Application of arginine as an efficient eluent in cation exchange chromatographic purification of a PEGylated peptide.

Pfizer, Inc., 700 Chesterfield Village Parkway, Chesterfield, MO 63017, USA.
Journal of Chromatography A (Impact Factor: 4.61). 06/2010; 1217(24):3783-93. DOI: 10.1016/j.chroma.2010.04.019
Source: PubMed

ABSTRACT A cation exchange chromatographic purification process step was developed for the purification of human PEGylated PYY 3-36 from the PEGylation reaction mixture. In this publication we describe experiments carried out to evaluate the chromatographic performance of arginine chloride as an effective cation exchange chromatography eluent. Using arginine we obtained improved recovery and resolution during chromatographic purification of a peptide PEGylation reaction mixture. The chromatographic elution performance of arginine was compared to other cationic amino acids and sodium chloride. Arginine provided higher yield and better resolution of product from other process impurities. The process was successfully scaled up to produce clinical supplies. The basis for improvement in process performance with arginine was characterized by examining the effect of buffer and concentration of the PEGylated peptide on hydrodynamic volume of the molecule in solution. These results were used to predict the behavior of the molecule in the chromatography process. The enhanced chromatographic performance could be attributed to changes in molecular size with concentration, higher eluent strength of arginine, and resulting changes in mass transfer resistance.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Protein binding equilibrium and mass transfer kinetics are studied for cation exchangers containing charged polymer grafts as well as for a macroporous matrix in pH 5 acetate buffers using sodium, tetra-n-butylammonium (TBAH), arginine, and calcium as counterions and a monoclonal antibody (mAb) as a model protein. Dynamic light scattering shows that there is no significant effect of the counterion type on the mAb aqueous diffusivity. The counterion type also does not affect substantially the structure of the polymer grafts, nor does it affect the stoichiometry of the protein ion exchange process. While no counterion effects are also observed on the protein mass transfer kinetics for the macroporous matrix, very large effects are seen for the polymer grafted matrices with protein adsorption rates increasing dramatically in the order Ca⁺⁺> Arg⁺> Na⁺> TBAH⁺. This order is the same order in which the relative protein binding strength decreases. Accordingly, the counterions leading to weaker protein binding also lead to faster protein diffusion. Although the quantitative aspects are different, the same trends hold for different proteins (lysozyme and lactoferrin) and for an agarose-based matrix also containing grafted polymers (Capto™ S). The underlying mechanism is qualitatively consistent with protein transport occurring through a hopping process driven by the adsorbed protein concentration within the apparently flexible network structure formed by the grafted polymers. From a practical viewpoint, the results show that improved protein adsorption kinetics in polymer-grafted cation exchanger and, hence, improved performance, can be obtained by selecting particular counterions.
    Journal of Chromatography A 07/2012; 1253:83-93. · 4.61 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.
    Biotechnology and Bioengineering 08/2011; 109(1):176-86. · 4.16 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30 kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by (125)I based cell tests. Proteins modified with 5 kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification.
    Journal of Chromatography A 03/2012; 1236:90-6. · 4.61 Impact Factor