PULSE SPR: A High Throughput Method to Evaluate the Domain Stability of Antibodies

Downstream
purification of therapeutic antibodies requires candidates
to be stable under various stress conditions, such as low pH. Current
approaches to assess the conformational or colloidal stability of
biologics may require significant amounts of material, and the testing
may occur over an extended period of time. Furthermore, typical methodologies
for early stability testing often do not directly address low pH stability,
but focus more on conditions suitable for long-term drug product storage.
Here we report a high-throughput method that measures protonation-induced
unfolding of ligand binding sites for stability evaluation by surface
plasmon resonance or PULSE SPR. This method, which requires only several
micrograms of sample, is highly sensitive to the structural integrity
of ligand binding sites and correlates well with thermal and chemical
conformational stability. Combined with ligands targeting different
regions of antibodies, this method allows a comprehensive assessment
of antibody domain stability. By applying PULSE SPR, we found that
antibodies with different complementarity-determining regions (CDRs),
frameworks, formats, and interchain disulfide bonds showed different
stabilities under acidic conditions. Additionally, biologics that
aggregated in solution also had poor low pH stability. Taken together,
PULSE SPR is a high-throughput and low sample consumption method that
can be adopted to evaluate antibody domain stability and aggregation
at low pH, which are two important aspects of therapeutic biologics.

治疗性抗体的下游纯化环节要求候选抗体在低pH等多种应激条件下保持稳定。当前用于评估生物制剂构象或胶体稳定性的方法往往需要消耗大量样品，且检测周期较长。此外，早期稳定性测试的典型方法通常无法直接覆盖低pH稳定性评估，而是更侧重于适配药物产品长期储存的实验条件。本研究报道一种高通量检测方法：通过表面等离子体共振（Surface Plasmon Resonance, SPR）或PULSE SPR技术，检测配体结合位点的质子化诱导解折叠以实现稳定性评估。该方法仅需数微克样品，对配体结合位点的结构完整性具有极高灵敏度，且与热致及化学诱导的构象稳定性呈现良好相关性。结合靶向抗体不同区域的配体使用时，该方法可实现抗体结构域稳定性的全面评估。通过应用PULSE SPR技术，本研究发现：具有不同互补决定区（Complementarity-Determining Regions, CDRs）、框架区、抗体格式及链间二硫键的抗体在酸性条件下表现出各异的稳定性；此外，溶液中发生聚集的生物制剂其低pH稳定性也普遍较差。综上，PULSE SPR是一种高通量、低样品消耗的检测方法，可用于评估低pH条件下的抗体结构域稳定性与聚集情况，而这两点正是治疗性生物制剂的两项核心评价指标。