Evaluating the effect of in-process material on the binding mechanisms of surrogate viral particles to a multi-modal anion exchange resin
Introduction
Regulatory agencies require assurance biopharmaceuticals intended for human use are free of viruses and other potential pathogens. For clinical and commercial license applications for biologics, the potential for downstream unit operations to provide viral clearance is evaluated utilizing scaled down models (ICH, 1999). In order to claim viral clearance for an individual unit operation, the basis for viral removal or inactivation should be orthogonal to the other claimed operations.
Newer generations of commercially available chromatography resins utilize a combination of fundamentally different mechanisms (e.g., electrostatic and hydrophobic interactions) to partition the target protein from impurities. These media commonly are described as mixed-mode or multi-modal resins. A key regulatory question arises whether a multi-modal chromatography step can be considered orthogonal to other single-mode chromatography steps such as IEX or HIC columns if they at least partially rely on the same type of ligand:virus interaction.
Previous studies (Brown et al., 2017) have shown that models for mammalian viruses (i.e. bacteriophage) PR772 and PP7 can be bound to a multi-modal resin, Capto Adhere, via interactions with both the hydrophobic and the anionic functional groups on the ligand while ΦX174’s binding was dominated by the anionic moiety. These conclusions were based on a step-wise experimental approach using solute surface analytics and resin screening experiments. The surface analytics measured the relative hydrophobicity (Johnson et al., 2017) of the phage in conjunction with previously measured estimated charge characteristics of the particles (Brorson et al., 2008). Together, those studies hypothesized the affinity of the particles to hydrophobic and charged functional groups a priori as well as showing the similarities in properties to the model mammalian viruses. The hypothesized binding was confirmed via independently demonstrating the affinity of the particles to single mode resins (i.e. AEX and HIC) in comparison to a multi-modal resin across a range of NaCl concentrations and pH values.
Our study advances that initial research by demonstrating how process intermediates containing a model antibody and typical process impurities impact phage interaction with multi-modal resins.
Section snippets
Materials
Capto Adhere chromatographic resin was purchased from GE Healthcare. The bulk resin was packed in a 0.66 cm i.d. Omnifit glass column which was purchased from Thermo Fisher Scientific. An Akta Avant 25 FPLC system operated with Unicorn software version 6.1 was used for chromatographic separations. The bacteriophage PR772, PP7, and ΦX174 were produced from strains purchased from ATCC (ATCC Catalog #: BAA-769-B1, 15692-B4, and 13706-B1 respectively) which were propagated and titered as described
Distribution co-efficient screening
The first step of our studies was to determine k-values at various pH and conductivity set points for the interaction of our model mAbs with the Capto Adhere resin. This approach allowed for determination of conditions under which the antibody would run in flowthrough mode (Curtis et al., 2003) (Strauss et al., 2009) and weak partitioning mode (Kelley et al., 2008) (Iskra et al., 2015). The higher the k-value, the stronger the affinity of the mAb to the resin. We performed highly parallel
Discussion
All bioprocessing intermediates contain some level of process residuals such as HCP, HMW species, or DNA. The goal of a flowthrough downstream column is to remove these from the feed stream via a binding mechanism, and at the same time provide viral clearance. Accumulation of these residuals on anion exchange resin impacts viral clearance (Miesegaes et al., 2010).
This study demonstrates the robustness of multi-modal chromatography to clear two of the three bacteriophage species by multi-modal
Conclusion
Through the guidance of bacteriophage removal studies using buffer only and in-process material, the binding mechanisms of bacteriophage capture onto a multi-modal resin were found to be a complex mixture of hydrophobic and charge based interactions. For PP7 and PR772, the binding mechanism for adsorption onto the multi-modal resin was confirmed to be one of synergetic binding by the positively charged quaternary amine and the hydrophobic aromatic phenyl group. Removal of these species could be
Disclaimer
This article reflects the views of the authors and should not be construed to represent FDA’s views or policies. The authors declare that they have no competing interests nor endorse specific resins.
Acknowledgements
The authors would like to acknowledge Jessica Dement-Brown for her assistance in protein analytics. This project was supported in part by an appointment to the Research Participation Program at FDA/CDER/OBP administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the DoE and FDA. Funding was also provided by the CDER Regulatory Science & Review Enhancement Program (RSR) 15-14 and the CDER/OPQ Research Review Coordinating Committee (RRCC).
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