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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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Analysis of mAb aggregate formation, DNA content, host cell and medium components in the supernatant of a mAb2-producing CHO cell line. Supernatant was analyzed for mAb aggregate formation directly after inoculation and after 144 h cultivation (A + B). Amount of mAb2 monomer, dimer and oligomers was obtained from biological triplicates ± standard deviation. B is an enlargement of A. DNA content of collected SEC fractions was analyzed using NanoDrop 1000 spectrophotometer (C). For determination of HCPs and culture medium components, SEC fractions were pooled, concentrated and analyzed using SDS-PAGE under reducing conditions with subsequent silver staining (D).
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Fig4: Analysis of mAb aggregate formation, DNA content, host cell and medium components in the supernatant of a mAb2-producing CHO cell line. Supernatant was analyzed for mAb aggregate formation directly after inoculation and after 144 h cultivation (A + B). Amount of mAb2 monomer, dimer and oligomers was obtained from biological triplicates ± standard deviation. B is an enlargement of A. DNA content of collected SEC fractions was analyzed using NanoDrop 1000 spectrophotometer (C). For determination of HCPs and culture medium components, SEC fractions were pooled, concentrated and analyzed using SDS-PAGE under reducing conditions with subsequent silver staining (D).

Mentions: The cultivation showed typical growth, substrate and metabolite concentrations for mAb production (Additional file 2: Figure S1). Directly after inoculation neither monomer nor mAb aggregates were detected (Figure 4A + B). Chromatograms of the SE-HPLC analysis obtained from the supernatant of the three shake flasks showed identical results (Figure 4B). Retention times of the signals obtained immediately after the start of cultivation (14–16 min) corresponded to the elution times obtained in the analysis of the cell-free supernatant from a culture of a non-producing CHO DG44 host cell line (Figure 3H), indicating that the signals were caused by host cell impurities and medium components such as DNA, lipids, HCPs, secreted cellular metabolites and excess nutrients [10,34]. To ensure that these cell culture contaminants do not interfere with quantification of the mAb2 monomer and aggregates in the CHO supernatant, the corresponding chromatography fractions were collected and analyzed for DNA (Figure 4C) and HCPs (Figure 4D). DNA analysis of the collected SE-HPLC fractions revealed that the CHO mAb2 supernatant contained a significant amount of DNA (up to 15 ng μL−1). The finally formulated recombinant mAb product has to contain less than 10 ng/dose DNA [35]. Therefore the impurities have to be removed from the cell culture supernatant after harvest using several DSP operations [22]. However, DNA was detected only in the fractions containing host cell and culture medium components and not observed in the SEC fractions used for mAb aggregate quantification. In order to determine the amount of HCPs, the corresponding SEC fractions were pooled, concentrated and separated using reducing SDS-PAGE with subsequent silver staining (Figure 4D). The supernatant of the non-producing CHO cells (DG44) and the supernatant of the mAb2-producing CHO cells (CHO mAb2) both showed a huge amount of signals, indicating presence of HCPs in the supernatant. The strongest bands in the mAb2-producing CHO supernatant were caused by the heavy chain (around 50 kDa) and the light chain (around 25 kDa) of reduced mAb2, also visible in the purified mAb2 control (Ctr). Analysis of the collected SEC fractions revealed that the mAb2 monomer and aggregate fraction contained mainly heavy and light chain, indicating that nearly no HCPs were present. The monomer and aggregate fraction showed a signal at 150 kDa caused by unreduced mAb2 as well as bands between heavy and light chain, which were also visible in the control, whereas the fractions collected from host cell and culture medium components showed additional signals. It is known that mammalian cells secrete HCPs together with the product [36]. Nevertheless, the mAb2 monomer and aggregate fractions collected from the SE-HPLC analysis contained no detectable amounts of DNA and were nearly free of HCP enabling mAb aggregate quantification directly in the supernatant of CHO cells using the SEC columns tested in this work. However, the results also revealed the presence of a large amount of DNA and cell culture components in the supernatant of CHO cell culture and emphasize the importance of the use of appropriate SEC columns for aggregate analysis.Figure 4


Direct analysis of mAb aggregates in mammalian cell culture supernatant.

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

Analysis of mAb aggregate formation, DNA content, host cell and medium components in the supernatant of a mAb2-producing CHO cell line. Supernatant was analyzed for mAb aggregate formation directly after inoculation and after 144 h cultivation (A + B). Amount of mAb2 monomer, dimer and oligomers was obtained from biological triplicates ± standard deviation. B is an enlargement of A. DNA content of collected SEC fractions was analyzed using NanoDrop 1000 spectrophotometer (C). For determination of HCPs and culture medium components, SEC fractions were pooled, concentrated and analyzed using SDS-PAGE under reducing conditions with subsequent silver staining (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Analysis of mAb aggregate formation, DNA content, host cell and medium components in the supernatant of a mAb2-producing CHO cell line. Supernatant was analyzed for mAb aggregate formation directly after inoculation and after 144 h cultivation (A + B). Amount of mAb2 monomer, dimer and oligomers was obtained from biological triplicates ± standard deviation. B is an enlargement of A. DNA content of collected SEC fractions was analyzed using NanoDrop 1000 spectrophotometer (C). For determination of HCPs and culture medium components, SEC fractions were pooled, concentrated and analyzed using SDS-PAGE under reducing conditions with subsequent silver staining (D).
Mentions: The cultivation showed typical growth, substrate and metabolite concentrations for mAb production (Additional file 2: Figure S1). Directly after inoculation neither monomer nor mAb aggregates were detected (Figure 4A + B). Chromatograms of the SE-HPLC analysis obtained from the supernatant of the three shake flasks showed identical results (Figure 4B). Retention times of the signals obtained immediately after the start of cultivation (14–16 min) corresponded to the elution times obtained in the analysis of the cell-free supernatant from a culture of a non-producing CHO DG44 host cell line (Figure 3H), indicating that the signals were caused by host cell impurities and medium components such as DNA, lipids, HCPs, secreted cellular metabolites and excess nutrients [10,34]. To ensure that these cell culture contaminants do not interfere with quantification of the mAb2 monomer and aggregates in the CHO supernatant, the corresponding chromatography fractions were collected and analyzed for DNA (Figure 4C) and HCPs (Figure 4D). DNA analysis of the collected SE-HPLC fractions revealed that the CHO mAb2 supernatant contained a significant amount of DNA (up to 15 ng μL−1). The finally formulated recombinant mAb product has to contain less than 10 ng/dose DNA [35]. Therefore the impurities have to be removed from the cell culture supernatant after harvest using several DSP operations [22]. However, DNA was detected only in the fractions containing host cell and culture medium components and not observed in the SEC fractions used for mAb aggregate quantification. In order to determine the amount of HCPs, the corresponding SEC fractions were pooled, concentrated and separated using reducing SDS-PAGE with subsequent silver staining (Figure 4D). The supernatant of the non-producing CHO cells (DG44) and the supernatant of the mAb2-producing CHO cells (CHO mAb2) both showed a huge amount of signals, indicating presence of HCPs in the supernatant. The strongest bands in the mAb2-producing CHO supernatant were caused by the heavy chain (around 50 kDa) and the light chain (around 25 kDa) of reduced mAb2, also visible in the purified mAb2 control (Ctr). Analysis of the collected SEC fractions revealed that the mAb2 monomer and aggregate fraction contained mainly heavy and light chain, indicating that nearly no HCPs were present. The monomer and aggregate fraction showed a signal at 150 kDa caused by unreduced mAb2 as well as bands between heavy and light chain, which were also visible in the control, whereas the fractions collected from host cell and culture medium components showed additional signals. It is known that mammalian cells secrete HCPs together with the product [36]. Nevertheless, the mAb2 monomer and aggregate fractions collected from the SE-HPLC analysis contained no detectable amounts of DNA and were nearly free of HCP enabling mAb aggregate quantification directly in the supernatant of CHO cells using the SEC columns tested in this work. However, the results also revealed the presence of a large amount of DNA and cell culture components in the supernatant of CHO cell culture and emphasize the importance of the use of appropriate SEC columns for aggregate analysis.Figure 4

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