Basic principles of stability

An understanding of the principles of degradation, as well as the statistical tools for measuring product stability, is essential to management of product quality. Key to this is management of vaccine potency. Vaccine shelf life is best managed through determination of a minimum potency release requirement, which helps assure adequate potency throughout expiry. Use of statistical tools such a least squares regression analysis should be employed to model potency decay. The use of such tools provides incentive to properly design vaccine stability studies, while holding stability measurements to specification presents a disincentive for collecting valuable data. The laws of kinetics such as Arrhenius behavior help practitioners design effective accelerated stability programs, which can be utilized to manage stability after a process change. Design of stability studies should be carefully considered, with an eye to minimizing the variability of the stability parameter. In the case of measuring the degradation rate, testing at the beginning and the end of the study improves the precision of this estimate. Additional design considerations such as bracketing and matrixing improve the efficiency of stability evaluation of vaccines.     

Goals of stability evaluation throughout the vaccine life cycle

Stability studies play a critical role in assuring product quality at all points in the vaccine life cycle. At and after licensure, stability studies on quality attributes (including potency) provide a critical link between marketed and clinically evaluated vaccine product, addressing important regulatory concerns by assuring that product quality is maintained throughout the dating period. During development, stability studies are done to assure product quality and to obtain the data needed to support licensure. Stability studies may also be performed after licensure to assure that product continues to perform as it did pre-licensure, as well as to evaluate the effect on product quality of deliberately introduced manufacturing changes. At each phase in the product life cycle, it is important to consider the goals of stability evaluation and to perform appropriate statistical analyses in order to assure and reach appropriate conclusions about product quality.     

Stability of vaccines – Bridging from stability data to continuous safety and efficacy throughout shelf life – An always reliable approach?

Stability studies are important tools to reliably ensure that efficacy and safety of medicinal products will remain unchanged from release of drug product until the end of shelf life. For complex medicinal products such as biological medicinal products, including vaccines, design and conduct of such studies requires particularly careful considerations in order to ensure that technical data resulting from stability studies are indeed indicative for unchanged clinical performance. Ideally, relevance of specifications controlled by stability studies as well as definition of shelf life should be justified by acceptable clinical data obtained with product at the end of the shelf life claimed.     

Evaluating Current Practices in Shelf Life Estimation

The current International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) methods for determining the supported shelf life of a drug product, described in ICH guidance documents Q1A and Q1E, are evaluated in this paper. To support this evaluation, an industry data set is used which is comprised of 26 individual stability batches of a common drug product where most batches are measured over a 24 month storage period. Using randomly sampled sets of 3 or 6 batches from the industry data set, the current ICH methods are assessed from three perspectives. First, the distributional properties of the supported shelf lives are summarized and compared to the distributional properties of the true shelf lives associated with the industry data set, assuming the industry data set represents a finite population of drug product batches for discussion purposes. Second, the results of the ICH “poolability” tests for model selection are summarized and the separate shelf life distributions from the possible alternative models are compared. Finally, the ICH methods are evaluated in terms of their ability to manage risk. Shelf life estimates that are too long result in an unacceptable percentage of nonconforming batches at expiry while those that are too short put the manufacturer at risk of possibly having to prematurely discard safe and efficacious drug product. Based on the analysis of the industry data set, the ICH-recommended approach did not produce supported shelf lives that effectively managed risk. Alternative approaches are required.     

口服脊髓灰质炎减毒活疫苗(人二倍体细胞)的稳定性研究

目的评价细胞工厂工艺连续生产的口服脊髓灰质炎减毒活疫苗(人二倍体细胞)的稳定性。方法疫苗在-20℃放置24个月,检测病毒滴度、外观、抗生素残留量、无菌性,及对病毒血清型进行鉴别;2~8℃放置12个月检测疫苗稳定性;室温放置7周、37℃放置7 d检测加速热稳定性并冻融的稳定性。结果该疫苗-20℃可贮存24个月以上,2~8℃有效期可延长至12个月,且冻融不会影响疫苗的稳定性。结论疫苗质量稳定,各项检测结果均符合《中华人民共和国药典》三部(2010版)及企业《口服脊髓灰质炎减毒活疫苗(人二倍体细胞)注册标准》。     

The Application of the Accelerated Stability Assessment Program (ASAP) to Quality by Design (QbD) for Drug Product Stability

An isoconversion paradigm, where times in different temperature and humidity-controlled stability chambers are set to provide a fixed degradant level, is shown to compensate for the complex, non-single order kinetics of solid drug products. A humidity-corrected Arrhenius equation provides reliable estimates for temperature and relative humidity effects on degradation rates. A statistical protocol is employed to determine best fits for chemical stability data, which in turn allows for accurate estimations of shelf life (with appropriate confidence intervals) at any storage condition including inside packaging (based on the moisture vapor transmission rate of the packaging and moisture sorption isotherms of the internal components). These methodologies provide both faster results and far better predictions of chemical stability limited shelf life (expiry) than previously possible. Precise shelf-life estimations are generally determined using a 2-week, product-specific protocol. Once the model for a product is developed, it can play a critical role in providing the product understanding necessary for a quality by design (QbD) filing for product approval and enable rational control strategies to assure product stability. Moreover, this Accelerated Stability Assessment Program (ASAP) enables the coupling of product attributes (e.g., moisture content, packaging options) to allow for flexibility in how control strategies are implemented to provide a balance of cost, speed, and other factors while maintaining adequate stability.     

On the Shelf Life of Pharmaceutical Products

This article proposes new terminology that distinguishes between different concepts involved in the discussion of the shelf life of pharmaceutical products. Such comprehensive and common language is currently lacking from various guidelines, which confuses implementation and impedes comparisons of different methodologies. The five new terms that are necessary for a coherent discussion of shelf life are: true shelf life, estimated shelf life, supported shelf life, maximum shelf life, and labeled shelf life. These concepts are already in use, but not named as such. The article discusses various levels of "product" on which different stakeholders tend to focus (e.g., a single-dosage unit, a batch, a production process, etc.). The article also highlights a key missing element in the discussion of shelf life-a Quality Statement, which defines the quality standard for all key stakeholders. Arguments are presented that for regulatory and statistical reasons the true product shelf life should be defined in terms of a suitably small quantile (e.g., fifth) of the distribution of batch shelf lives. The choice of quantile translates to an upper bound on the probability that a randomly selected batch will be nonconforming when tested at the storage time defined by the labeled shelf life. For this strategy, a random-batch model is required. This approach, unlike a fixed-batch model, allows estimation of both within- and between-batch variability, and allows inferences to be made about the entire production process. This work was conducted by the Stability Shelf Life Working Group of the Product Quality Research Institute.     

Kinetics of pramlintide degradation in aqueous solution as a function of temperature and pH

The stability of the 37-amino acid peptide pramlintide, in aqueous solution, was studied as a function of pH and temperature. Samples of pramlintide formulated as a parenteral product were exposed to elevated temperatures and to realistic storage conditions for as long as 30 months. Pramlintide degradation was monitored by three high-performance liquid chromatography (HPLC) methods: a reversedphase (RP-HPLC) and a strong-cation exchange (SCX-HPLC) method for percentage purity determination by area normalization, plus a second RP-HPLC method for potency determinationversus external standards. The pH-rate profile for pramlintide shows increasing degradation rate constants with increasing pH over the range pH=3.5 to 5.0. The Arrhenius expression for pramlintide degradation at pH=4.0 over the temperature range 5°C to 50°C is In(k₀)=37.39−21.900/RT, where k₀ is the zero-order rate constant (in %/mo) for pramlintide degradation. The pramlintide parenteral product formulated at pH=4.0 is extremely stable, with percentage purity and percentage potency loss of only approximately 2% over 30 months at 5°C. The formulated pramlintide drug product has acceptable shelf life for long-term storage at 5°C and up to a 30-day patient use when stored at ambient temperature.     

Cell culture media impact on drug product solution stability

To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf‐life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell‐free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG₄ monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998–1008, 2016     

Supercritical CO2 interpolymer complex encapsulation improves heat stability of probiotic bifidobacteria

The probiotic industry faces the challenge of retention of probiotic culture viability as numbers of these cells within their products inevitably decrease over time. In order to retain probiotic viability levels above the therapeutic minimum over the duration of the product’s shelf life, various methods have been employed, among which encapsulation has received much interest. In line with exploitation of encapsulation for protection of probiotics against adverse conditions, we have previously encapsulated bifidobacteria in poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex microparticles under supercritical conditions. The microparticles produced had suitable characteristics for food applications and also protected the bacteria in simulated gastrointestinal fluids. The current study reports on accelerated shelf life studies of PVP:PVAc-CA encapsulated Bifidobacterium lactis Bb12 and Bifidobacterium longum Bb46. Samples were stored as free powders in glass vials at 30 °C for 12 weeks and then analysed for viable counts and water activity levels weekly or fortnightly. Water activities of the samples were within the range of 0.25–0.43, with an average a _w  = 0.34, throughout the storage period. PVP:PVAc-CA interpolymer complex encapsulation retained viable levels above the recommended minimum for 10 and 12 weeks, for B. longum Bb46 and B. lactis Bb12, respectively, thereby extending their shelf lives under high storage temperature by between 4 and 7 weeks. These results reveal the possibility for manufacture of encapsulated probiotic powders with increased stability at ambient temperatures. This would potentially allow the supply of a stable probiotic formulation to impoverished communities without proper storage facilities recommended for most of the currently available commercial probiotic products.     

Stability of Poly(ε-caprolactone) Microparticles Containing Brucella ovis Antigens as a Vaccine Delivery System Against Brucellosis

In previous works, our research group has successfully proved the use of subcellular vaccines based on poly(ε-caprolactone) (PEC) microparticles containing an antigenic extract of Brucella ovis (HS) against experimental brucellosis in both mice and rams. However, the successful exploitation of pharmaceutical products, and therefore of this product as veterinary vaccine, requires preservation of both biological activity and native structure in all steps of development from purification to storage. In this context, we have carried out an accelerated stability study to evaluate the relative stability of HS when loading in PEC microparticles. For this purpose, freeze-dried microparticles were stored at 40 ± 1°C and 75% RH as a preliminary analysis of a stability testing. The results showed that both physico-chemical (size, morphology, antigen content, release profile) and biological (integrity and antigenicity of the HS) properties were preserved after 6 months of storage. On the contrary, after 1 year of storage, the HS release profile was dramatically affected probably due to a progressive loss of the polymer microstructure. In addition, the degradation and loss of the antigenicity of the HS components was also evident by SDS-PAGE and immunoblotting analysis. In fact, after 12 months of storage, only the integrity and antigenicity of two of the major protective proteins of the HS antigenic complex were preserved.     

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

ABSTRACT

Protein therapeutic higher order structure (HOS) is a quality attribute that can be assessed to help predict shelf life. To model product shelf-life values, possible sample-dependent pathways of degradation that may affect drug efficacy or safety need to be evaluated. As changes in drug thermal stability over time can be correlated with an increased risk of HOS perturbations, the effect of long-term storage on the product should be measured as a function of temperature. Here, complementary high-resolution mass spectrometry methods for HOS analysis were used to identify storage-dependent changes of biotherapeutics (bevacizumab (Avastin), trastuzumab (Herceptin), rituximab (Rituxan), and the NIST reference material 8671 (NISTmAb)) under accelerated or manufacturer-recommended storage conditions. Collision-induced unfolding ion mobility-mass spectrometry data showed changes in monoclonal antibody folded stability profiles that were consistent with the appearance of a characteristic unfolded population. Orthogonal hydrogen-deuterium exchange-mass spectrometry data revealed that the observed changes in unfolding occurred in parallel to changes in HOS localized to the periphery of the hinge region. Using intact reverse-phase liquid chromatography-mass spectrometry, we identified several mass species indicative of peptide backbone hydrolysis, located between the variable and constant domains of the heavy chain of bevacizumab. Taken together, our data highlighted the capability of these approaches to identify age- or temperature-dependent changes in biotherapeutic HOS.     

An annual vaccine: Seasonal influenza

The accelerated development process for annual vaccines such as seasonal influenza presents unique challenges for the evaluation of vaccine stability. Real-time real-condition studies provide limited information at the time of registration, while regulators seek evidence that the current vaccine will perform satisfactorily in the field. Participants in the IABS Workshop on Stability Evaluation of Vaccines, a Life Cycle Approach, were offered a case study from the development of the 2007 influenza vaccine. The case study was introduced with preliminary data from the long-term study, as well as results from the completed year long study. The manufacturer also offered a proposed protocol for stability evaluation of vaccines developed in subsequent seasons. Participants were asked to answer a series of questions posed by the regulator, and critique the proposed stability protocol according to the principles described during the workshop.     

Shelf life and safety aspects of chilled cooked and peeled shrimps (Pandalus borealis) in modified atmosphere packaging

AIMS: To evaluate the growth of Listeria monocytogenes and shelf life of cooked and peeled shrimps in modified atmosphere packaging (MAP). METHODS AND RESULTS: Storage trials with naturally contaminated cooked and peeled MAP shrimps (Pandalus borealis) were carried out at 2, 5 and 8 degrees C. Challenge tests at the same conditions were performed after inoculation with Listeria monocytogenes. Both storage trials and challenge tests were repeated after 4 months of frozen storage (-22 degrees C). Brochothrix thermosphacta and Carnobacterium maltaromaticum were responsible for sensory spoilage of cooked and peeled MAP shrimps. In challenge tests, growth of L. monocytogenes was observed at all of the storage temperatures studied. At 5 and 8 degrees C the concentration of L. monocytogenes increased more than a 1000-fold before the product became sensory spoiled whereas this was not observed at 2 degrees C. Frozen storage had only a minor inhibiting effect on growth of L. monocytogenes in the thawed product. CONCLUSIONS: To prevent L. monocytogenes becoming a safety problem, cooked and peeled MAP shrimps should be distributed at 2 degrees C and with a maximum shelf life of 20-21 d. At higher temperatures shelf life is significantly reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: Information is provided to establish shelf life of cooked and peeled MAP shrimps.     

Light and storage time influence the microbial quality of minimally processed rocket

Ready-to-eat minimally processed fruits and vegetables are an ideal substrate for the growth of microorganisms, including human pathogens and mycotoxin-producing species, which question their quality and safety for customers. While we are aware of the importance of production workflows in structuring the products' microbial communities, we still know little about the factors that shape microbiomes during the timeframe products are available to customers, and beyond this timeframe. Here, we study the influence of storage light condition (light or dark) on microbiological and physico-chemical parameters of minimally processed rocket leaves at different shelf life timepoints (the day the product becomes available to consumers, expiration date, 3 days after the expiration date). Our results suggest that the total microbial load increases from the day the product becomes available to consumers, to the expiry date and after the product's expiration. However, when studying the composition of the fungal microbiome, we did not observe significant changes in its structure as the effect of product shelf life or storage light condition. We also found that products stored under light condition had a higher total bacterial load compared to those stored in darkness. Our results might be helpful in crafting improved workflows for product's storage during its shelf life, which might ultimately lead to a re-evaluation of storage times resulting in reduced food waste due to product spoilage or expiration.     

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C_H2 and C_H3 domains.     

Assessing distributions of estimated drug shelf lives in stability analysis

A stability study is usually conducted to ensure that a drug product can meet the approved specifications prior to its expiration dating period (shelf life). Several approaches for determination of drug shelf life assuming random batches have been proposed. In this paper, we examine sampling distributions of the estimated shelf lives proposed by Shao and Chow (1994, Biometrics 50, 753-763). An application to some stability data from the pharmaceutical industry is presented.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load,Lag Period,and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.     

Effect of γ-Irradiation on the Microflora of Freshwater Fish: I. Microbial Load, Lag Period, and Rate of Growth on Yellow Perch (Perca flavescens) Fillets

Microbial flora were compared in irradiated and nonirradiated yellow perch fillets. These studies included effects of irradiation on the total microbial population, the lag phase, and rate of growth in this freshwater fishery product. The work was conducted concurrently with sensory and chemical evaluation, and constituted part of an investigation designed to evaluate the effect of substerilization doses (0.3 and 0.6 Mrad) of Co⁶⁰ γ rays on the storage life of yellow perch fillets at 1.0 or 6.0 C. In five storage tests, total plate counts prior to irradiation did not exceed 8.7 × 10⁵ per gram of sample; this count was reduced nearly 100% by irradiation with either 0.3 or 0.6 Mrad. Progressively lower maximal bacterial populations and lengthened lag phases were obtained as more radiation was used. The growth rate of the population did not appear to decrease significantly. Microbial data obtained in these studies confirmed the sensory and chemical studies, by indicating that irradiation can significantly extend the refrigerated shelf life of freshwater fish.