Center for Biotechnology & Interdisciplinary Studies, Department of Chemical & Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Mol Pharm. 2012 Apr 2;9(4):744-51. doi: 10.1021/mp200448j. Epub 2012 Feb 17.
Monoclonal antibodies display highly variable solution properties such as solubility and viscosity at elevated concentrations (>50 mg/mL), which complicates antibody formulation and delivery. To understand this complex behavior, it is critical to measure the underlying protein self-interactions that govern the solution properties of antibody suspensions. We have evaluated the pH-dependent self-association behavior of three monoclonal antibodies using self-interaction chromatography for a range of pH values commonly used in antibody formulations (pH 4.4-6). At low ionic strength (<25 mM), we find that each antibody is more associative at near-neutral pH (pH 6) than at low pH (pH 4.4). At high ionic strength (>100 mM), we observe the opposite pH-dependent pattern of antibody self-association. Importantly, this inversion in self-association behavior is not unique to multidomain antibodies, as similar pH-dependent behavior is observed for some small globular proteins (e.g., ribonuclease A and α-chymotrypsinogen). We also find that the opalescence of concentrated antibody solutions (90 mg/mL) is minimized at low ionic strength at pH 4.4 and high ionic strength at pH 6, in agreement with the self-interaction measurements conducted at low antibody concentrations (5 mg/mL). Our results highlight the complexity of antibody self-association and emphasize the need for systematic approaches to optimize the solution properties of concentrated antibody formulations.
单克隆抗体在高浓度（>50mg/mL）下显示出高度可变的溶液性质，如溶解度和粘度，这使得抗体的配方和输送变得复杂。为了理解这种复杂的行为，测量控制抗体悬浮液溶液性质的潜在蛋白质自相互作用是至关重要的。我们使用自相互作用色谱法评估了三种单克隆抗体在抗体配方中常用的一系列 pH 值（pH4.4-6）下的 pH 依赖性自缔合行为。在低离子强度（<25mM）下，我们发现每种抗体在近中性 pH（pH6）下比在低 pH（pH4.4）下更具缔合性。在高离子强度（>100mM）下，我们观察到相反的 pH 依赖性抗体自缔合模式。重要的是，这种自缔合行为的反转不仅限于多结构域抗体，因为一些小的球形蛋白（如核糖核酸酶 A 和α-糜蛋白酶原）也观察到类似的 pH 依赖性行为。我们还发现，在低离子强度 pH4.4 和高离子强度 pH6 下，浓缩抗体溶液（90mg/mL）的光学不透明度最小，这与在低抗体浓度（5mg/mL）下进行的自相互作用测量结果一致。我们的结果突出了抗体自缔合的复杂性，并强调需要系统的方法来优化浓缩抗体配方的溶液性质。
