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

研究人员：Paul Dalby(生化工程，UCL)，Paul Matejtschuk(NIBSC，HPA)我们的目标是建立一个自动化的微型平台，以评估治疗性蛋白质、疫苗和细胞配方，并确定物理性质测量、强制降解研究和液体和冻干配方的长期货架期之间是否存在相关性或缺乏意义：为了最大限度地减少生物制剂的化学和物理降解，可以在复杂的配方中添加赋形剂。冻干或液体配方的优化目前是经验的，需要许多时间和样本消耗的实验。为了节省时间和材料，初始配方筛选通常测量熔融温度(生物热稳定性-TM和玻璃化转变-Tg)、聚集倾向(B22值)或在高温下被迫降解形成的聚集体(光散射，SEC)等性质。使用这种测量作为长期(1-2年)储存稳定性的指标的有效性仍然存在争议，因为降解机制可能很复杂。我们最近建立了精确的微型方法，使少量生物制品承受生物过程压力，如蛋白质复性、搅拌和冷冻干燥[1，2](EPSRC与NIBSC合作)，并快速评估它们的热稳定性[3，4]、活性[1，2，5]和聚集倾向[1，6]。最近BBSRC/BRIC及其后续项目(BB/FOF/272)建立了微流控技术来测量85000倍以下蛋白质的热稳定性[7]。我们的目标是集成微尺度技术来同时评估多种生物过程压力和分子特性，获得一系列生物制品和配方的大量数据，然后识别或反驳初始(TM、TG、B22通过DLS，活性)、中期(强制降解)和长期(保质期)特性之间的潜在相关性。工作计划：第一年--在NIBSC进行分析和生物处理技能培训。在工艺瓶中配制和冷冻干燥一系列生物制品[8，9]。评价其生物活性和聚集性。在UCL和NISBC学习现有的微尺度和能源化技术，并在工艺规模的小瓶中进行验证。集成微型技术，以允许并行评估配方对液体和冻干样品稳定性以及样品特性的影响。第2年-结合微尺度技术和实验设计(DOE)，为各种蛋白质、疫苗和细胞的配方对产品TM、TG、B22、聚集性、对冷冻干燥和强制降解(液体和冷冻干燥)的耐受性的影响得出平行的表面响应模型。第3年-评估10毫升瓶装最佳和选定次佳液体和冻干配方的长期储存(70至45℃下0-12个月)。在UCL和NIBSC使用标准的高效液相、DLS和LC MS技术测量生物活性、聚集体和错误折叠形式、氧化和脱酰胺。将数据与相当的短期房地产测量和yr2的中期强迫退化研究进行比较，以确定或反驳它们之间的相关性。确定用于预测具有长期稳定性的10毫升小瓶配方的最佳微尺度DOE、测量和生物过程应激策略。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 2007 Biotech Letts 29：1759 6.Ahmad SS，Dalby 2010 Biotech Bioeng 108：3228.哈伯德·A，贝文·S，Matejtschuk P 2007肛门生物化学387：2503 9.Matejtschuk P等2009生物化学37：1-7

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