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Research ArticleResearch

Bioprocess: Robustness with Respect to Mycoplasma Species

Talia Faison, Julie Wang, Sarah Johnson, Matthew Brown, Meng-Jung Chiang, Sherri Dolan, Carl Breuning, Sai Rashmika Velugula-Yellela, Scott Lute, Erica J. Fratz-Berilla and Kurt Brorson
PDA Journal of Pharmaceutical Science and Technology March 2020, 74 (2) 201-212; DOI: https://doi.org/10.5731/pdajpst.2018.009613
Talia Faison
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Julie Wang
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Sarah Johnson
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Matthew Brown
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Meng-Jung Chiang
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Sherri Dolan
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Carl Breuning
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Sai Rashmika Velugula-Yellela
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Scott Lute
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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Erica J. Fratz-Berilla
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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  • For correspondence: Erica.berilla@fda.hhs.gov
Kurt Brorson
Division II/Office of Biotechnology Products/Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20903.
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    Figure 1

    A typical protein A purification chromatogram with peaks labeled with the corresponding fraction collected. The pH probe was taken off-line following elution to protect it during column regeneration and sanitization steps. Background for column chromatography and mycoplasma contamination studies, adapted from Wang et al., 2017, with permission.

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    Figure 2

    Growth kinetics of mycoplasma species at known contamination levels in simulated idle protein A column and in CHO cell coculture. (A-C) On-resin growth studies for model mycoplasma species. Plot legends are: LC = Load Control spike (no resin); MSS F = MabSelect SuRe, fouled; MSS N = MabSelect SuRe, naïve; PS F = ProSep-vA Ultra, fouled; PS N = ProSep-vA Ultra, naïve. M. arginini colony counts (C) for ProSep samples on day 3 exceeded countable plate range and are approximated. (D) Representative graph of mycoplasma growth curves in CHO coculture using 1 L spinner flasks. Data generated from the average of 1–4 replicate cultures per mollicute species, no data available for A. laidlawii on days 5–6.

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    Figure 3

    Sensitivity of mycoplasma to low-pH inactivation2. Differences in buffer (100 mM acetate) pH and contact time tolerance for M. orale compared to A. laidlawii. A. laidlawii is easily cleared to undetectable levels within 60 min at pH 3.8 whereas ≥2 Log10 CFU M. orale remain after exposure to even lower pH. A. laidlawii data from Wang et al., 2017, used with permission.

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    TABLE I

    Chromatography Steps and Buffer Componentsa

    Chromatography StepBuffer Composition
    Load controlHCCF
    Flow-throughHCCF
    PBSb washPBS, pH 7.2
    Salt washPBS + 1 M NaCl, pH 7.2
    Elution100 mM acetate, pH 2.9
    • ↵a Harvested cell culture fluid (HCCF) load was spiked with either M. orale or M. arginini in the current experiments and fractions were collected to measure the partitioning of total mycoplasma CFU present during protein A purification. Elution fraction was in-line neutralized with 3 M tris (pH 8.6). Background for column chromatography and mycoplasma contamination studies, adapted from Wang et al., 2017, with permission.

    • ↵b PBS is phosphate-buffered saline.

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    TABLE II

    Protein A Chromatographya

    Chromatography Parameter PatternFlow Rate (mL/Min)Salt Wash (Added to PBS)bElution pH (100 mM Glycine)Regeneration (6 M Urea)
    Best case11 M NaCl3.5Yes
    Center point1575 mM NaCl3.75No
    Worst case1150 mM NaCl4.0No
    • ↵a Best-case, center point, and worst-case factors previously determined as affecting the clearance of A. laidlawii were applied to M. orale and M. arginini in this study.

    • b PBS is phosphate-buffered saline.

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    TABLE III

    Chromatography Steps and Buffers to Prepare Resin for On-Resin Growth Experimentsa

    Chromatography StepBufferpHColumn Volumes
    Equilibration25 mM Tris + 100 mM NaCl7.52
    LoadHCCF725
    Wash25 mM Tris + 100 mM NaCl7.5≤3
    Secondary Wash25 mM Tris + 1 M NaCl7.54
    Wash25 mM Tris + 100 mM NaCl7.53
    Elution0.1 M Acetic Acid2.94
    Equilibration25 mM Tris + 100 mM NaCl7.55
    • ↵a Protein A chromatography media with silica (ProSep-vA Ultra) or agarose (MabSelect SuRe) backbones were cycled as indicated or used fresh to create “fouled” and “naive” resin conditions for mycoplasma spike.

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    TABLE IV

    Carryover Studiesa

    SpeciesChromatography Parameter Conditions1Load Control (log10 CFU)bFlow-through Burden (log10 CFU)Salt Wash Burden (log10 CFU)Elution LRVcCarryover (CFU/mL)
    A. laidlawiiBest case7.3 ± 0.16.9 ± 0.14.7 ± 0.15.9 ± 0.3<10
    Center point7.1 ± 0.26.7 ± 0.14.2 ± 0.44.7 ± 0.4<10
    Worst case7.3 ± 0.16.9 ± 0.14.3 ± 0.13.3 ± 0.225
    M. oraleBest case8.07.85.54.0700
    Center point7.3 ± 0.17.1 ± 0.25.1 ± 0.24.3 ± 0.91.9 × 103
    Worst case7.5 ± 0.47.5 ± 0.45.1 ± 0.93.2 ± 1.22.5 × 103
    Medium Column Worst case8.4 ± 0.17.6 ± 0.55.9 ± 0.52.8 ± 0.11.5 × 104
    M. argininiCenter point7.9 ± 0.37.7 ± 0.15.2 ± 0.45.0 ± 0.4105
    Worst case7.6 ± 0.57.5 ± 0.54.4 ± 0.53.3 ± 0.4205
    • ↵a Mycoplasma burden in input (load control), output (eluate), as well as flow-through and salt wash fractions were collected and measured for titer. Log reduction value (LRV) was calculated from the mycoplasma burden in the elution fractions. Carryover was measured from early effluent from a second cycle1. Best case was performed once as proof of concept for M. orale because the focus of these studies was to identify the conditions that would have poor mycoplasma clearance. Only center point and worst-case experiments were performed with M. arginini because of its higher LRVs compared to M. orale. Adapted from Wang et al., 2017, with permission.

    • ↵b CFU is colony-forming units.

    • ↵c LRV is log reduction value.

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    TABLE V

    Buffer Species Sensitivity of M. orale to Low-pH Inactivationa

    Test Article CompositionTreatment Time (Min)LRVb
    M. orale100 mM glycine pH 3.850.5 ± 0.1
    601.9 ± 0.6
    M. orale100 mM glycine pH 3.550.9 ± 0.3
    603.3 ± 0.4
    M. orale100 mM acetate pH 3.850.7 ± 0.2
    602.2 ± 0.5
    M. orale100 mM acetate pH 3.551.2 ± 0.3
    604.1 ± 0.5
    • ↵a Antibody-containing eluate was titrated to pH 3.5 or 3.8, spiked with M. orale and incubated for 60 min. Samples were taken at time 0 min (sample load concentration), 5 min, and 60 min, then tittered.

    • b LRV is log reduction value.

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    TABLE VI

    Solvent/Detergent (S/D) Studiesa

    SpeciesS/D CompositionTreatment Time (Min)LRVb
    A. laidlawii0.001% TNBPc50.10 ± 0.08
    0.005% Tween 80600.82 ± 0.34
    A. laidlawii0.1% TNBP55.11 ± 1.81
    0.5% Tween 8060>5.95 ± 0.36
    M. orale0.001% TNBP50.1 ± 0.1
    0.005% Tween 80600.1 ± 0.1
    M. orale0.1% TNBP51.1
    0.5% Tween 80603.8
    M. arginini0.1% TNBP52.9
    0.5% Tween 8060>3.3
    • ↵a Viability of A. laidlawii, M. orale, and M. arginini after S/D treatment. Mycoplasma-spiked harvested cell culture fluid was treated with standard S/D concentration of 0.1% Tri(n-butyl) phosphate (TNBP) and 0.5% Tween 80 solution; 100x diluted S/D was used as a sub-optimal comparator control. Complete inactivation of M. arginini was observed after 60 min. A. laidlawii data from Wang et al., 2017, used with permission.

    • b LRV is log reduction value. > indicates no visible mycoplasma colonies present.

    • ↵c TNBP is Tri(n-butyl) phosphate.

    • View popup
    TABLE VII

    Ultraviolet C (UVc) Irradiation Studies of A. Laidlawii and M. oralea

    Test Article CompositionUVc Dose DeliveryLoad Titer (log10 CFU)bLRVc
    1% A. laidlawii 1X Media100 J/m26.5>5.5
    300 J/m26.5>5.5
    1% A. laidlawii 3X Media30 J/m26.52.1
    100 J/m26.2>5.2
    240 J/m26.2>5.2
    1% M. orale 1X Media100 J/m24.8>3.8
    300 J/m24.8>3.8
    1% M. orale 3X Media100 J/m24.7>3.7
    283 J/m24.7>3.7
    • ↵a Negative control irradiation dose performed on 3X media with A. laidlawii at 30 J/ m2. UVc treatment was dose independent in complete mycoplasma clearance.

    • ↵b CFU is colony-forming unit.

    • ↵c LRV is log reduction value. > indicates no visible mycoplasma colonies present.

    • View popup
    TABLE VIII

    Media and Viral Filter Retention of A. laidlawii with Virosart Filtersa

    Filter TypeTest Article CompositionVolume Filtered (mL)Loading (L/m2)Load Titer (log10 CFU)bLRVc
    Virosart Media Filter0.5% A. laidlawii 1X OptiCHO881756.1>5.1
    921836.2>5.2
    Virosart Media Filter0.1% A. laidlawii 1X OptiCHO2254505.3>4.3
    2224435.6>4.6
    Virosart HF Viral Filter0.5% A. laidlawii PBS + 1g/L BSA1533076.1>5.1
    1503006.1>5.1
    Virosart CPV Viral Filter0.1% A. laidlawii PBS + 1g/L BSA601205.2>4.2
    601205.2>4.2
    • ↵a Small-pore viral filters in general were effective barriers against mycoplasma penetration yielding at least 4.2 LRV clearance. Even at a higher load density, the second-generation filter (Virosart HF) shows substantial mycoplasma retention (5.1 Log10 CFU).

    • ↵b CFU is colony-forming unit.

    • ↵c LRV is log reduction value. > indicates no visible mycoplasma colonies present.

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PDA Journal of Pharmaceutical Science and Technology: 74 (2)
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Bioprocess: Robustness with Respect to Mycoplasma Species
Talia Faison, Julie Wang, Sarah Johnson, Matthew Brown, Meng-Jung Chiang, Sherri Dolan, Carl Breuning, Sai Rashmika Velugula-Yellela, Scott Lute, Erica J. Fratz-Berilla, Kurt Brorson
PDA Journal of Pharmaceutical Science and Technology Mar 2020, 74 (2) 201-212; DOI: 10.5731/pdajpst.2018.009613

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Bioprocess: Robustness with Respect to Mycoplasma Species
Talia Faison, Julie Wang, Sarah Johnson, Matthew Brown, Meng-Jung Chiang, Sherri Dolan, Carl Breuning, Sai Rashmika Velugula-Yellela, Scott Lute, Erica J. Fratz-Berilla, Kurt Brorson
PDA Journal of Pharmaceutical Science and Technology Mar 2020, 74 (2) 201-212; DOI: 10.5731/pdajpst.2018.009613
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Keywords

  • Mycoplasma
  • Acholeplasma laidlawii
  • Mycoplasma arginini
  • Mycoplasma orale
  • Bioprocessing
  • Protein A chromatography
  • Chromatography column/media
  • Solvent/detergent
  • Low-pH hold
  • Spike/clearance
  • Cell culture
  • Viral filter
  • Media filter
  • Ultraviolet-C irradiation

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