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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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Stability and traceability of mAb aggregate standards. The stability of NaCl-induced mAb1 (A + B) and mAb2 (C + D) aggregates was investigated. B is an enlargement of A, D is an enlargement of C. (E) Stability of FT-induced mAb1 using MAbPac SEC-1. (F) Stability of FT-induced mAb2 aggregates using Yarra SEC-4000. (G) Traceability of NaCl-induced mAb2 aggregates using MAbPac SEC-1. (H) Traceability of FT-induced mAb2 aggregates using Yarra SEC-4000 under cell culture conditions.
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Fig3: Stability and traceability of mAb aggregate standards. The stability of NaCl-induced mAb1 (A + B) and mAb2 (C + D) aggregates was investigated. B is an enlargement of A, D is an enlargement of C. (E) Stability of FT-induced mAb1 using MAbPac SEC-1. (F) Stability of FT-induced mAb2 aggregates using Yarra SEC-4000. (G) Traceability of NaCl-induced mAb2 aggregates using MAbPac SEC-1. (H) Traceability of FT-induced mAb2 aggregates using Yarra SEC-4000 under cell culture conditions.

Mentions: To evaluate stability of NaCl-induced aggregates, mAb1 and mAb2 aggregates were induced using 500 mmol L−1 NaCl and analyzed after storage at RT for up to 72 h (Figure 3A-D). MAb1 dimer content increased already after 2 h from 1.3% to 3.3% (Figure 3B) and mAb2 dimer increased within 72 h from 6.5% to 10.2% (Figure 3D), revealing that the aggregation reaction was not finished within this time period. These observations are in good accordance with other reports [26,31,32]. Stability analysis of FT-induced aggregates revealed that the aggregates formed were readily reversible (Figure 3E + F). MAb1 tetramer increased from 11% to 26% after 2 h storage at RT (Figure 3E). FT-induced aggregates of mAb2 seemed to be reversible. Increasing storage time at room temperature resulted in mAb2 monomer recovery with a corresponding decrease in dimer and oligomers (Figure 3F). After 1 h storage at RT, FT-induced mAb2 dimer decreased from 14% to 4% and the tetramer from 2.5% to 0.3%, whereas mAb2 monomer increased from 82.6% to 95.7%. This shift in aggregate distribution was also observed by Philo, who reported that incubation of Protein X at 29°C after FT led to a drop in dimer and larger aggregate content, with a corresponding increase in monomer [33]. Since Philo used Protein X, our results are the first report, to the best of our knowledge, which showed the reversibility of FT-induced aggregates for mAbs. Since the aggregates induced by NaCl and FT were unstable, all aggregate standards were prepared freshly for further experiments.Figure 3


Direct analysis of mAb aggregates in mammalian cell culture supernatant.

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

Stability and traceability of mAb aggregate standards. The stability of NaCl-induced mAb1 (A + B) and mAb2 (C + D) aggregates was investigated. B is an enlargement of A, D is an enlargement of C. (E) Stability of FT-induced mAb1 using MAbPac SEC-1. (F) Stability of FT-induced mAb2 aggregates using Yarra SEC-4000. (G) Traceability of NaCl-induced mAb2 aggregates using MAbPac SEC-1. (H) Traceability of FT-induced mAb2 aggregates using Yarra SEC-4000 under cell culture conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Stability and traceability of mAb aggregate standards. The stability of NaCl-induced mAb1 (A + B) and mAb2 (C + D) aggregates was investigated. B is an enlargement of A, D is an enlargement of C. (E) Stability of FT-induced mAb1 using MAbPac SEC-1. (F) Stability of FT-induced mAb2 aggregates using Yarra SEC-4000. (G) Traceability of NaCl-induced mAb2 aggregates using MAbPac SEC-1. (H) Traceability of FT-induced mAb2 aggregates using Yarra SEC-4000 under cell culture conditions.
Mentions: To evaluate stability of NaCl-induced aggregates, mAb1 and mAb2 aggregates were induced using 500 mmol L−1 NaCl and analyzed after storage at RT for up to 72 h (Figure 3A-D). MAb1 dimer content increased already after 2 h from 1.3% to 3.3% (Figure 3B) and mAb2 dimer increased within 72 h from 6.5% to 10.2% (Figure 3D), revealing that the aggregation reaction was not finished within this time period. These observations are in good accordance with other reports [26,31,32]. Stability analysis of FT-induced aggregates revealed that the aggregates formed were readily reversible (Figure 3E + F). MAb1 tetramer increased from 11% to 26% after 2 h storage at RT (Figure 3E). FT-induced aggregates of mAb2 seemed to be reversible. Increasing storage time at room temperature resulted in mAb2 monomer recovery with a corresponding decrease in dimer and oligomers (Figure 3F). After 1 h storage at RT, FT-induced mAb2 dimer decreased from 14% to 4% and the tetramer from 2.5% to 0.3%, whereas mAb2 monomer increased from 82.6% to 95.7%. This shift in aggregate distribution was also observed by Philo, who reported that incubation of Protein X at 29°C after FT led to a drop in dimer and larger aggregate content, with a corresponding increase in monomer [33]. Since Philo used Protein X, our results are the first report, to the best of our knowledge, which showed the reversibility of FT-induced aggregates for mAbs. Since the aggregates induced by NaCl and FT were unstable, all aggregate standards were prepared freshly for further experiments.Figure 3

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