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. 2020 Sep;117(9):2802-2815.
doi: 10.1002/bit.27436. Epub 2020 Jun 4.

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

Erica J Fratz-Berilla  1 , Phillip Angart  1 , Ryan J Graham  2   3 , David N Powers  1 , Adil Mohammad  2 , Casey Kohnhorst  4 , Talia Faison  1 , Sai Rashmika Velugula-Yellela  1 , Nicholas Trunfio  5 , Cyrus Agarabi  1
Affiliations

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

Erica J Fratz-Berilla et al. Biotechnol Bioeng. 2020 Sep.

Abstract

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.

Keywords: Chinese hamster ovary (CHO) cell culture; biomanufacturing; bioprocessing; critical quality attribute (CQA); monoclonal antibody (mAb); mycoplasma; partial least squares (PLS) regression; principal component analysis (PCA).

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Conflict of interest statement

The authors declare that there are no conflict of interests.

Figures

Figure 1
Figure 1
Charge distributions in mAb purified from control perfusion bioreactors and those contaminated with Mycoplasma arginini by perfusion day. Data represent the mean of three technical replicates and error bars represent ±1 standard deviation of the mean. Statistical significance is defined by a two‐tailed paired t test between corresponding control bioreactor perfusion days (Day 2‐High and Day 3‐High) or perfusion day before mycoplasma spike (Day 9‐Low and Day 12‐High) (*p < .01). For complete datasets on CHO cell growth and mycoplasma growth, please see Fratz‐Berilla et al. (2019) [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Purity and aggregation in mAb purified from control perfusion bioreactors and those contaminated with Mycoplasma arginini by perfusion day. Data represent the mean of three technical replicates and error bars represent ±1 standard deviation of the mean. Statistical significance is defined by two‐tailed paired t test between corresponding control bioreactor perfusion days (Day 2‐High and Day 3‐High) or perfusion day before mycoplasma spike (Day 9‐Low and Day 12‐High) (*p < .01) [Color figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Glycan profiles in mAb purified from control perfusion bioreactors and those contaminated with Mycoplasma arginini by perfusion day. High mannose species, especially those with 6 mannoses or more, increase in the M. arginini‐contaminated bioreactors [Color figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Mannose 5 species in mAb purified from control perfusion bioreactors and those contaminated with Mycoplasma arginini by perfusion day. Solid bars indicate perfusion days in which the bioreactor is uncontaminated and striped bars indicate the presence of contamination. The top panel compares Day 2‐High and Day 2‐Control, the middle panel compares Day 3‐High and Day 3‐Control, and the bottom panel compares Day 9‐Low and Day 12‐High [Color figure can be viewed at wileyonlinelibrary.com]
Figure 5
Figure 5
Mannose 6–9 species in mAb purified from control perfusion bioreactors and those contaminated with Mycoplasma arginini by perfusion day. Solid bars indicate perfusion days in which the bioreactor is uncontaminated and striped bars indicate the presence of contamination. The top panel compares Day 2‐High and Day 2‐Control, the middle panel compares Day 3‐High and Day 3‐Control, and the bottom panel compares Day 9‐Low and Day 12‐High [Color figure can be viewed at wileyonlinelibrary.com]
Figure 6
Figure 6
PCA loadings plots of Day 9‐Low and Day 12‐High. Culture data included are concentration of mycoplasma (red), IgG titer (gray), nutrients (glucose and glutamine—green), arginine (yellow), and waste (ammonium, lactate, and glutamate—purple). Product quality data included are glycan species (G0F‐N, G0F, G0‐N, G0, G1Fa, G1Fb—gray; M5, M6, M7, M8, high mannose—blue), aggregate and nonaggregate (main) species (gray; * distinguishes aggregate data) and charge variant species (orange). IgG, immunoglobulin G; PCA, principal component analysis [Color figure can be viewed at wileyonlinelibrary.com]
Figure 7
Figure 7
PLS models of NIR spectra to predicted mycoplasma contamination in Day 9‐Low and Day 12‐High. NIR spectra can predict mycoplasma contaminations at high (4–8 log [CFU/ml]) concentrations, but some false positives occur at low mycoplasma concentrations. The batch age predictions indicate a strong linear correlation and lack of polarized clustering between the predicted and observed batch age, which provides evidence of reliability of the saturated mycoplasma clusters in the prediction models. CFU, colony‐forming unit; NIR, near‐infrared; PLS, partial least square
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