The effects of spatial organization of hydrophobic and charged moieties on multimodal (MM) cation-exchange ligands were examined by studying protein retention behavior on two commercial chromatographic media, Capto™ MMC and Nuvia™ cPrime™. Proteins with extended regions of surface-exposed aliphatic residues were found to have enhanced retention on the Capto MMC system as compared to the Nuvia cPrime resin. The results further indicated that while the Nuvia cPrime ligand had a strong preference for interactions with aromatic groups, the Capto MMC ligand appeared to interact with both aliphatic and aromatic clusters on the protein surfaces. These observations were formalized into a new set of protein surface property descriptors, which quantified the local distribution of electrostatic and hydrophobic potentials as well as distinguishing between aromatic and aliphatic properties. Using these descriptors, high-performing quantitative structure-activity relationship (QSAR) models (R(2)>0.88) were generated for both the Capto MMC and Nuvia cPrime datasets at pH 5 and pH 6. Descriptors of electrostatic properties were generally common across the four models; however both Capto MMC models included descriptors that quantified regions of aliphatic-based hydrophobicity in addition to aromatic descriptors. Retention was generally reduced by lowering the ligand densities on both MM resins. Notably, elution order was largely unaffected by the change in surface density, but smaller and more aliphatic proteins tended to be more affected by this drop in ligand density. This suggests that modulating the exposure, shape and density of the hydrophobic moieties in multimodal chromatographic systems can alter the preference for surface exposed aliphatic or aromatic residues, thus providing an additional dimension for modulating the selectivity of MM protein separation systems.

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There has been a recent drive in commercial large-scale production of biotechnology products to convert current batch mode processing to continuous processing manufacturing. There have been reports of model systems capable of adapting and linking upstream and downstream technologies into a continuous manufacturing pipeline. However, in many of these proposed continuous processing model systems, viral safety has not been comprehensively addressed. Viral safety and detection is a highly important and often expensive regulatory requirement for any new biological product. To ensure success in the adaption of continuous processing to large-scale production, there is a need to consider the development of approaches that allow for seamless incorporation of viral testing and clearance/inactivation methods. In this review, we outline potential strategies to apply current viral testing and clearance/inactivation technologies to continuous processing, as well as modifications of existing unit operations to ensure the successful integration of viral clearance into the continuous processing of biological products. Biotechnol. Bioeng. 2017;114: 21-32. © 2016 Wiley Periodicals, Inc.

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We propose a standard protocol for integrity testing the residence-time distribution (RTD) in a "Jig in a Box" design (JIB)-a previously described tortuous-path, tubular, low-pH, continuous viral inactivation reactor, ensuring that biopharmaceutical products will be incubated for the required minimum residence time, t . t is the time by which just 0.001% of the total product containing virus has exited the incubation chamber (i.e., t ). This t is selected to ensure a >4-log reduction in viral load. As current tracers and in-line analytical technologies may not be able to detect tracers at the 0.001% level, an alternative approach is required. The authors describe a method for deriving t from t (i.e., the time at which 0.5% of the product has emerged from the reactor outlet) and an experimentally confirmed offset value, t = t -t . The authors also evaluate tracer candidates-including 100-nm-diameter gold nanoparticles, dextrose, monoclonal antibody, and riboflavin-for pre-process acceptability and the effects of viscosity, molecular diffusion coefficient, and particle size. The authors show that a JIB will yield t and RTDs that are nearly identical for multiple tracers due to Dean vortex induced mixing. Results indicate that almost any small-molecule tracer that is generally recognized as safe can be used in pre-use integrity testing of a continuous viral inactivation reactor under the Deans values (De) of 119-595.