The influence of grafted polymer architecture and fluid hydrodynamics on protein separation by entropic interaction chromatography

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 +/- 0.005 chains/nm(2)) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities.     

Protein chromatography in neat organic solvents

Pure formamide and ethylene glycol are used instead of water as processing media for protein chromatography. A number of common proteins are freely soluble in these solvents and most do not undergo irrersible inactivation in them. Batch adsorption studies reveal that proteins readily adsorbed to various ion-exchangers in formamide and ethyline glycol and subsequently can be completely desorbed by adding inorganic salts (LiCl and NH(4)NO(3)) to the solvents. The idea of protein separations in formamide and ethylene glycol is illustrated by column chromatography and preparative separation of mixtures of (i) oxidized A and B chains of insulin and (ii) lysozyme and ribonuclease on the anion-exchanger triethylaminoethycellulose and the cation-exchanger phosphocellulose, respectively.     

Analysis of brush-particle interactions using self-consistent-field theory

Non-specific protein adsorption can be reduced by attaching polymer chains by one end to a sorbent surface. End-grafted polymer modified surfaces have also found application in size-based chromatographic bioseparations. To better understand how to tailor surfaces for these applications, a numerical SCF model has been used to calculate theoretical results for the polymer density distribution of interacting polymer chains around a solute particle positioned at a fixed distance from a surface. In addition, the excess energy required to move the particle into the polymer chains (interaction energy) is calculated using a statistical mechanical treatment of the lattice model. The effect of system variables such as particle size, chain length, surface density and Flory interaction parameters on density distributions and interaction energies is also studied. Calculations for the interaction of a solute particle with a surface covered by many polymer chains (a brush) show that the polymer segments will fill in behind the particle quite rapidly as it moves toward the surface. When there is no strong energetic attraction between the polymer and solute we predict that the interaction energy will be purely repulsive upon compression due to losses in conformational entropy of the polymer chains. Above a critical chain length, which depends upon particle size, a maximum in the force required to move the particle toward the surface is observed due to an engulfment of the particle as chains attempt to access the free volume behind the particle. If an attraction exists between the polymer and solute, such that a minimum in the interaction energy is seen, the optimum conditions for solute repulsion occur at the highest surface density attainable. Long chain length can lead to increased solute concentration within the polymer layer due to the fact that an increased number of favourable polymer–solute contacts are able to occur than with short chains at a similar entropic penalty.     

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

Instrumentation for advanced microseparations in pharmaceutical analysis and proteomics

Small-volume chromatographic columns are only able to generate narrow peaks when flow rates, injection volume and instrument components, such as detector, connecting tubing and fittings, are matched to the peak dispersion from the column. Criteria for the proper design of chromatographic instrumentation are therefore derived from a general model on total dispersion. The performance of such a system is then experimentally evaluated from applications run on narrow-bore, small-volume columns. In order to achieve flow rates that match the dimensions of such columns, a new concept for electronic flow control (EFC) is introduced. A theoretical optimization of column efficiency and throughput is discussed and the results verified with practical examples on short, narrow-bore columns packed with small, porous and superficially porous particles. For complex sample mixtures, the concept of peak capacity is introduced and applied to orthogonal separation principles in multiple chromatographic dimensions through column switching techniques.     

Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: comparison between polymer brush coating and surface functionalization

In the present article, open tubular-IMAC columns, functionalized by iminodiacetic acid (IDA) for the immobilization of Fe(3+), were prepared by in situ chemical modification of fused silica capillary using two chemistries, polymer brush coating and surface functionalization. One column was based on a poly-(glycidyl methacrylate) brush (GMA) and the other on 3-glycidoxypropyltrimethoxysilane (GLYMO). Phosphopeptide enrichment on the open tubular columns was evaluated on an α(S1), α(S2) mixture and β casein peptides. The optimized enrichment protocol includes sample loading in a slightly acidic solution made with pure deionized water, a washing step with 10% acetonitrile, 0.1% formic acid, and an elution step with 50% acetonitrile, 0.1% phosphoric acid at pH 8.0. MALDI-TOF spectra generated from eluted fractions show several phosphorylated peptides. For example, 7 phosphorylated peptides of the α(S1), α(S2) casein mixture were identified, including a pentaphosphorylated peptide. In terms of selectivity, the two proposed chemistries exhibit different behaviors: the GMA-IDA-Fe(3+) IMAC polymer brush column elutes all phosphorylated peptides in one fraction independently of phosphorylation degree, whereas the GLYMO-IMAC polymer brush provides longer elution times for higher phosphorylation states. In particular, the pentaphosphorylated peptide was eluted after a 30 min elution versus 5 min for monophosphorylated species (isocratic gradient).     

High-throughput screening of chromatographic separations: II. Hydrophobic interaction

A high-throughput screen (HTS) was developed to evaluate the selectivity of various hydrophobic interaction chromatography (HIC) resins for separating a mAb from aggregate species. Prior to the resin screen, the solubility of the protein was assessed to determine the allowable HIC operating region by examining 384 combinations of pH, salt, and protein concentration. The resin screen then incorporated 480 batch-binding and elution conditions with eight HIC resins in combination with six salts. The results from the screen were reproducible, and demonstrated quantitative recovery of the mAb and aggregate. The translation of the HTS batch-binding data to lab-scale chromatography columns was tested for four conditions spanning the range of product binding and selectivity. After accounting for the higher number of theoretical plates in the columns, the purity and recovery of the lab-scale column runs agreed with the HTS results demonstrating the predictive power of the filterplate system. The HTS data were further analyzed by the calculation of pertinent thermodynamic parameters such as the partition coefficient, K(P), and the separation factor, alpha. The separation factor was used to rank the purification capabilities of the resin and salt conditions explored.     

Polyelectrolyte brushes grafted from cellulose nanocrystals using Cu-mediated surface-initiated controlled radical polymerization

Herein we report the synthesis of cellulose nanocrystals (CNCs) grafted with poly(acrylic acid) (PAA) chains of different lengths using Cu-mediated surface initiated-controlled radical polymerization (SI-CRP). First, poly(tert-butylacrylate) (PtBA) brushes were synthesized; then, subsequent acid hydrolysis was used to furnish PAA brushes tethered onto the CNC surfaces. The CNCs were chemically modified to create initiator moieties on the CNC surfaces using chemical vapor deposition (CVD) and continued in solvent phase in DMF. A density of initiator groups of 4.6 bromine ester groups/nm(2) on the CNC surface was reached, suggesting a dense functionalization and a promising starting point for the controlled/living radical polymerization. The SI-CRP of tert-butylacrylate proceeded in a well-controlled manner with the aid of added sacrificial initiator, yielding polymer brushes with polydispersity values typically well below 1.12. We calculated the polymer brush grafting density to almost 0.3 chains/nm(2), corresponding to high grafting densities and dense polymer brush formation on the nanocrystals. Successful rapid acid hydrolysis to remove the tert-butyl groups yielded pH-responsive PAA-polyelectrolyte brushes bound to the CNC surface. Individually dispersed rod-like nanoparticles with brushes of PtBA or PAA were clearly visualized by AFM and TEM imaging.     

A simple radiochemical assay for prostaglandin synthetase

A simple radiochemical procedure for prostaglandin synthetase is described, in which incubation mixtures are applied to small disposable columns of Bio-Sil A (silica gel) resin and eluted with two different solvent mixtures to achieve a separation of unmetabolized substrates from prostaglandin products.     

Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.     

Partitioning of individual flexible polymers into a nanoscopic protein pore

Polymer dynamics are of fundamental importance in materials science, biotechnology, and medicine. However, very little is known about the kinetics of partitioning of flexible polymer molecules into pores of nanometer dimensions. We employed electrical recording to probe the partitioning of single poly(ethylene glycol) (PEG) molecules, at concentrations near the dilute regime, into the transmembrane beta-barrel of individual protein pores formed from staphylococcal alpha-hemolysin (alphaHL). The interactions of the alpha-hemolysin pore with the PEGs (M(w) 940-6000 Da) fell into two classes: short-duration events (tau approximately 20 micro s), approximately 85% of the total, and long-duration events (tau approximately 100 micro s), approximately 15% of the total. The association rate constants (k(on)) for both classes of events were strongly dependent on polymer mass, and values of k(on) ranged over two orders of magnitude. By contrast, the dissociation rate constants (k(off)) exhibited a weak dependence on mass, suggesting that the polymer chains are largely compacted before they enter the pore, and do not decompact to a significant extent before they exit. The values of k(on) and k(off) were used to determine partition coefficients (Pi) for the PEGs between the bulk aqueous phase and the pore lumen. The low values of Pi are in keeping with a negligible interaction between the PEG chains and the interior surface of the pore, which is independent of ionic strength. For the long events, values of Pi decrease exponentially with polymer mass, according to the scaling law of Daoud and de Gennes. For PEG molecules larger than approximately 5 kDa, Pi reached a limiting value suggesting that these PEG chains cannot fit entirely into the beta-barrel.     

Molecular and crystal structures of chitosan/HI type I salt determined by X-ray fiber diffraction

The three-dimensional structure of chitosan/HI type I salt was determined by the X-ray fiber diffraction technique and linked-atom least-squares refinement method. Two polymer chains and four iodide ions (I(-)) crystallized in a monoclinic unit cell with dimensions a = 9.46(2), b = 9.79(2)], c (fiber axis)=10.33(2)A, beta = 105.2(2) degrees and a space group P2(1). Chitosan chains adopted an extended twofold helical conformation that was stabilized by O-3...O-5 hydrogen bonds, and the O-6 atom adopted nearly gt orientation. Polymer chains zigzag along the b-axis and directly connect to each other by N-2...O-6 hydrogen bonds. Two columns of iodide ions were shown to pack at the bending points of the zigzag sheets, and their locations are closely related to those of water columns in the hydrated chitosan. The iodide ions stabilized the salt structure by forming hydrogen bonds with the N-2 and O-6 atoms of the polymer chains together with an electrostatic interaction between N-2 and the iodide ions.     

Solving multicomponent chiral separation challenges using a new SFC tandem column screening tool

A tool for improved tandem column chiral supercritical fluid chromatography (SFC) method development screening was prepared by modification of a commercial analytical SFC instrument with two different software-controllable, six position high-pressure column selection valves, each controlling a bank of five different columns and a pass through line. The resulting instrument, which has the ability to screen 10 different individual columns and 25 different tandem column arrangements, is a useful tool for facilitating the screening of tandem column SFC arrangements for separation of complex mixtures of stereoisomers or other multicomponent mixtures. Strategies for optimal use of the instrument are discussed, and several examples of the use of the instrument in developing tandem SFC methods for resolution of multicomponent mixtures are presented.     

Development and application of an automated, low-volume chromatography system for resin and condition screening

A small-volume chromatography system was developed for rapid resin and parameter screening and applied to the purification of a therapeutic monoclonal antibody from a key product-related impurity. Accounting for constraints in peripheral volume, gradient formation, column integrity, and fraction collection in microtiter plates, the resulting system employed 2-mL columns and was successfully integrated with plate-based methods for rapid sample analysis (e. g., use of automated liquid handlers, plate readers, and HPLC). Several cation-exchange chromatography resins were screened using automated programs and tailored gradients for the combination of a particular resin and a given antibody feedstock produced during Phase 1 development. Results from the tailored gradient runs were used to select a resin, and to arrive at efficient stepwise elution schedules for the chosen resin. By maintaining a constant residence time, final operating parameters were successfully scaled to representative bed heights and column diameters up to 2.6 cm (106 mL). This approach significantly improved throughput while reducing development time and material consumption.     

Terminally functionalized thermoresponsive polymer brushes for simultaneously promoting cell adhesion and cell sheet harvest

For preparing cell sheets effectively for cell sheet-based regenerative medicine, cell-adhesion strength to thermoresponsive cell culture surfaces need to be controlled precisely. To design new thermoresponsive surfaces via a terminal modification method, thermoresponsive polymer brush surfaces were fabricated through the surface-initiated reversible addition-fragmentation chain transfer (RAFT) radical polymerization of N-isopropylacrylamide (IPAAm) on glass substrates. The RAFT-mediated grafting method gave dithiobenzoate (DTB) groups to grafted PIPAAm termini, which can be converted to various functional groups. In this study, the terminal carboxylation of PIPAAm chains provided high cell adhesive property to thermoresponsive surfaces. Although cell adhesion is generally promoted by a decrease in the grafted PIPAAm amount, the decrease also decelerated thermally-induced cell detachment, whereas the influence of terminal modification was negligible on the cell detachment. Consequently, the terminally modified PIPAAm brush surfaces allowed smooth muscle cells (SMCs) to simultaneously adhere strongly and detach themselves rapidly. In this study, SMCs were unable to reach a confluent monolayer on as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm(2)) without terminal carboxylation due to their insufficient cell-adhesion strength. On the other hand, though a decrease in the PIPAAm amount allowed SMCs to form a confluent cell monolayer on the PIPAAm brush surface, the SMCs were unable to be harvested as a monolithic cell sheet by low-temperature culture at 20 °C. Because of their unique property, only terminal-carboxylated PIPAAm brush surfaces achieved rapid harvesting of complete cell sheets by low-temperature culturing.     

Transport and binding characterization of a novel hybrid particle impregnated membrane material for bioseparations

The transport and binding properties of a novel hybrid particle-nonwoven membrane medium are described. In this construct, a polymeric chromatographic resin is entrapped between two layers of a nonwoven polypropylene membrane. The membrane-supported resin medium offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with PIM displayed excellent flow distribution and had interstitial porosities of 0.48 ± 0.01, 25-60% larger than those typical of a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate in 30 column volumes while maintaining a pressure drop significantly lower than that of a packed bed with a similar amount of resin. The dynamic binding capacity of bovine serum albumin (BSA) to the chromatographic resin entrapped in the PIM packed column was essentially the same as that observed with the same volume of resin in a packed bed. The General Rate (GR) model of chromatography was used to analyze experiments indicating the breakthrough behavior of the PIM columns is predictable, and very similar to those of a normal packed bed. These results suggest that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin and could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing.     

Restricted diffusion effect on the binding of proteins to porous polymer resins as studied by repetitive injection method

A solution of bovine serum albumin (BSA) is repeatedly injected into a column packed with highly porous and hydrophobic polymer resins at appropriate intervals. The injected BSA is thoroughly retained in the column for 10 injections and, afterwards, starts to be eluted from the column gradually. Taking into consideration the restricting effect of already bound BSA upon the diffusion of newly injected BSA into the pores of the polymer resins, we can interpret the BSA elution profile from columns packed with polymer resin of various pore sizes and porosities. The effects of the binding rate constant and BSA concentration upon the elution profiles of BSA are also analyzed. Formyl groups are introduced into the polymers as a binding site with proteins, and the elution profiles of BSA from the column packed with the formylated resin are also analyzed.     

Osmotic properties of poly(ethylene glycols): quantitative features of brush and bulk scaling laws

From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander-de Gennes theory of polymer brushes successfully fits the intermembrane distance versus applied osmotic stress data of Kenworthy et al. for poly(ethylene glycol)-grafted multilamellar liposomes. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent monomer density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semidilute regime of bulk polymer solutions). The condition that attached polymers behave as semidilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of semidilute polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander-de Gennes brush scaling laws to the Kenworthy et al. data yield effective monomer lengths of 3.3-3.6 A, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal mole fraction of poly(ethylene glycol)-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.