Continuous low pH viral inactivation: Operation and scaling strategy informs viral clearance study.

A continuous viral inactivation (CVI) tubular reactor was designed for low pH viral inactivation within a continuous downstream system across multiple scales of operation. The reactors were designed to provide a minimum residence time of >60 min. The efficacy of this tubular reactor was tested with xenotropic murine leukemia virus (X-MuLV) through pulse injection experiments. It was determined that the minimum residence time of the small-scale reactor design, when operated at the target process flow rate, occurred between 63 and 67 min. Inactivation kinetics were compared between continuous operation and standard batch practices using three monoclonal antibodies. The quantification of the virus log reduction values (LRV) was similar between the two modes of operation and most of the acid-treated samples had virus concentrations below the limit of detection. However, residual infectivity was still present in the endpoint batch samples of two experiments while the continuous samples always remained below the limit of detection. This provides the foundation for leveraging a standard batch-based model to quantify the LRV for a CVI unit operation.

Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches.

The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization.

Systematic Interpolation Method Predicts Antibody Monomer-Dimer Separation by Gradient Elution Chromatography at High Protein Loads.

A previously developed empirical interpolation (EI) method is extended to predict highly overloaded multicomponent elution behavior on a cation exchange (CEX) column based on batch isotherm data. Instead of a fully mechanistic model, the EI method employs an empirically modified multicomponent Langmuir equation to correlate two-component adsorption isotherm data at different salt concentrations. Piecewise cubic interpolating polynomials are then used to predict competitive binding at intermediate salt concentrations. The approach is tested for the separation of monoclonal antibody monomer and dimer mixtures by gradient elution on the cation exchange resin Nuvia HR-S. Adsorption isotherms are obtained over a range of salt concentrations with varying monomer and dimer concentrations. Coupled with a lumped kinetic model, the interpolated isotherms predict the column behavior for highly overloaded conditions. Predictions based on the EI method shows good agreement with experimental elution curves for protein loads up to 40 mg mL-1 column or about 50% of the column binding capacity. The approach can be extended to other chromatographic modalities and to more than two components.

Gradient elution behavior of proteins in hydrophobic interaction chromatography with a U-shaped retention factor curve under overloaded conditions.

The gradient elution hydrophobic interaction chromatography of a monoclonal antibody that exhibits U-shaped retention as a function of the ammonium sulfate concentration is investigated for overloaded conditions at protein loads up to 30% of the column equilibrium binding capacity. Protein load and gradient slope affect both elution peak shape and protein recovery during the gradient. Higher protein loads result in tailing peaks with near 100% recovery that transition to fronting peaks and incomplete recovery as the protein load is reduced. The gradient slope also affects peak shape and recovery. Tailing peaks with lower recovery are obtained with sharper gradients and the most tailing peak and lowest recovery are obtained when step elution rather than gradient is implemented. Modeling the chromatographic elution based on independently determined adsorption isotherms as a function of protein and ammonium sulfate concentration predicts results in agreement with the experimental trends confirming that the unusual chromatographic behavior observed is due to the U-shaped protein binding as a function of the ammonium sulfate concentration. Although less pronounced than in the dilute limit, the U-shaped binding still produces peak shapes and recovery losses as a function of gradient slope that differ from those seen for systems where retention is a monotonic function of salt concentration.

Gradient elution behavior of proteins in hydrophobic interaction chromatography with U-shaped retention factor curves.

Protein retention in hydrophobic interaction chromatography is described by the solvophobic theory as a function of the kosmostropic salt concentration. In general, an increase in salt concentration drives protein partitioning to the hydrophobic surface while a decrease reduces it. In some cases, however, protein retention also increases at low salt concentrations resulting in a U-shaped retention factor curve. During gradient elution the salt concentration is gradually decreased from a high value thereby reducing the retention factor and increasing the protein chromatographic velocity. For these conditions, a steep gradient can overtake the protein in the column, causing it to rebind. Two dynamic models, one based on the local equilibrium theory and the other based on the linear driving force approximation, are presented. We show that the normalized gradient slope determines whether the protein elutes in the gradient, partially elutes, or is trapped in the column. Experimental results are presented for two different monoclonal antibodies and for lysozyme on Capto Phenyl (High Sub) resin. One of the mAbs and lysozyme exhibit U-shaped retention factor curves and for each, we determine the critical gradient slope beyond which 100% recovery is no longer possible. Elution with a reverse gradient is also demonstrated at low salt concentrations for these proteins. Understanding this behavior has implications in the design of gradient elution since the gradient slope impacts protein recovery.

Systematic interpolation method predicts protein chromatographic elution with salt gradients, pH gradients and combined salt/pH gradients.

A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high-throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high-throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column.

Surface induced three-peak elution behavior of a monoclonal antibody during cation exchange chromatography.

A monoclonal antibody exhibits a two- or three-peak elution behavior when loaded on the CEX resin POROS XS and eluted with a salt gradient. Two peaks are observed without a hold step while a third more strongly retained peak becomes noticeable with a hold time as low as 10min. As the hold time is increased further, the first peak gradually disappears, the second peak initially increases and then decreases, and the third peak continuously increases. Dynamic light scattering shows that the third peak contains significant levels of aggregates formed in the column. Circular dichroism, HX-MS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HX-MS analysis indicate that the aggregates derive from an unfolded intermediate that is slowly formed while the protein is bound to the resin. Aggregate formation does not occur on a different CEX resin, Nuvia HR-S, with similar particle size but with a more homogenous structure or when the sodium acetate load buffer is replaced with arginine acetate. The two early eluting peaks observed for POROS XS regardless of hold time are shown to comprise exclusively monomeric species. A set of biophysical measurements as well as mechanistic modeling support the hypothesis that these two peaks form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics.

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model.

Predicting protein elution for overloaded ion exchange columns requires models capable of describing protein binding over broad ranges of protein and salt concentrations. Although approximate mechanistic models are available, they do not always have the accuracy needed for precise predictions. The aim of this work is to develop a method to predict protein chromatographic behavior from batch isotherm data without relying on a mechanistic model. The method uses a systematic empirical interpolation (EI) scheme coupled with a lumped kinetic model with rate parameters determined from HETP measurements for non-binding conditions, to numerically predict the column behavior. For two experimental systems considered in this work, predictions based on the EI scheme are in excellent agreement with experimental elution profiles under highly overloaded conditions without using any adjustable parameters. A qualitative study of the sensitivity of predicting protein elution profiles to the precision, granularity, and extent of the batch adsorption data shows that the EI scheme is relatively insensitive to the properties of the dataset used, requiring only that the experimental ranges of protein and salt concentrations overlap those under which the protein actually elutes from the column and possess a ± 10% measurement precision.