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

微型冻干和液体治疗制剂，用于研究强制降解与长期保质期之间的关系

• 批准号: BB/J003824/1
• 负责人: Paul Dalby
• 金额: $ 13.24万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ003824%2F1
• 关键词: Microscale; freeze; dried; liquid; formulations

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

研究者：保罗·达尔比（生物化学工程，伦敦大学学院），保罗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，疫苗和细胞制剂，并确定任何相关性液体和冻干制剂的物理性质测量、强制降解研究和长期有效期之间的差异（或缺乏差异）为了最大限度地减少生物制剂的化学和物理降解，可以在复杂制剂中添加辅料。目前，冻干或液体制剂的优化是经验性的，需要大量时间和消耗样本的实验。为了节省时间和材料，初始配方筛选通常测量诸如熔融温度（生物热稳定性- Tm和玻璃化转变- Tg）、聚集倾向（B22值）或通过在高温下强制降解形成的聚集体（光散射，SEC）的性质。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，使少量生物制剂经受生物过程应力，如蛋白质重折叠、搅拌和冷冻干燥[1，2]（EPSRC与NIBSC的学生关系），并快速评估其热稳定性[3，4]、活性[1，2，5]和聚集倾向[1，6]。最近的BBSRC/BRIC和后续项目（BB/FOF/272）建立了微流体技术来测量85000倍以下蛋白质的热稳定性[7]。我们的目标是整合微尺度技术，同时评估多种生物过程应力和分子特性，获得一系列生物制剂和制剂的数据，然后确定或反驳初始（Tm，Tg，B22，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1年-在NIBSC进行分析和生物工艺技能培训。在工艺小瓶中配制并冷冻干燥一系列生物制剂[8，9]。评价其生物活性和聚集性。在UCL和NISBC学习现有的微量和DoE技术，并在工艺规模的小瓶中验证它们。集成微尺度技术，以允许平行评估制剂对液体和冻干样品稳定性以及样品性质的影响。第2年-结合联合收割机微尺度技术和实验设计（DoE），推导出各种蛋白质、疫苗和细胞的制剂对产品Tm、Tg、B22、聚集、冻干耐受性和强制降解（液体和冻干）影响的平行表面响应模型。第3年-评估10 ml小瓶中最佳和选定次佳液体和冻干制剂的长期储存（70至45 ℃下0-12个月）。在UCL和NIBSC通过标准HPLC、DLS和LCMS技术测量生物活性、聚集体和错误折叠形式、氧化和脱酰胺。将数据与第2年的等效短期性能测量和中期强制降解研究进行比较，以确定或反驳它们之间的相关性。确定最佳的微量DoE、测量和生物工艺应力策略，以预测具有长期稳定性的10 ml小瓶制剂。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.米勒OJ，希伯特EG，英格拉姆CU，Lye GJ，Dalby PA 2007生物技术学报29：1759 6. Ahmad SS，Dalby PA 2010 Biotech Bioeng 108：322 7. Gaudet M，Remtulla N，杰克逊SE，主ERG，Bracewell DG，Aeppli G，Dalby PA 2010蛋白质科学19：1544 8. Hubbard A，Bevan S，Matejtschuk P 2007 Anal Bioanal Chem 387：2503 9. Matejtschuk P等2009 Biologicals 37：1-7