Direct analysis of mAb aggregates in mammalian cell culture supernatant.
Direct analysis of mAb aggregates in mammalian cell culture supernatant.
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DOI：
10.1186/s12896-014-0099-3
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发表时间：
2014-11-29
影响因子：
3.5
通讯作者：
Hesse F
中科院分区：
文献类型：
--
作者：
Paul AJ;Schwab K;Hesse F
单克隆抗体（mAb）生产过程中的蛋白质聚集可能发生在上游和下游加工（DSP）中。目前用于确定细胞培养期间聚集体形成的方法包括尺寸排阻色谱法（SEC），其具有先前的亲和色谱步骤以去除干扰细胞培养组分。预纯化步骤本身已经可以影响蛋白质聚集，因此不一定反映细胞培养物中存在的真实的聚集体含量。为了直接分析中国仓鼠卵巢（CHO）细胞培养物上清液中mAb聚集体的形成，我们建立了一个方案，该方案允许使用SEC进行聚集体定量，而无需伪造的预纯化步骤。使用3 μm硅胶SEC色谱柱或专为mAb聚集体分析定制的SEC色谱柱，可将mAb单体和聚集体与干扰细胞培养物组分分离，从而可直接测定上清液中的聚集体。单克隆抗体产生的CHO DG 44细胞系的抗体聚集体分析证明了该方法的可行性。令人惊讶的是，CHO细胞的上清液由超过75%的mAb二聚体和更大的寡聚体组成，代表了比迄今为止文献中报道的显著更高的聚集体含量。本研究强调，使用适当的SEC色谱柱和适当的mAb聚集体标准品直接在细胞培养物上清液中进行聚集体定量是可行的，而不会通过先前的亲和色谱法进行伪造。此外，我们的研究结果表明，聚集体的形成应直接在细胞培养中解决，而不仅仅是DSP中的问题。本文的在线版本（doi：10.1186/s12896-014-0099-3）包含补充材料，可供授权用户使用。
Protein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP). Current methods to determine aggregate formation during cell culture include size exclusion chromatography (SEC) with a previous affinity chromatography step in order to remove disturbing cell culture components. The pre-purification step itself can already influence protein aggregation and therefore does not necessarily reflect the real aggregate content present in cell culture. To analyze mAb aggregate formation directly in the supernatant of Chinese hamster ovary (CHO) cell culture, we established a protocol, which allows aggregate quantification using SEC, without a falsifying pre-purification step. The use of a 3 μm silica SEC column or a SEC column tailored for mAb aggregate analysis allows the separation of mAb monomer and aggregates from disturbing cell culture components, which enables aggregate determination directly in the supernatant. Antibody aggregate analysis of a mAb-producing CHO DG44 cell line demonstrated the feasibility of the method. Astonishingly, the supernatant of the CHO cells consisted of over 75% mAb dimer and larger oligomers, representing a substantially higher aggregate content than reported in literature so far. This study highlights that aggregate quantification directly in the cell culture supernatant using appropriate SEC columns with suitable mAb aggregate standards is feasible without falsification by previous affinity chromatography. Moreover, our results indicate that aggregate formation should be addressed directly in the cell culture and is not only a problem in DSP. The online version of this article (doi:10.1186/s12896-014-0099-3) contains supplementary material, which is available to authorized users.
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