Waters Corporation, Instrument/Core Research/Fundamentals, 34 Maple Street, Milford, MA 01757, USA.
J Chromatogr A. 2023 Jul 19;1701:464050. doi: 10.1016/j.chroma.2023.464050. Epub 2023 May 9.
The increasing demand for the characterization of large biomolecules such as monoclonal antibodies, double-stranded deoxyribonucleic acid (dsDNA), and virus-like particles (VLPs) is raising fundamental questions pertaining to their absorption (ingress) and escape (egress) kinetics from fully porous particles. The exact expression of their concentration profiles is derived as a function of time and radial position across a single sub-3 μm Bridge-Ethylene-Hybrid (BEH) Particle present in size exclusion chromatography (SEC) columns. The boundary condition at the external surface area of the particle is a rectangular concentration profile mimicking the passage of the chromatographic zone. Four different BEH Particles were considered in the calculations depending on the molecular size of the analyte: 2.0 μm 100 Å BEH Particles for small molecules, 2.0 μm 200 Å BEH Particles for monoclonal antibodies, 2.0 μm 300 Å BEH Particles for dsDNA (100 base pairs), and 2.5 μm 900 Å BEH Particles for virus-like particles (VLPs). The calculated concentration profiles of small molecules and monoclonal antibodies confirm that all BEH Particles present in the column reach quasi-instantaneously thermodynamic equilibrium with the bulk mobile phase during the passage of the chromatographic band. This is no longer the case for larger biomolecules such as dsDNA or VLPs, especially when the SEC particle is located near the column inlet and for high velocities. The kinetics of biomolecule egress is slower than its kinetics of ingress leading to pronounced peak tailing. The mean concentration of the largest biomolecules in the SEC particles remains always smaller than the maximum bulk concentration. This persistent and transient intra-particle diffusion regime has direct implications on the theoretical expressions of the observed retention factors and plate heights. Classical theories of chromatography assume uniform spatial distribution of the analyte in the particle volume: this hypothesis is not verified for the largest biomolecules. These results imply that non-porous particles or monolithic structures are the most promising stationary phases for the separation and purification of the largest biomolecules in life science.
对于诸如单克隆抗体、双链脱氧核糖核酸 (dsDNA) 和病毒样颗粒 (VLPs) 等大型生物分子的特性描述的需求不断增加，这就提出了一些基本问题，涉及它们从全多孔颗粒中的吸收（进入）和逃逸（流出）动力学。作为时间和穿过单个亚 3μm 桥接乙氧基杂化 (BEH) 颗粒的径向位置的函数，推导出它们浓度分布的精确表达式。颗粒外表面的边界条件是一个模拟色谱区通过的矩形浓度分布。根据分析物的分子大小，计算中考虑了四种不同的 BEH 颗粒：2.0μm 100Å BEH 颗粒用于小分子，2.0μm 200Å BEH 颗粒用于单克隆抗体，2.0μm 300Å BEH 颗粒用于 dsDNA（100 个碱基对），2.5μm 900Å BEH 颗粒用于病毒样颗粒 (VLPs)。小分子和单克隆抗体的计算浓度分布证实，在色谱带通过期间，柱中存在的所有 BEH 颗粒都与体相流动相迅速达到热力学平衡。对于较大的生物分子，如 dsDNA 或 VLPs，情况不再如此，尤其是当 SEC 颗粒位于柱入口附近且流速较高时。生物分子流出的动力学比进入的动力学慢，导致明显的峰尾。最大生物分子在 SEC 颗粒中的平均浓度始终小于最大体相浓度。这种持续和瞬态的颗粒内扩散状态对观察到的保留因子和板高的理论表达式有直接影响。色谱的经典理论假设分析物在颗粒体积中的空间分布均匀：对于最大的生物分子，这一假设并不成立。这些结果表明，对于生命科学中最大生物分子的分离和纯化，非多孔颗粒或整体结构是最有前途的固定相。
