Protein Binding Kinetics in Multimodal Systems: Implications for Protein Separations.

In this work, quartz crystal microbalance with dissipation (QCM-D) was employed to study the kinetic processes involved in the interaction of proteins with self-assembled monolayers (SAMs) of multimodal (MM) ligands. SAMs were fabricated to mimic two chromatographic multimodal resins with varying accessibility of the aromatic moiety to provide a well-defined model system. Kinetic parameters were determined for two different proteins in the presence of the arginine and guanidine and a comparison was made with chromatographic retention data. The results indicated that the accessibility of the ligand's aromatic moiety can have an important impact on the kinetics and chromatographic retention behavior. Interestingly, arginine and guanidine had very different effects on the protein adsorption and desorption kinetics in these MM systems. For cytochrome C, arginine resulted in a significant decrease and increase in the adsorption and desorption rates, respectively, while guanidine produced a dramatic increase in the desorption rate, with minimal effect on the adsorption rate. In addition, at different concentrations of arginine, two distinct kinetic scenarios were observed. For α-chymotrypsin, the presence of 0.1 M guanidine in the aromatic exposed ligand system produced an increase in the adsorption rate and only a moderate increase in the desorption rate, which helped to explain the surprising increase in the chromatographic salt elution concentration. These results demonstrate that protein adsorption kinetics in the presence of different mobile phase modifiers and MM ligand chemistries can play an important role in contributing to selectivity in MM chromatography.

Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding.

Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al. Langmuir 2014, 30 (44), 13205-13216). We present a clear confirmation of those studies by performing single-molecule force spectroscopy (SMFS) measurements of unbinding complemented with molecular dynamics (MD) calculations of the adsorption free energy of ubiquitin in two distinct orientations with self-assembled monolayers (SAMs) functionalized with "Capto" ligands. These orientations were maintained in the SMFS experiments by tethering ubiquitin mutants to SAM surfaces through strategically located cysteines, thus exposing the desired faces of the protein. Analogous orientations were maintained in MD simulations using suitable constraining methods. Remarkably, despite differences between the finer details of experimental and simulation methodologies, they confirm a clear preference for the previously hypothesized binding face of ubiquitin. Furthermore, MD simulations provided significant insights into the mechanism of protein binding onto this multimodal surface. Because SMFS and MD simulations both directly probe protein-surface interactions, this work establishes a key link between experiments and simulations at molecular scale through the determination of protein face-specific binding energetics. Our approach may have direct applications in biophysical systems where face- or orientation-specific interactions are important, such as biomaterials, sensors, and biomanufacturing.

Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Model-based process development for the purification of a modified human growth hormone using multimodal chromatography.

This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone. Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins. Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal. Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling. The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification.

Methods development in multimodal chromatography with mobile phase modifiers using the steric mass action model.

The ability to predict downstream protein purification processes is of great value in the biopharmaceutical industry; saving time, cost and resources. While many complex models exist, the appropriate use of simple models can be a useful tool for rapidly designing and optimizing processes as well as for risk analysis and establishing parameter ranges. In this study, the steric mass action isotherm is success-fully employed to predict the chromatographic behavior of a multimodal anionic Capto adhere systemin the presence of various mobile phase modifiers. An experimental protocol consisting of only a few column experiments is shown to be sufficient to establish the model. Proof of concept is carried out using human insulin and bovine serum albumin which have varying degrees of hydrophobicity, charge and size. Finally, the model predictions are verified under various experimental conditions and the unique selectivity of this multimodal system is explored and compared with traditional anion exchange resins.The simple model approach described here represents a rapid and useful method for model based process development of multimodal chromatography.

Model-based risk analysis of coupled process steps.

A section of a biopharmaceutical manufacturing process involving the enzymatic coupling of a polymer to a therapeutic protein was characterized with regards to the process parameter sensitivity and design space. To minimize the formation of unwanted by-products in the enzymatic reaction, the substrate was added in small amounts and unreacted protein was separated using size-exclusion chromatography (SEC) and recycled to the reactor. The quality of the final recovered product was thus a result of the conditions in both the reactor and the SEC, and a design space had to be established for both processes together. This was achieved by developing mechanistic models of the reaction and SEC steps, establishing the causal links between process conditions and product quality. Model analysis was used to complement the qualitative risk assessment, and design space and critical process parameters were identified. The simulation results gave an experimental plan focusing on the "worst-case regions" in terms of product quality and yield. In this way, the experiments could be used to verify both the suggested process and the model results. This work demonstrates the necessary steps of model-assisted process analysis, from model development through experimental verification.