Maintenance of vaccine stability through annual stability and comparability studies.

Evaluation of vaccine stability does not end with licensure of the product. An annual stability program helps assure continued quality of product throughout the dating period, while comparability studies are performed after a process or facilities change in order to demonstrate that the change has not impacted the stability characteristics of the product. Careful attention to the design and analysis of post licensure studies helps mitigate the risk of missing a meaningful shift in the degradation rate of a vaccine, as well as the possibility of incorrectly earmarking a stability shift when the product remains acceptable.

Vaccine stability study design and analysis to support product licensure.

Stability evaluation supporting vaccine licensure includes studies of bulk intermediates as well as final container product. Long-term and accelerated studies are performed to support shelf life and to determine release limits for the vaccine. Vaccine shelf life is best determined utilizing a formal statistical evaluation outlined in the ICH guidelines, while minimum release is calculated to help assure adequate potency through handling and storage of the vaccine. In addition to supporting release potency determination, accelerated stability studies may be used to support a strategy to recalculate product expiry after an unintended temperature excursion such as a cold storage unit failure or mishandling during transport. Appropriate statistical evaluation of vaccine stability data promotes strategic stability study design, in order to reduce the uncertainty associated with the determination of the degradation rate, and the associated risk to the customer.

Detection of HPV in Norwegian cervical biopsy specimens with type-specific PCR and reverse line blot assays.

BACKGROUND: Although the association of HPV16 and HPV18 DNA with cervical cancers has been well studied, the prevalence of these types in high-grade cervical intraepithelial neoplasias (CIN2/3) may differ from the prevalence found in cervical cancer specimens. OBJECTIVE: To determine the prevalence of specific HPV types found in high-grade CIN2/3 biopsy samples. STUDY DESIGN: One thousand eight hundred and forty-eight cervical biopsy specimens were obtained from Norwegian women. HPV16 and HPV18 type and gene-specific PCR assays were performed amplifying a portion of the E6, E7 and L1 genes. In addition, the reverse line blot assay was performed on a subset of specimens, in which a portion of L1 was amplified and hybridized to strips coated with complimentary HPV sequences. RESULTS: The prevalences of HPV16 and HPV18 in the 1848 biopsy cohort were 32.3% and 6.0%, respectively. HPV16 was detected in 47.5% and HPV18 in 5.9% of diagnosed CIN2/3 specimens. Approximately 12% of the CIN2/3 specimens contained two HPV types and 2.5% contained three HPV types. CONCLUSIONS: The prevalence of HPV16 increases from grades CIN1 to CIN2 to CIN3. The prevalence of HPV18 did not change significantly with increasing CIN grade. The majority of infections diagnosed as CIN2/3 contained a single HPV type. The type-specific PCR assay had increased detection sensitivity over the reverse line blot assay.

Assessing the reproducibility of an analytical method.

The reproducibility of a validated analytical method may require reassessment because of various reasons, such as the transfer between laboratories or companies, changes in the instruments or software platforms (or both), or changes in critical reagents, among others. This paper is a demonstration of an assay bridging study in evaluating reproducibility. The approach is simple but very informative and offers many advantages over existing approaches.

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model.

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

Using QPCR to assign infectious potencies to adenovirus based vaccines and vectors for gene therapy: toward a universal method for the facile quantitation of virus and vector potency.

The assignment of infectious potency to test articles of adenovirus has been conducted mainly using classical end-point dilution methods, which rely on virus induced cytopathology to reveal the presence of infectious virus. These assays suffer the disadvantages of labor intensity, duration, throughput restriction and variability. In the course of our development of an Ad5 based HIV vaccine for clinical evaluation, we sought a facile method for the assignment of potency to the numerous test articles generated during the development of bioprocesses for bulk manufacture, downstream purification and formulation. In this paper we describe a quantitative PCR based potency assay (QPA) which uses QPCR to quantitate adenovirus genomes replicated 24h after the inoculation of a test article on 293 cell monolayers, and then relates that mass to potency by interpolation to a standard curve of replicated adenovirus genomes constructed with a reference adenovirus standard to which infectious potency has been previously assigned in the classical end-point dilution assay. The QPA assay for adenovirus is simple and rapid, with a throughput capacity adequate to the potency assay demands of bioprocess development, and with a precision expressed as a root variability of 16.8% R.S.D., allowing for close discriminations of the products of alternative process configurations. The adenovirus QPA principle can be applied to the quantitation of infectious potency of both RNA and DNA viruses and we report briefly on the development of QPA assays for measles and mumps. QPA assays owing to their simplicity and easy automation, rapidity, capacity and precision hold promise to become widely practiced methods for the quantitation of the potency of live virus vaccines and other recombinant virus vectors.

Monitoring the stability of human vaccines.

Postmarketing stability studies of vaccines that tend to be close to their clinical specification at the end of the expiration dating period may require enhanced annual monitoring. In addition, an early assessment of product stability prior to completion of each individual study is desired. However, predictive measures of individual lots may produce early indication of failure. In many cases, these prove to be false alarms. For such products, continued product quality after marketing should, therefore, depend less on evaluating individual observations or individual lot projections, and more on assuring that the underlying stability profile of the product as a whole has not changed. We propose a monitoring procedure and an index of the average quality of vaccine lots currently on the market. We explore the statistical properties of the index for several experimental designs for sampling of marketed lots, and we describe an optimality property of the index.