Mechanistic Studies and Formulation Mitigations of Adeno-associated Virus Capsid Rupture During Freezing and Thawing: Mechanisms of Freeze/Thaw Induced AAV Rupture.

Gene therapies delivered using adeno-associated virus (AAV) vectors are showing promise for many diseases. Frozen AAV drug products are exposed to freeze-thaw (F/T) cycles during manufacturing, storage, and distribution. In this work we studied the mechanisms of AAV capsid rupture during F/T. We found that exposure to interfaces, exacerbated by F/T, and the mechanical force of excipient devitrification correlated with AAV capsid rupture during F/T. There was no impact of pH shifts, cryo-concentration, or cold-denaturation. Results were similar for AAV8 and AAV9. With these mechanistic insights we identified three formulation mitigation approaches. Addition of ≥0.0005% w/v poloxamer 188 (P188) eliminated substantial recovery losses (up to ∼60% without P188) and minimized rupture to ≤1% per F/T cycle. Elimination of exothermic devitrification events during rewarming, either by formulating with a low buffer concentration, or by adding a cryoprotectant further reduced rupture during F/T. Rupture of AAV9 was <0.2% per F/T cycle in a formulation with 1 mM phosphate, 4.4 mM dextrose, electrolytes, and 0.001% P188 at pH 7.2. Rupture of AAV8 was not detected when formulated with 4% sucrose, 100 mM salt, and 0.001% P188 at pH 7.4. These results provide insights into effective strategies for stabilizing AAVs against rupture during F/T.

Impact of Time Out of Intended Storage and Freeze-thaw Rates on the Stability of Adeno-associated Virus 8 and 9.

There are an increasing number of clinical studies evaluating different adeno-associated virus (AAV) serotypes as vectors for gene therapy. Long-term frozen storage can maximize the stability of AAV. Freeze-thaw (F/T) cycles and exposures to room temperature (RT) and refrigerated conditions occur during manufacturing, labeling, and clinical use. In this work we exposed AAV8 and AAV9 at low and high concentrations to five F/T cycles compounded with RT and refrigerated holds in a 'daisy chain' time out of intended storage (TOIS) stability study, which may be a best practice in early development. We also evaluated the impact of 5 F/T cycles for multiple permutations of fast and slow cooling and rewarming rates. The quality attributes of AAV8 and AAV9 remained within acceptable ranges after the daisy chain TOIS and F/T rate studies. Potency and concentration were unchanged within method variability. There was a minor increase in non-encapsidated ('free') DNA released from AAV8 after F/T in a phosphate-buffered saline formulation. DNA release during F/T was minimized in a formulation with a low buffer concentration and was not detected in a formulation containing sucrose. We conclude that AAV8 and AAV9 have stability profiles that are suitable for manufacturing and clinical development.

Structure of the AAVhu.37 capsid by cryoelectron microscopy.

Adeno-associated viruses (AAVs) are used as in vivo gene-delivery vectors in gene-therapy products and have been heavily investigated for numerous indications. Over 100 naturally occurring AAV serotypes and variants have been isolated from primate samples. Many reports have described unique properties of these variants (for instance, differences in potency, target cell or evasion of the immune response), despite high amino-acid sequence conservation. AAVhu.37 is of interest for clinical applications owing to its proficient transduction of the liver and central nervous system. The sequence identity of the AAVhu.37 VP1 to the well characterized AAVrh.10 serotype, for which no structure is available, is greater than 98%. Here, the structure of the AAVhu.37 capsid at 2.56 Šresolution obtained via single-particle cryo-electron microscopy is presented.

Scientific considerations in the review and approval of generic enoxaparin in the United States.

In 2010, the US Food and Drug Administration (FDA) approved a generic low-molecular-weight heparin without clinical safety or efficacy data under the Abbreviated New Drug Application (ANDA) pathway. To enable a determination of active ingredient sameness of generic and innovator enoxaparin products, the FDA developed a scientifically rigorous approach based on five criteria: first, equivalence of physicochemical properties; second, equivalence of heparin source material and mode of depolymerization; third, equivalence in disaccharide building blocks, fragment mapping and sequence of oligosaccharide species; fourth, equivalence in biological and biochemical assays; and finally, equivalence of in vivo pharmacodynamic profile. In addition to fulfillment of these criteria, FDA also used in vitro, ex vivo and model animal data to ensure there was no increased immunogenicity risk of the generic enoxaparin product relative to the brand name product. The approval of the highly complex enoxaparin product using this framework under the ANDA pathway represents a major development. It also suggests that analytical and scientific advancements may in certain cases allow the elimination of unnecessary in vivo testing in animals and humans.

Results of a survey of biological drug and device industries inspected by FDA under the Team Biologics Program.

The Product Quality Research Institute, in conjunction with the Food and Drug Administration, conducted an anonymous, electronic survey of the biological products manufacturing industry inspected by Team Biologics, with emphasis in obtaining industry input on inspection and compliance aspects of program operations. Representatives from all of the product-specific manufacturing industries inspected under the Team Biologics Program responded to this survey (vaccines; fractionated plasma proteins and recombinant analogs; allergenics; therapeutics and in-vivo diagnostics; and in-vitro diagnostics, including blood grouping reagents). Data and written feedback was obtained regarding each firm's interactions and experiences of Team Biologics inspections at its facilities over the past three years. The three areas most impacted by Team Biologic inspections were "Production and Process Controls", "Failure Investigations" and "Facility / Equipment Controls". Overall assessment of the program was generally positive with 68% identifying a positive impact on the sites operations and 88% assessed the inspections as being conducted fairly. The findings and conclusions of this report will be utilized by the FDA to evaluate and further assess the impact of the Team Biologics Program and to implement any necessary changes. This report provides useful information to companies currently manufacturing licensed biologic products subject to Team Biologics inspections and also to those companies anticipating these inspections for future product manufacturing.

The FDA's assessment of follow-on protein products: a historical perspective.

The scientific and regulatory issues that are associated with the possible introduction of 'follow-on' versions of protein drug products are the topic of considerable debate at present. Because of the differences between protein drug products and small-molecule drugs, the development of follow-on versions of protein products presents more complex scientific challenges than those presented by the development of generic versions of small-molecule drugs. Here, with a view to illustrating the Food and Drug Administration's (FDA's) scientific reasoning and experience in this area, we discuss past examples of the FDA's actions involving the evaluation of various types of follow-on and second-generation protein products and within-product manufacturing changes. The FDA believes its evaluation of the safety and effectiveness of follow-on protein products will evolve as scientific and technological advances in product characterization and manufacturing continue to reduce some of the complexity and uncertainty that are inherent in the manufacturing of protein products.