Biologics Development, Bristol Myers Squibb, 38 Jackson Road, Devens, MA 01434.
Biologics Development, Bristol Myers Squibb, 38 Jackson Road, Devens, MA 01434.
J Chromatogr A. 2021 Apr 26;1643:462008. doi: 10.1016/j.chroma.2021.462008. Epub 2021 Mar 18.
Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants.
生物制品（如单克隆抗体 [mAbs]）的电荷变体通常在稳定性和生物活性方面起着重要作用。然而，这些电荷变体的特性分析具有挑战性，主要是因为缺乏高效和有效的分离方法。在这项工作中，我们提出了一种新颖的使用方法，即将一种已建立的、高生产力的连续色谱法，即多柱逆流溶剂梯度纯化（MCSGP），用于创建更丰富的产品，以便更好地用于分析特性分析。我们展示了这种分离方法的原理，并将其与传统的批处理高效液相色谱（HPLC）或快速蛋白液相色谱（FPLC）方法进行了比较，以分离不同 mAb 的电荷变体为例。在大多数情况下，我们能够表明，与传统的分级方法相比，使用相同的分离条件，MCSGP 方法能够提供更高的纯度和数量的样品。在一个这样的例子中，使用 MCSGP 方法制备的样品在 10 小时的处理时间内达到了 95%的纯度，而使用 FPLC 和 HPLC 方法制备的样品在 48 小时和 300 小时的处理时间内分别达到了 78%和 87%的纯度。我们进一步评估了使用阳离子交换色谱（CEX）和阴离子交换色谱（AEX）树脂的盐和 pH 梯度的电荷变体富集策略，以提供更有效的分离，并在富集后减少样品处理量。结果，我们发现我们能够利用不同的梯度来改变某些带电物质的富集能力。最后，我们总结了本工作中使用的 mAb 电荷变体，并强调了使用连续色谱法富集电荷变体对分析特性分析的益处。该方法为电荷变体的富集提供了新的选择，并促进了电荷变体的分析特性分析。
