Microscale freeze-dried and liquid formulations of therapeutics to investigate the relationship between forced degradation and long-term shelf life

Investigators: Paul Dalby (Biochemical Engineering, UCL), Paul Matejtschuk (NIBSC, HPA) We aim to establish an automated microscale platform to assess therapeutic protein, vaccine and cell formulations, and identify any correlations (or lack of) between physical property measurements, forced degradation studies, and the long-term shelf life of liquid and freeze-dried formulations Significance: To minimise the chemical and physical degradation of biologics, excipients can be added in complex formulations. The optimisation of freeze-dried or liquid formulations is currently empirical, and requires many time and sample-consuming experiments. To save time and materials, initial formulation screens often measure properties such as melting temperatures (biologic thermostability - Tm & glass transitions - Tg), aggregation propensity (B22 values), or aggregate formed by forced degradation at elevated temperatures (light scattering, SEC). The validity of using such measurements as indicators for long term (1-2 yr) storage stability is still debated as degradation mechanisms can be complex. We recently established accurate microscale methods to subject small quantities of biologics to bioprocess stresses such as protein refolding, agitation and freeze-drying [1,2] (EPSRC studentship with NIBSC), and rapidly evaluate their thermostability [3,4], activity [1,2,5] and propensity to aggregate [1,6]. Recent BBSRC/BRIC and follow-on (BB/FOF/272) projects established microfluidic techniques to measure the thermostability of 85000-fold less protein [7]. We aim to integrate the microscale techniques to simultaneously evaluate multiple bioprocess stresses and molecular properties, obtain a body of data for a range of biologics and formulations, then identify or disprove potential correlations between initial (Tm, Tg, B22 by DLS, activity), medium-term (forced degradation) and long-term (shelf life) properties. Workplan: Year 1 - training in analytical and bioprocess skills at NIBSC. Formulate and freeze dry a range of biologics [8,9] in process vials. Evaluate their biological activity and aggregation. Learn existing microscale and DoE techniques at UCL and NISBC, and validate them in process scale vials. Integrate microscale techniques to allow parallel evaluations of the impact of formulations on liquid and freeze dried sample stability, and sample properties. Year 2 - combine microscale techniques and Design of Experiments (DoE) to derive parallel surface response models for the impact of formulations on product Tm, Tg, B22, aggregation, tolerances to freeze-drying and forced degradation (liquid and freeze-dried), for a wide range of proteins, vaccines and cells. Year 3 - evaluate long-term storage (0-12 months at 70 to 45oC) of optimal and selected sub-optimal liquid and freeze-dried formulations in 10 ml vials. Measure biological activity, aggregates and misfolded forms, oxidation and deamidation by standard HPLC, DLS and LCMS techniques at UCL and NIBSC. Compare data to equivalent short-term property measurements, and medium term forced degradation studies from yr2, to identify or disprove correlations between them. Determine the best microscale DoE, measurement and bioprocess stress strategy for predicting 10 ml vial formulations with long-term stability. 1. Mannall GJ, Myers JP, Liddell J, Titchener-Hooker NJ, Dalby PA 2009 Biotech Bioeng 103:329 2. Grant Y, Matejtschuk P, Dalby PA 2009 Biotech Bioeng 104:957 3. Aucamp JP, Cosme AM, Lye GJ, Dalby PA 2005 Biotech Bioeng 89: 599 4. Aucamp JP, Martinez-Torres RJ, Hibbert EG, Dalby PA 2008 Biotech Bioeng 99:1303 5. Miller OJ, Hibbert EG, Ingram CU, Lye GJ, Dalby PA 2007 Biotech Letts 29:1759 6. Ahmad SS, Dalby PA 2010 Biotech Bioeng 108:322 7. Gaudet M, Remtulla N, Jackson SE, Main ERG, Bracewell DG, Aeppli G, Dalby PA 2010 Protein Science 19:1544 8. Hubbard A, Bevan S, Matejtschuk P 2007 Anal Bioanal Chem 387:2503 9. Matejtschuk P et al 2009 Biologicals 37:1-7