Article Figures & Data
Figures
- 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.
- 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.
- 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.
Tables
Chromatography Step Buffer Composition Load control HCCF Flow-through HCCF PBSb wash PBS, pH 7.2 Salt wash PBS + 1 M NaCl, pH 7.2 Elution 100 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.
Chromatography Parameter Pattern Flow Rate (mL/Min) Salt Wash (Added to PBS)b Elution pH (100 mM Glycine) Regeneration (6 M Urea) Best case 1 1 M NaCl 3.5 Yes Center point 1 575 mM NaCl 3.75 No Worst case 1 150 mM NaCl 4.0 No ↵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.
- TABLE III
Chromatography Steps and Buffers to Prepare Resin for On-Resin Growth Experimentsa
Chromatography Step Buffer pH Column Volumes Equilibration 25 mM Tris + 100 mM NaCl 7.5 2 Load HCCF 7 25 Wash 25 mM Tris + 100 mM NaCl 7.5 ≤3 Secondary Wash 25 mM Tris + 1 M NaCl 7.5 4 Wash 25 mM Tris + 100 mM NaCl 7.5 3 Elution 0.1 M Acetic Acid 2.9 4 Equilibration 25 mM Tris + 100 mM NaCl 7.5 5 ↵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.
Species Chromatography Parameter Conditions1 Load Control (log10 CFU)b Flow-through Burden (log10 CFU) Salt Wash Burden (log10 CFU) Elution LRVc Carryover (CFU/mL) A. laidlawii Best case 7.3 ± 0.1 6.9 ± 0.1 4.7 ± 0.1 5.9 ± 0.3 <10 Center point 7.1 ± 0.2 6.7 ± 0.1 4.2 ± 0.4 4.7 ± 0.4 <10 Worst case 7.3 ± 0.1 6.9 ± 0.1 4.3 ± 0.1 3.3 ± 0.2 25 M. orale Best case 8.0 7.8 5.5 4.0 700 Center point 7.3 ± 0.1 7.1 ± 0.2 5.1 ± 0.2 4.3 ± 0.9 1.9 × 103 Worst case 7.5 ± 0.4 7.5 ± 0.4 5.1 ± 0.9 3.2 ± 1.2 2.5 × 103 Medium Column Worst case 8.4 ± 0.1 7.6 ± 0.5 5.9 ± 0.5 2.8 ± 0.1 1.5 × 104 M. arginini Center point 7.9 ± 0.3 7.7 ± 0.1 5.2 ± 0.4 5.0 ± 0.4 105 Worst case 7.6 ± 0.5 7.5 ± 0.5 4.4 ± 0.5 3.3 ± 0.4 205 ↵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.
Test Article Composition Treatment Time (Min) LRVb M. orale100 mM glycine pH 3.8 5 0.5 ± 0.1 60 1.9 ± 0.6 M. orale100 mM glycine pH 3.5 5 0.9 ± 0.3 60 3.3 ± 0.4 M. orale100 mM acetate pH 3.8 5 0.7 ± 0.2 60 2.2 ± 0.5 M. orale100 mM acetate pH 3.5 5 1.2 ± 0.3 60 4.1 ± 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.
Species S/D Composition Treatment Time (Min) LRVb A. laidlawii 0.001% TNBPc 5 0.10 ± 0.08 0.005% Tween 80 60 0.82 ± 0.34 A. laidlawii 0.1% TNBP 5 5.11 ± 1.81 0.5% Tween 80 60 >5.95 ± 0.36 M. orale 0.001% TNBP 5 0.1 ± 0.1 0.005% Tween 80 60 0.1 ± 0.1 M. orale 0.1% TNBP 5 1.1 0.5% Tween 80 60 3.8 M. arginini 0.1% TNBP 5 2.9 0.5% Tween 80 60 >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.
Filter Type Test Article Composition Volume Filtered (mL) Loading (L/m2) Load Titer (log10 CFU)b LRVc Virosart Media Filter 0.5% A. laidlawii 1X OptiCHO 88 175 6.1 >5.1 92 183 6.2 >5.2 Virosart Media Filter 0.1% A. laidlawii 1X OptiCHO 225 450 5.3 >4.3 222 443 5.6 >4.6 Virosart HF Viral Filter 0.5% A. laidlawii PBS + 1g/L BSA 153 307 6.1 >5.1 150 300 6.1 >5.1 Virosart CPV Viral Filter 0.1% A. laidlawii PBS + 1g/L BSA 60 120 5.2 >4.2 60 120 5.2 >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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