Cation-exchange Displacement Chromatography Research Articles

Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography

In this report, we have demonstrated the isolation and enrichment of charge variants of a monoclonal antibody IgG1 using cation exchange displacement chromatography. We successfully achieved the separation of acidic, main and basic charge variants with high recovery (>70%) and purity (>90%) by using a commercially available stationary phase in conjunction with a commercially available displacer. In addition, we have isolated and enriched a trace methionine-oxidized variant of the monoclonal antibody allowing a secondary means of identification of this variant while providing sufficient enrichment for further analysis, stability tests and potency determination. Further characterization of the displacement trains by SEC indicate the possibility of enrichment of high and low molecular weight species. Glycan analysis of the displacement fractions indicates minimal variation in glycan distribution patterns among a wide spectrum of charge variants. These results provide a case study demonstrating the utility of cation exchange displacement chromatography as a viable approach to isolate and enrich antibody charge variants for enhanced molecular characterization.

Charge variants in IgG1

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and α-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 μm, 1000 Å) and a monolithic column (UNO™ S1). Both columns are strong cation exchangers, carrying SO 3 −-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100–200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20 000 g mol −1, which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.

Isotope effects in the V(IV)-malate complex formation system

Cation-exchange displacement chromatography of VO 2+ was carried out for studying vanadium isotope fractionation in the malate complex formation system. After the chromatographic elution, the heavier isotope 51V was found to be enriched at the front boundary of the VO 2+ band, which indicates that the heavier isotope is preferentially fractionated into the malate complex in the aqueous solution phase. The isotope separation coefficient ε (= α−1) for 50V and 51V was obtained as 1.0×10 −4 for the malate system at 298 K. This value is smaller than the ones for UO 2 2+ and Cu 2+ in the same ligand systems. Binuclear complex species in UO 2 2+ and Cu 2+ solutions may be attributed to the larger isotope effects in the complex formation system. Through comparison of the reported separation coefficient values of ε per unit mass differences ( ε/Δ M) of the alkali, alkaline earth and transition elements in the complex formation systems, it is suggested that the stronger the complex of the concerned element, the more the isotopic fractionation can obtain.

Separation of vanadium isotopes by ion-exchange chromatography

Cation-exchange displacement chromatography of VO 2+ was carried out for studying vanadium isotope effects in carboxylate ligand-exchange systems. The heavier isotope 51V was enriched in the carboxylate complex solution. The isotope separation coefficients ϵ(= α−1) for 50V/ 51V were 2.2·10 −4 and 2.4·10 −4 for citrate and lactate systems at 298 K, respectively. These values are much larger than those obtained in a previous study on the malate system. The existence of binuclear complexes of VO 2+ may create the conditions for larger isotope fractionation. From the viewpoint of the process development of isotope separation, the heights equivalent to a theoretical plate of these processes were analyzed and found to be very small in each system due to the homogeneous, small and highly porous resin used. Citrate may be better than the other tested systems for the vanadium isotope separation.

The Effect of Temperature on Uranium Isotope Effects Studied by Cation Exchange Displacement Chromatography

The uranium isotope effect in the exchange system uranyl(VI)-malate ligand at 288-343 K has been studied by ion exchange displacement chromatography. At all temperatures 235U is enriched at the front of the uranium band. The single stage separation coefficient, (Ɛ = α - 1), increased from (0.9 ± 0.1) × 10-4 at 288 K to (2.9 ± 0.3) × 10-4 at 343 K. The equilibrium constant of the isotope exchange reaction equaled the separation factor at the current experimental conditions. The increase of the separation coefficient with temperature, which is in contrast to the uranium(IV)-ligand exchange systems, can be explained by the introduction of the field shift effect.

Mass influences in the performance of oligomeric poly(diallyldimethylammonium chloride) as displacer for cation-exchange displacement chromatography of proteins

A novel type of linear polyelectrolyte, namely poly-DADMAC [poly(diallyldimethylammonium chloride)], was prepared and studied as a displacer for cation-exchange displacement chromatography of proteins. In contrast to the commercially available polymers of that chemistry, the novel type of poly-DADMAC introduced here is characterized by a homogeneous linear structure, a narrow distribution of the (adjustable) molar mass as well as by a defined and homogeneous affinity for the stationary phase. Five poly-DADMACs of different size (17 900 to 88 000 g/mol) were prepared and compared with regard to their stationary phase affinity and protein separation potential, taking a mixture of basic proteins, namely lysozyme, cytochrome C, and ribonuclease A (from bovine pancreas), as an example. The steric mass action model was employed to aid method development. Under the chosen conditions (low ionic strength of the mobile phase guaranteeing strong binding of both the proteins and the displacer) the poly-DADMAC with the lowest molar masses proved to be the most efficient displacers for the basic proteins with a stationary phase affinity constant of 5.3·10 16 and a steric factor of 224. Using this substance as displacer, a sample mixture containing up to three proteins was separated and the proteins recovered at high yields (80–97%) and in high purity and concentration.

Polycations as displacer in high-performance bioseparation

Displacement chromatography is an interesting but up to now rarely used type of preparative biochromatography. The lack of well-engineered and accessible displacer contributes to this phenomenon. In this paper a novel type of displacer is introduced for cation-exchange displacement chromatography, which will soon become commercially available. The molecule is a well-defined PolyDADMAC [poly(diallyldimethylammonium chloride)] with a molar mass of less than 35 000 g/mol, an exclusively linear structure and a molar mass polydispersity of less than 1.5. A method for synthesizing such a polymer at high yields is described. The PolyDADMAC is shown to be an efficient displacer of basic proteins from strong cation-exchange columns.

Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants

Removal of low level impurities that are closely related to the bioproduct is a commonly encountered challenge in the purification of biopharmaceuticals. These separations are typically carried out by using shallow linear salt gradients at relatively low column loadings, significantly limiting the throughput of the purification process. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brain-derived neurotrophic factor, rHuBDNF. The utility of displacement chromatography is compared to gradient elution for the removal of variants of the rHuBDNF. The results demonstrate that displacement chromatography is capable of achieving high yields and purity at high column loadings. Displacements developed on 20 μm and 50 μm cation-exchange resins are shown to provide 8-fold and 4.5-fold increases in production rates, respectively, when compared to an existing linear gradient elution operation. These results demonstrate the efficacy of displacement chromatography for the purification of therapeutic proteins from complex feed streams.

Cation-exchange displacement chromatography of proteins with protamine displacers: effect of induced salt gradients.

Protamine was investigated for its utility as a protein displacer in cation-exchange systems. Although the protamine solution contained several variants of the molecule, the high affinity of all of the components in this heterogeneous biopolymer enabled it to act as an efficient protein displacer. To facilitate parameter estimation of the protamine, a preliminary purification was carried out by preparative elution chromatography. Chromatographic parameters of both the feed proteins and protamine displacer were obtained for use in a multicomponent steric mass action ion-exchange displacement model. Model simulations were compared to displacement results under both moderate and intense induced salt gradient conditions. In both cases, excellent agreement was obtained between the displacement experiments and theoretical predictions. In addition, these studies serve to dramatize the importance of induced salt gradients in ion-exchange displacement systems.

Displacement separations by continuous annular chromatography

AbstractContinuous displacement chromatography separations are obtained using a continuous annular chromatograph (CAC). The apparatus consists of a rotating annular bed. At fixed positions at the top of the bed, four solutions are continuously supplied: the feed mixture to be separated, a displacer solution, a regenerant, and a rinse. The four solutions flow concurrently through the bed with little mixing. If the bed is sufficiently deep, a fully developed displacement train forms and the separated components are continuously recovered at various angular locations at the bottom of the bed. Mixtures of amino acid were separated by cation exchange displacement chromatography in a laboratory‐scale CAC. Separation and concentration of dilute mixtures was obtained with a continuous separation performance comparable with the performance of a conventional chromatograph. A mathematical model is developed to describe the separation and provide guidance in design and optimization.