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Direct analysis of mAb aggregates in mammalian cell culture supernatant.

Paul AJ, Schwab K, Hesse F - BMC Biotechnol. (2014)

Bottom Line: Protein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Biotechnology (IAB), Biberach University of Applied Sciences, 88400, Biberach, Germany. paul@hochschule-bc.de.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

SEC analysis of mAb2 cell culture samples. CHO cell culture supernatant analyzed using a common SEC (TSKgel G3000SWXL) column (A). Analysis of purified mAb2 compared to SFM4CHO medium using MAbPac SEC-1 (B) and CHO DG44 supernatant using Yarra SEC-4000 (C).
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Fig1: SEC analysis of mAb2 cell culture samples. CHO cell culture supernatant analyzed using a common SEC (TSKgel G3000SWXL) column (A). Analysis of purified mAb2 compared to SFM4CHO medium using MAbPac SEC-1 (B) and CHO DG44 supernatant using Yarra SEC-4000 (C).

Mentions: 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]. This approach is time-consuming, laborious and can critically influence the aggregation status. Direct analysis of cell culture samples containing mAb using SEC is complicated, since components such as DNA, lipids and HCPs can interfere with the analysis of the product [10]. Accordingly, analysis of CHO mAb2 fermentation supernatant samples using a common SEC column (TSKgel G3000SWXL) was insufficient and therefore confirmed that separation of mAb monomer from other supernatant signals disturbed aggregate detection (Figure 1A). Since analysis of cell culture samples using SEC appeared to be a problem of separation capacity, columns promising higher separation efficiencies were tested. Using a SEC column specifically designed for mAb analysis (MAbPac SEC-1) showed that cell culture medium components eluted later than purified mAb monomer (Figure 1B). Serum-free media often contain growth factors, lipoproteins and other factors as growth-promoting supplements. It was confirmed that none of the media components interfered with the analysis of the mAb2 monomer as these components showed higher retention times. Furthermore, analysis of the supernatant from the CHO DG44 culture using a 3 μm SEC column (Yarra SEC-4000) revealed that also the cell culture components in the supernatant eluted later than the mAb monomer (Figure 1C). These host cell components, i.e. cellular proteins, DNA and other components usually complicate the detection of target proteins [22]. Our results indicated that cell culture and medium impurities eluted later than the mAb monomer using columns providing sufficiently high separation efficiency. Therefore, both columns were tested for their applicability for the analysis of mAb aggregates in cell culture samples.Figure 1


Direct analysis of mAb aggregates in mammalian cell culture supernatant.

Paul AJ, Schwab K, Hesse F - BMC Biotechnol. (2014)

SEC analysis of mAb2 cell culture samples. CHO cell culture supernatant analyzed using a common SEC (TSKgel G3000SWXL) column (A). Analysis of purified mAb2 compared to SFM4CHO medium using MAbPac SEC-1 (B) and CHO DG44 supernatant using Yarra SEC-4000 (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256052&req=5

Fig1: SEC analysis of mAb2 cell culture samples. CHO cell culture supernatant analyzed using a common SEC (TSKgel G3000SWXL) column (A). Analysis of purified mAb2 compared to SFM4CHO medium using MAbPac SEC-1 (B) and CHO DG44 supernatant using Yarra SEC-4000 (C).
Mentions: 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]. This approach is time-consuming, laborious and can critically influence the aggregation status. Direct analysis of cell culture samples containing mAb using SEC is complicated, since components such as DNA, lipids and HCPs can interfere with the analysis of the product [10]. Accordingly, analysis of CHO mAb2 fermentation supernatant samples using a common SEC column (TSKgel G3000SWXL) was insufficient and therefore confirmed that separation of mAb monomer from other supernatant signals disturbed aggregate detection (Figure 1A). Since analysis of cell culture samples using SEC appeared to be a problem of separation capacity, columns promising higher separation efficiencies were tested. Using a SEC column specifically designed for mAb analysis (MAbPac SEC-1) showed that cell culture medium components eluted later than purified mAb monomer (Figure 1B). Serum-free media often contain growth factors, lipoproteins and other factors as growth-promoting supplements. It was confirmed that none of the media components interfered with the analysis of the mAb2 monomer as these components showed higher retention times. Furthermore, analysis of the supernatant from the CHO DG44 culture using a 3 μm SEC column (Yarra SEC-4000) revealed that also the cell culture components in the supernatant eluted later than the mAb monomer (Figure 1C). These host cell components, i.e. cellular proteins, DNA and other components usually complicate the detection of target proteins [22]. Our results indicated that cell culture and medium impurities eluted later than the mAb monomer using columns providing sufficiently high separation efficiency. Therefore, both columns were tested for their applicability for the analysis of mAb aggregates in cell culture samples.Figure 1

Bottom Line: Protein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP).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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Biotechnology (IAB), Biberach University of Applied Sciences, 88400, Biberach, Germany. paul@hochschule-bc.de.

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus