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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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Molecular weight determination of mAb aggregate standards. MAb2 aggregate standards were induced using either NaCl (A) or FT (B) and analyzed using SEC-MALS.
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Fig2: Molecular weight determination of mAb aggregate standards. MAb2 aggregate standards were induced using either NaCl (A) or FT (B) and analyzed using SEC-MALS.

Mentions: Increased salt concentration reduces the colloidal stability by suppressing electrostatic repulsion, leading to aggregation [25]. We used this mechanism to induce aggregation of the two model proteins. The use of different NaCl concentrations induced the formation of small aggregates as well as large aggregates (Table 1). In comparison to pH-shift induced aggregation, fewer aggregates were formed using NaCl, but it is worth mentioning that NaCl-induction specifically formed one smaller HMW species. Using SEC-MALS, the molecular weight of the NaCl-induced HMW species was determined as a 300 kDa mAb2 dimer (Figure 2A). In addition, the light scattering signal revealed the presence of a large aggregate population, which was not visible using UV detection. With increasing NaCl concentrations mAb1 monomer content decreased and dimer increased from 0.6% (w/o NaCl) to 2.0% (1.5 mol L−1 NaCl). Similar to mAb1 also mAb2 monomer decreased with increasing NaCl concentration, whereas the dimer content increased more than fourfold from 1.1% (w/o NaCl) to 4.6% (1.5 mol L−1 NaCl). In contrast to mAb2, mAb1 also formed some degradation products. Our results correlate with the results of Fesinmeyer et al., who reported that even in milli-molar concentrations, salts promote mAb aggregate formation [26]. They suggested anion binding as possible reason for aggregation by lowering mAb conformational stability and reduced valence. Analysis with DLS revealed that mAb2 also formed large aggregates (Table 1). NaCl-induction increased the aggregate size up to an average diameter of around 75 nm (1.5 mol L−1 NaCl). However, mAb1 did not form large aggregates. Our work showed that increasing ionic strength by 100 mmol L−1 NaCl is sufficient to induce mAb dimers, and at concentrations higher than 500 mmol L−1 also to induce larger mAb aggregates. Cation concentration in the culture broth increases during the fermentation process due to pH control [27]. Consequently, osmolality in the bioreactor increases over time thus NaCl-induced aggregates are likely to be present in the cell culture broth. Since 500 mmol L−1 NaCl induced a significant amount of small aggregates (3.0% dimer) without forming large HMW species of mAb2, this concentration was used to induce mAb2 dimers for stability studies and the generation of aggregate standards.Figure 2


Direct analysis of mAb aggregates in mammalian cell culture supernatant.

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

Molecular weight determination of mAb aggregate standards. MAb2 aggregate standards were induced using either NaCl (A) or FT (B) and analyzed using SEC-MALS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Molecular weight determination of mAb aggregate standards. MAb2 aggregate standards were induced using either NaCl (A) or FT (B) and analyzed using SEC-MALS.
Mentions: Increased salt concentration reduces the colloidal stability by suppressing electrostatic repulsion, leading to aggregation [25]. We used this mechanism to induce aggregation of the two model proteins. The use of different NaCl concentrations induced the formation of small aggregates as well as large aggregates (Table 1). In comparison to pH-shift induced aggregation, fewer aggregates were formed using NaCl, but it is worth mentioning that NaCl-induction specifically formed one smaller HMW species. Using SEC-MALS, the molecular weight of the NaCl-induced HMW species was determined as a 300 kDa mAb2 dimer (Figure 2A). In addition, the light scattering signal revealed the presence of a large aggregate population, which was not visible using UV detection. With increasing NaCl concentrations mAb1 monomer content decreased and dimer increased from 0.6% (w/o NaCl) to 2.0% (1.5 mol L−1 NaCl). Similar to mAb1 also mAb2 monomer decreased with increasing NaCl concentration, whereas the dimer content increased more than fourfold from 1.1% (w/o NaCl) to 4.6% (1.5 mol L−1 NaCl). In contrast to mAb2, mAb1 also formed some degradation products. Our results correlate with the results of Fesinmeyer et al., who reported that even in milli-molar concentrations, salts promote mAb aggregate formation [26]. They suggested anion binding as possible reason for aggregation by lowering mAb conformational stability and reduced valence. Analysis with DLS revealed that mAb2 also formed large aggregates (Table 1). NaCl-induction increased the aggregate size up to an average diameter of around 75 nm (1.5 mol L−1 NaCl). However, mAb1 did not form large aggregates. Our work showed that increasing ionic strength by 100 mmol L−1 NaCl is sufficient to induce mAb dimers, and at concentrations higher than 500 mmol L−1 also to induce larger mAb aggregates. Cation concentration in the culture broth increases during the fermentation process due to pH control [27]. Consequently, osmolality in the bioreactor increases over time thus NaCl-induced aggregates are likely to be present in the cell culture broth. Since 500 mmol L−1 NaCl induced a significant amount of small aggregates (3.0% dimer) without forming large HMW species of mAb2, this concentration was used to induce mAb2 dimers for stability studies and the generation of aggregate standards.Figure 2

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