A Novel, Q‐PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A Chromatography

Abstract

Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

Characterizing and enhancing virus removal by protein A chromatography

“…(Zhang et al, 2009;2014 through but decreased during washes. The studies performed here do not directly demonstrate such direct association but it is the simplest mechanism consistent with the data.…”

“…These methods have been used widely to study the separation of the product-and processrelated impurities Kelley et al, 2008;Sisodiya, Lequieu, Rodriguez, McDonald, & Lazzareschi, 2012;Wensel, Kelley, & Coffman, 2008). The limited application of HTS for virus clearance studies is likely due to the cost, complexity, and need for specialized labs, all of which can be overcome in scale-down protein A models using quantitative methods to measure CHO endogenous, noninfectious RVLP (M. Zhang et al, 2009Zhang et al, , 2014 | 847 hydrogen-bond, or combinations of these functional groups that reverse the effect. The limited application of HTS for virus clearance studies is likely due to the cost, complexity, and need for specialized labs, all of which can be overcome in scale-down protein A models using quantitative methods to measure CHO endogenous, noninfectious RVLP (M. Zhang et al, 2009Zhang et al, , 2014 | 847 hydrogen-bond, or combinations of these functional groups that reverse the effect.…”

Quality by design approach for viral clearance by protein a chromatography

“…For RVLP, the viral RNA was extracted using Qiagen EZ1 virus mini kit v2.0 with the EZ1/EZ1 advanced robotic system, according to vendor instructions. Free viral nucleic acids not associated with intact virus particles were removed based on the digestion procedures described earlier (Lute et al, ; Zhang et al, ) with slight modifications (10 μL DNase I, 40 μL of DNase I buffer, and 350 μL sample volume).…”

“…Such traditional full studies can be extremely expensive and time‐consuming to perform if using virus‐spiking to determine viral clearance capacity. Previous publications report comparable clearance when using QPCR assays that quantify either CHO endogenous retrovirus‐like particles (RVLP) or spiked X‐MuLV, and that retrovirus removal by Protein A could be evaluated by RVLP removal using samples from a Protein A process characterization studies (Zhang et al, ). In this report, we propose an experimentally determined QbD‐based approach for assessing viral clearance by Protein A chromatography.…”

Conference Summary: Gaps, Lessons Learned, and Areas for Improvement

“…This was first reported in 2009 by M. Zhang of Genentech Inc. (6) and expanded upon at the 2011 Symposium by M. Zhang (Session IV). It was reported that RVLP removal by protein A chromatography is comparable to that measured from spiked MuLV, and that RVLP removal determination can use full-scale samples.…”