UV-Vis) spectrophotometry presents an attractive alternative but suffers from an unexplained nonlinear concentration-absorbance relationship, precluding its reliable use. Here, we reveal that molecular behavior of pneumococcal polysaccharide conjugates including the solution conformation dynamics is the principal cause of this quantification anomaly. We demonstrate that polysaccharide chains undergo concentration-dependent conformational transitions: from an extended state with enhanced absorbance at low concentrations, to a protein-coated form, and finally to aggregates at high concentrations. This molecular behavior, characterized by dynamic light scattering and diffusion-ordered NMR spectroscopy, directly causes nonlinear absorbance and spectral shifts. By modeling this relationship with a sine wave-based function, we identified a stable quantitative window and established a robust UV-Vis method for direct polysaccharide quantification. The developed method is proven to have acceptable recovery in conjugate samples with VLP carrier proteins that are in a stable conformation. Our findings provide a mechanistic understanding of a long-standing analytical challenge in vaccinology. The developed method, validated for its precision, accuracy, enables rapid and simple quantification of polysaccharide content with minimal sample consumption. This work not only facilitates accelerated conjugate vaccine development but also offers a generalizable framework for analyzing complex biomolecular therapeutics.

The results of a retrospective analysis of data on rabies virus neutralizing antibody titers of occupationally at-risk individuals receiving vaccination with Abhayrab®, a Vero cell culture-derived inactivated rabies vaccine, are communicated. While rabies vaccines generally elicit strong immunity, specific data on the long-term efficacy of Abhayrab® was previously lacking. The data on neutralizing antibody titers of occupationally immunized individuals receiving a three-dose prophylactic intramuscular regimen (0.5 mL on days 0, 7, and 28) were analyzed retrospectively to evaluate the longevity of humoral response. The data were also used to assess the impact of sex of the individual, their marital status, and age on the humoral response. Our analyses demonstrated that pre-exposure prophylaxis with Abhayrab® elicited protective antibody titers persisting for up to 12 years. The vaccine successfully induced protection in individuals up to 69 years of age. Further, immune responses were not unfavorably influenced by age, sex, or marital status of the individual, and the vaccine maintained a high safety profile. The results of the analyses confirm that a single prophylactic course of Abhayrab® provides broad, long-lived protection. These findings reinforce the clinical importance of preventive vaccination for at-risk populations and support the vaccine's sustained efficacy over a decade post-administration.

Here we report the results of the collaborative studies to establish a WHO International Reference Reagent (IRR) for Diphtheria Antitoxin for use in Flocculation Test (coded 23/102) and a WHO IRR for Tetanus Antitoxin for use in Flocculation Test (coded 21/230). The candidate IRRs were evaluated using the Ramon flocculation test method against the WHO International Standard (IS) for Diphtheria Toxoid for use in Flocculation Test and the IS for Tetanus Toxoid for use in Flocculation Test, accordingly. The results demonstrated that the IRRs are fit for purpose for use in the flocculation test, and the results of an accelerated thermal degradation study indicated that the materials will have good long-term stability. There are no units assigned to these reference reagents since they must be pre-calibrated against the respective toxoid IS in each laboratory before use.

In May 2025, the International Alliance for Biological Standardization (IABS) held a workshop on analytical and bioprocess platforms for biological product development and commercialization. This workshop explored the use of platform approaches and technologies in product development and registration. The topics discussed included the creation, verification, and maintenance of platform technologies, their lifecycle management, their application in regulatory submissions, and the transition from early clinical trials to commercial approval. The participants highlighted the importance of clearly defining the boundaries and applicability of platform approaches in pharmaceutical development. A consistent and open dialogue between industry stakeholders, regulatory reviewers, and health authorities is essential to ensure a shared understanding and alignment in defining the platform landscape.