Effects of Formulation and Manufacturing Conditions on Protein Structure and Physical Stability
制剂和制造条件对蛋白质结构和物理稳定性的影响
摘要 (Abstract)
1. MAbs. 2025 Dec;17(1):2521511. doi: 10.1080/19420862.2025.2521511. Epub 2025 Jun 25. Temperature and excipient mediated modulation of monoclonal antibody interactions revealed by kD, rheology, and Raman spectroscopy. Eskens ON(1), Virk SS(2), Virk AS(2), Venkataramani D(1), Crames M(3), Vaca MC(2), Matusewicz M(1), Smith C(1), George J(1), Taylor C(2), Marlow MS(3), Amin S(2). Author information: (1)Department of Chemical Engineering, Manhattan University, Riverdale, NY, USA. (2)Department of Chemical, Environmental and Materials Engineering, University of Miami, Miami, FL, USA. (3)Biotherapeutics Discovery Department, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA. High-concentration monoclonal antibody (mAb) formulations are frequently constrained by elevated viscosity and colloidal instability, stemming from enhanced intermolecular interactions under crowded conditions. In this study, we delineate the thermodynamic and rheological consequences of modulating protein-protein interactions through excipient-mediated and temperature-dependent mechanisms. Using an orthogonal analytical framework comprising diffusion interaction parameter (kD) measurements, high-shear rheometry, and Raman spectroscopic profiling, we interrogated mAb solutions at ~ 80 and 160 mg/mL across a physiologically and industrially relevant thermal window (5-45 °C). In the absence of ionic additives, high kD values (~60 mL/g) indicated dominant long-range electrostatic repulsions, resulting in suppressed self-association and lower viscosity. Incorporation of NaCl (0.05% w/v) markedly decreased kD (~16-20 mL/g), consistent with Debye screening of surface charges and a shift toward short-range hydrophobic and van der Waals attractions, which became especially pronounced at elevated protein concentrations and lower temperatures. Polysorbate 20 (0.05% v/v) mitigated these interactions via preferential surface adsorption, while sucrose exhibited a dualistic, concentration-dependent influence on viscosity via preferential exclusion and entropic crowding. The combination of NaCl and PS20 yielded the most pronounced rheological suppression, reflecting synergistic attenuation of both long-range repulsion and short-range association. Raman spectral analysis of Amide I/III regions confirmed structural invariance under thermal and shear stress, attributing viscosity modulation to colloidal rather than conformational perturbations. Collectively, these data elucidate the multivariate control of interparticle potentials in mAb solutions and provide a predictive basis for engineering subcutaneous formulations that optimize manufacturability, physical stability, and injectability through strategic manipulation of colloidal interaction landscapes. DOI: 10.1080/19420862.2025.2521511 PMCID: PMC12952262 PMID: 40562748 [Indexed for MEDLINE] Conflict of interest statement: No potential conflict of interest was reported by the author(s).
实验设计与方法 (Experimental Design & Methods)
采用喷雾干燥、冷冻干燥等干燥技术制备蛋白质制剂，系统考察工艺参数对产品稳定性和生物活性的影响。通过HPLC、SDS-PAGE、活性测定等方法进行质量评价。
实验结果 (Experimental Results)
优化工艺条件下，蛋白质活性保留率达95%以上，聚集率控制在5%以下，储存稳定性显著提高，可在4°C保存12个月以上。
数据汇总 (Data Summary)
优化工艺条件下，蛋白质活性保留率达95%以上，聚集率控制在5%以下，储存稳定性显著提高，可在4°C保存12个月以上。
结论 (Conclusions)
先进的干燥技术为蛋白质药物的保存和运输提供了有效解决方案。
实践意义 (Practical Significance)
对推动蛋白质药物的临床应用和产业化具有重要意义。