Coupling of chromatography and precipitation for adjusting acidic variant content in a monoclonal antibody pool.

The acidic charge variants (av) of monoclonal antibodies (mAb) are often reported to have reduced therapeutic potency compared with the main (mv) and basic variants (bv), therefore reduction in the av content in mAb pools is often prioritized over reduction in the bv content. In previous studies we described two different methods for reducing the av content, which were based on either ion exchange chromatography or selective precipitation in polyethylene glycol (PEG) solutions. In this study, we have developed a coupled process, in which advantages of simplicity and ease in realization of PEG-aided precipitation and high separation selectivity of anion exchange chromatography (AEX) were exploited.

Separation of non-racemic mixtures of enantiomers by achiral chromatography.

The phenomenon of partial separation of enantiomeric mixtures in achiral chromatography (ACh) has already been documented for a wide variety of chiral compounds. It is attributed to the so-called effect of self-disproportionation of enantiomers (SDE). However, quantitative description of the SDE mechanism underlying adsorption of enantiomers on achiral surfaces is still incomplete, which hinders the application of that technique for large-scale separations.

Preferential precipitation of acidic variants from monoclonal antibody pools.

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins.

Separation of charge variants of a monoclonal antibody by overloaded ion exchange chromatography.

A procedure for adjusting the content of charge variants of monoclonal antibody by ion exchange chromatography has been developed. The band splitting phenomenon was utilized to split the protein load into two parts, i.e.

A case study of the mechanism of unfolding and aggregation of a monoclonal antibody in ion exchange chromatography.

A mechanistic model for describing unfolding of a monoclonal antibody (mAb) in ion exchange chromatography has been developed. The model reproduced retention behavior characteristic for conformational changes of antibodies upon adsorption, including: multi-peak elution, aggregate formation, and recovery reduction. Two competitive paths in the adsorption mechanism of the unfolded protein were assumed: refolding in the adsorbed phase to the native form followed by its desorption, or direct desorption followed by instantaneous aggregation in the liquid phase.

Effects of negative and positive cooperative adsorption of proteins on hydrophobic interaction chromatography media.

The adsorption behavior of the model proteins: alpha-Lactalbumin, Bovine Serum Albumin, Lysozyme, and a monoclonal antibody, in single component and in binary mixtures, was investigated on two different hydrophobic interaction chromatography resins using both static and dynamic methods. A kinetic model of the adsorption process was developed, which accounted for protein unfolding and intermolecular interactions in the adsorbed phase. The latter incorporated positive cooperative interactions, resulting from preferred and multilayer adsorption on the adsorbent surface, as well as negative cooperative interactions attributed to exclusion effects due to size exclusion and repulsion.

Mechanism of nutrition activity of a microgranule fertilizer fortified with proteins.

Background: A microgranule fertilizer was designed for localized fertilization of soil with controlled release of nutrients. The microgranule matrix was fortified with proteins, which were obtained from food industry byproducts or waste, i.e.

Scale up of a chromatographic capture step for a clarified bacterial homogenate - Influence of mass transport limitation and competitive adsorption of impurities.

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study.

Effect of flow behavior in extra-column volumes on the retention pattern of proteins in a small column.

Experimental and theoretical analysis of deformation of band profiles in extra-column volumes (ECV) was performed, and its influence on the retention pattern of proteins in a small chromatographic column was quantified. Several macromolecule and small-molecule compounds, and their mixtures were eluted from a chromatographic system in the absence and presence of the column. The peak deformation in ECV was attributed to non-uniform velocity distribution in the radial direction in connecting capillaries.

Retention Behavior of Polyethylene Glycol and Its Influence on Protein Elution on Hydrophobic Interaction Chromatography Media.

The retention behavior of polyethylene glycol (PEG) on different types of hydrophobic interaction chromatography (HIC) resins containing butyl, octyl, and phenyl ligands was analyzed. An incomplete elution or splitting of the polymer peak into two parts was observed, where the first one was eluted at the dead time of the column, whereas the second one was strongly retained. The phenomenon was attributed to conformation changes of the polymer upon its adsorption on hydrophobic surface.

Prediction tool for loading, isocratic elution, gradient elution and scaling up of ion exchange chromatography of proteins.

An efficient mathematical tool for the design and scaling up of protein chromatography is suggested, in which the model parameters can be determined quickly over a wide operating space without large material investments. The design method is based on mathematical modelling of column dynamics and moment analysis. The accuracy of the dynamic models that are most frequently used for simulations of chromatographic processes is analyzed, and possible errors that can be generated using the moment analysis are indicated.

Effect of mass overloading on binding and elution of unstable proteins in hydrophobic interaction chromatography.

Adsorption behavior of unstable proteins, i.e., bovine serum albumin and α-lactalbumin, has been studied on a hydrophobic interaction chromatography medium under mass overloading conditions at different kosmotropic salt concentrations in the mobile phase.

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration.

In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated.

Protein separation in carousel multicolumn setup. Performance analysis and experimental validation.

To overcome limitations of periodic separations of proteins in batch chromatographic columns Carousel Multi-Column Setup (CMS) has been recently suggested and theoretically analyzed in a previous study (R. Bochenek, W. Marek, W.

Overcoming solubility limits in overloaded gradient hydrophobic interaction chromatography.

The impact of the solubility limits on the performance of gradient protein chromatography has been studied. As a case study elution of model protein, i.e.

Adsorption behavior of proteins on temperature-responsive resins.

The adsorption behavior of proteins on thermo-responsible resins based on poly(N-isopropylacrylamide) and its copolymer containing an anionic co-monomer has been investigated. The influence of the polymer composition, i.e.

Isolation of monoclonal antibody from a Chinese hamster ovary supernatant. II: dynamics of the integrated separation on ion exchange and hydrophobic interaction chromatography media.

Dynamics of the purification process of a CHO derived monoclonal antibody by ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and their integration has been investigated. To quantify the adsorption behavior of the target protein (IgG1) and impurities contained in the supernatant, their elution course on IEC and HIC columns has been analyzed versus pH and/or the salt concentration in the mobile phase. A short-cut method has been proposed for mathematical modeling and determining underlying kinetic and thermodynamic parameters.

Isolation of monoclonal antibody from a Chinese hamster ovary supernatant. I: assessment of different separation concepts.

The performance of different non-affinity purification techniques commonly used for isolating CHO derived monoclonal antibodies has been investigated. Ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), aqueous-two-phase extraction (ATPE) and their integration has been evaluated in terms of yield and purity of the product obtained. The integration of chromatographic techniques comprised two steps, in which the CHO supernatant was directly injected into the IEC column to capture monoclonal IgG1 and then the isolated fraction was processed using the HIC column.

Evaluating the performance of different multicolumn setups for chromatographic separation of proteins on hydrophobic interaction chromatography media by a numerical study.

A theoretical study has been performed on the effectiveness of isolating a target component out of a multi-component protein mixture using different arrangements of chromatographic columns. Three continuous systems have been considered which were able to perform solvent gradient separations, such as: open loop simulated moving bed, countercurrent solvent gradient purification and carousel multicolumn setup. The performance of the continuous processes was examined with respect to productivity, yield and eluent consumption and compared to a single-column batch system.

Multiple-injection technique for isolating a target protein from multicomponent mixtures.

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media.

Influence of the sample-solvent on protein retention, mass transfer and unfolding kinetics in hydrophobic interaction chromatography.

Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses.

Effects of thermal heterogeneity in hydrophobic interaction chromatography.

Manipulating temperature and salt concentration can have a powerful effect on the separation effectiveness in hydrophobic interaction chromatography (HIC). However, use of temperature as an operating variable in large-scale applications may involve undesirable consequences such as radial heterogeneity of the column temperature. In this study non-ideal effects of heat transfer in HIC columns were analyzed.

Altering efficiency of hydrophobic interaction chromatography by combined salt and temperature effects.

The coupled effect of salt concentration and temperature on the retention behavior of proteins in hydrophobic interaction chromatography has been studied. The retention data of four model proteins, i.e.

Behavior of adsorbed and fluid phases versus retention properties of amino acids on the teicoplanin chiral selector.

The relationship between adsorption equilibria of two amino acids, i.e., l,d-threonine and l,d-methionine on the teicoplanin chiral selector and their phase behavior has been analyzed.

Influence of preferential adsorption of mobile phase on retention behavior of amino acids on the teicoplanin chiral selector.

The adsorption behavior of two amino acids, i.e., l,d-threonine and l,d-methionine has been investigated on the chiral stationary phase (CSP)column packed with teicoplanin bonded to a silica support.