Abstract
BackgroundProtein 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.ResultsThe 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0099-3) contains supplementary material, which is available to authorized users.
Highlights
Protein aggregation during monoclonal antibody production can occur in upstream and downstream processing (DSP)
size exclusion chromatography (SEC)-based analysis of monoclonal antibodies (mAbs) aggregates in cell culture supernatant MAbs produced in mammalian cells are secreted into the cell culture supernatant, where formation of aggregates can be detected using SEC analysis after a Protein A capture step [4,15]
Our results indicated that cell culture and medium impurities eluted later than the mAb monomer using columns providing sufficiently high separation efficiency
Summary
Protein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP). The pre-purification step itself can already influence protein aggregation and 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. DSP offers the opportunity to remove aggregates, but this often leads to a reduction of protein yields Another strategy involves reducing the formation of aggregates in the cell culture [10]. Phillips et al showed that acidic pH values of Protein A elution led to substantial protein aggregation and precipitation [18] This implies that Protein A purification itself might influence the aggregation status, purified samples do not necessarily reflect the aggregation state of mAbs in cell culture
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