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 （UCL生化工程），Paul Matejtschuk （NIBSC， HPA）我们的目标是建立一个自动化的微尺度平台来评估治疗性蛋白质，疫苗和细胞制剂，并确定物理性质测量，强制降解研究以及液体和冻干制剂长期保质期之间的相关性（或缺乏相关性）。为了尽量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性的，需要许多时间和样品消耗实验。为了节省时间和材料，最初的配方筛选通常会测量诸如熔融温度（生物热稳定性- Tm和玻璃化转变- Tg），聚集倾向（B22值）或在高温下强制降解形成的聚集体（光散射，SEC）等特性。使用这些测量作为长期（1-2年）储存稳定性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尺度方法，将少量生物制剂置于生物过程压力下，如蛋白质重折叠、搅拌和冷冻干燥[1,2]（EPSRC与NIBSC的学生），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和后续（BB/FOF/272）项目建立了微流体技术来测量85000倍的蛋白质[7]的热稳定性。我们的目标是整合微尺度技术，同时评估多种生物过程压力和分子特性，获得一系列生物制剂和配方的数据，然后确定或反驳初始（Tm, Tg, B22）、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进行分析和生物工艺技能培训。在工艺瓶中配制和冷冻干燥一系列生物制剂[8,9]。评估它们的生物活性和聚集性。在UCL和NISBC学习现有的微尺度和DoE技术，并在工艺规模小瓶中进行验证。整合微尺度技术，允许平行评估配方对液体和冻干样品稳定性和样品性质的影响。第2年-结合微尺度技术和实验设计（DoE），得出平行的表面响应模型，以确定配方对产品Tm， Tg, B22，聚集，对冻干和强制降解（液体和冻干）的耐受性的影响，适用于各种蛋白质，疫苗和细胞。第3年-评估最佳和选定的次最佳液体和冻干制剂在10毫升小瓶中的长期储存（0-12个月，70至45摄氏度）。通过标准HPLC， DLS和LCMS技术测量UCL和NIBSC的生物活性，聚集体和错误折叠形式，氧化和脱酰胺。将数据与等效的短期属性测量数据和从r2开始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。确定最佳的微尺度DoE，测量和生物过程应力策略，以预测具有长期稳定性的10ml小瓶配方。1. 李建军，李建军，李建军，李建军。2009生物技术与工程学报(3):349 - 349。张建军，张建军，张建军，等。2009生物工程学报(4):344 - 344。李建军，李建军，李建军，李建军。2005生物技术与工程学报(3):533 - 544。张建军，张建军，张建军，等。2008生物技术与工程学报（英文版）刘建军，刘建军，李建军，李建军，李建军，李建军。杨建军，杨建军，杨建军，等。高德德M， Remtulla N, Jackson SE, Main ERG, Bracewell DG, Aeppli G, Dalby PA 2010。张建军，张建军，张建军，等。2007生物化学学报（自然科学版）。马志强等2009生物制品37 (1):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