Separation of closely related monoclonal antibody charge variant impurities using poly(ethylenimine)-grafted cation-exchange chromatography resin.

The removal of protein charge variants due to complex chemical and enzymatic modifications like glycosylation, fragmentation and deamidation presents a significant challenge in the purification of monoclonal antibodies (mAb) and complicates downstream processing. These protein modifications occur either in vivo or during fermentation and downstream processing. The presence of charge variants can lead to diminished biological activity, differences in pharmacokinetics, pharmacodynamics, stability and efficacy. Therefore, these different product variants should be appropriately controlled for the consistency of product quality and to ensure patient safety. This investigation focuses on the development of a chromatography step for the removal of the charge variants from a recombinant single-chain variable antibody fragment (scFv-Fc-Ab). Poly(ethyleneimine)-grafted cation-exchange resins (Poly CSX and Poly ABX) were evaluated and compared to traditional macroporous cation-exchange and tentacle cation-exchange resins. Linear salt gradient experiments were conducted to study the separation efficiency of scFv-Fc-Ab variants using different resins. A classical thermodynamic model was used to develop a mechanistic understanding of the differences in charge variant retention behaviour of different resins. High selectivity in separation of scFv-Fc-Ab charge variants is obtained in the Poly CSX resin.

Selective removal of closely related clipped protein impurities using poly(ethylenimine)- grafted anion-exchange chromatography resin.

Proteolytic degradation is a serious problem that complicates downstream processing during production of recombinant therapeutic proteins. It can lead to decreased product yield, diminished biological activity, and suboptimal product quality. Proteolytic degradation or protein truncation is observed in various expression hosts and is mostly attributed to the activity of proteases released by host cells. Since these clipped proteins can impact pharmacokinetics and immunogenicity in addition to potency, they need to be appropriately controlled to ensure consistency of product quality and patient safety. A chromatography step for the selective removal of clipped proteins from an intact protein was developed in this study. Poly(ethylenimine)-grafted anion- exchange resins (PolyQUAT and PolyPEI) were evaluated and compared to traditional macroporous anion-exchange and tentacled anion-exchange resins. Isocratic retention experiments were conducted to determine the retention factors (k') and charge factors (Z) were determined through the classical stoichiometric displacement model. High selectivity in separation of closely related clipped proteins was obtained with the PolyQUAT resin. A robust design space was established for the PolyQUAT chromatography through Design-Of-Experiments (DoE) based process optimization. Results showed a product recovery of up to 63% with purity levels >99.0%. Approximately, one-log clearance of host cell protein and two-logs clearance of host cell DNA were also obtained. The newly developed PolyQUAT process was compared with an existing process and shown to be superior with respect to the number of process steps, process time, process yield, and product quality.

Pervaporation from a dense membrane: roles of permeant-membrane interactions, Kelvin effect, and membrane swelling.

Dense polymeric membranes with extremely small pores in the form of free volume are used widely in the pervaporative separation of liquid mixtures. The membrane permeation of a component followed by its vaporization on the opposite face is governed by the solubility and downstream pressure. We measured the evaporative flux of pure methanol and 2-propanol using dense membranes with different free volumes and different affinities (wettabilities and solubilities) for the permeant. Interestingly, the evaporative flux for different membranes vanished substantially (10-75%) below the equilibrium vapor pressure in the bulk. The discrepancy was larger for a smaller pore size and for more wettable membranes (higher positive spreading coefficients). This observation, which cannot be explained by the existing (mostly solution-diffusion type) models ofpervaporation, suggests an important role for the membrane-permeant interactions in nanopores that can lower the equilibrium vapor pressure. The pore sizes, as estimated from the positron annihilation, ranged from 0.2 to 0.6 nm for the dry membranes. Solubilities of methanol in different composite membranes were estimated from the Flory-Huggins theory. The interaction parameter was obtained from the surface properties measured by the contact angle goniometry in conjunction with the acid-base theory of polar surface interactions. For the membranes examined, the increase in the "wet" pore volume due to membrane swelling correlates almost linearly with the solubility of methanol in these membranes. Indeed, the observations are found to be consistent with the lowering of the equilibrium vapor pressure on the basis of the Kelvin equation. Thus, a higher solubility or selectivity of a membrane also implies stronger permeant-membrane interactions and a greater retention of the permeant by the membrane, thus decreasing its evaporative flux. This observation has important implications for the interpretation of existing experiments and in the separation of liquid mixtures by pervaporation.