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This paper presents a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids (CCFs) without the requirement for tracking the retention behaviors of large sets of proteins. Null‐producing CCFs were fractionated using linear salt gradients at constant pH on a set of multimodal resins. Fractions were then analyzed by ultraperformance‐reversed phase liquid chromatography and the resulting chromatograms provided host cell protein (HCP) "fingerprints." Using these fingerprints, an inner product vector‐based approach was employed to quantify the degree of orthogonality between pairs of resins and operating conditions for these large HCP protein sets. To compare resin orthogonality behavior in different expression systems, the Chinese hamster ovary and Pichia pastoris null‐producing CCFs were examined. Orthogonality in multimodal systems was found to strongly depend on the expression system and the HCPs being screened. We also identified several unexpected pairs of multimodal resins within the same family that exhibited significant orthogonality. Furthermore, "self‐orthogonality" was evaluated between resins operated at different pHs, and important operating regimes were identified for maximizing orthogonal selectivities. The framework developed in this paper for calculating orthogonality without the need for labor‐intensive HCP tracking has important implications for efficient process development and resin/operating condition selection for both monoclonal antibody (mAb) polishing steps and non‐mAb processes. In addition, this study provides a tool to unlock the untapped potential of multimodal resins by aiding in their rational selection and incorporation. Finally, the orthogonality framework here can facilitate the development of sets of next‐generation multimodal resins specifically designed to provide highly orthogonal and efficient separations tailored for different expression systems. [ABSTRACT FROM AUTHOR]

This study presents the chromatographic purification of immunoglobulin G (IgG) from human plasma using a two‐column process integrating the peptide‐based adsorbents LigaGuard™, which captures non‐Ig plasma proteins in flow‐through mode, and LigaTrap™, which isolates IgG in bind‐and‐elute. Buffer composition and column loading were optimized for both adsorbents. Two process configurations were evaluated. In the first design, plasma was fed to a LigaGuard™ column to capture plasma proteins, the effluent was loaded on the LigaTrap™ column, and the bound IgG was eluted with 63.8% global recovery and 99.7% purity; in comparison, Protein G agarose afforded approximately 67% recovery and 97.2% purity. In the alternative design, the LigaGuard™ column was utilized to polish the LigaTrap™ elution stream, affording 82.3% global recovery and 98.8% purity. Collectively, these results demonstrate the potential of a fully chromatographic process for purifying polyclonal IgG from plasma feedstocks. [ABSTRACT FROM AUTHOR]

Cyanobacterial phycobilisomes are made up from phycobiliproteins held together by linker proteins. Incident photons are absorbed by the phycobiliproteins that carry linear tetrapyrrole chromophores with extended conjugated bond systems. The chemical structure of the chromophores also bestows the phycobiliproteins with excellent radical scavenging and antioxidant properties and phycobiliproteins have pharmaceutical value as such. We purified and characterized the phycobilisome and its major protein constituent C-phycocyanin from the freshwater filamentous cyanobacterium Aphanizomenon gracile. Isolated phycobilisomes were shown to be intact and contained the phycobiliproteins allophyco-cyanin and C-phycocyanin, but not phycoerythrin, together with the classic set of linker proteins. C-phycocyanin could be easily purified and was of analytical grade with spectral characteristics similar to that of other cyanobacterial species. To our best knowledge, this is the first study to report the purification and characterization of the Aphanizomenon gracile phycobilisome and C-phycocyanin.

Arginine in a free-state and as part of peptides and proteins shows distinct tendency to form clusters. In free-form, it has been found useful in cryoprotection, as a drug excipient for both solid and liquid formulations, as an aggregation suppressor, and an eluent in protein chromatography. In many cases, the mechanisms by which arginine acts in all these applications is either debatable or at least continues to attract interest. It is quite possible that arginine clusters may be involved in many such applications. Furthermore, it is possible that such clusters are likely to behave as intrinsically disordered polypeptides. These considerations may help in understanding the roles of arginine in diverse applications and may even lead to better strategies for using arginine in different situations. • Arginine shows distinct tendency to form clusters. • In free-form, it has multiple applications in biotechnology and cellular biology. • The mechanisms of multiple arginine actions continue to attract interest. • It is quite possible that arginine clusters may be involved in many such applications. • It is likely that such clusters behave as intrinsically disordered polypeptides. [ABSTRACT FROM AUTHOR]

The green-light absorbing proteorhodopsin (GPR) is the prototype of bacterial light-driven proton pumps. It has been the focus of continuous research since its discovery 20 years ago and has sparked the development and application of various biophysical techniques. However, a certain controversy and ambiguity about the oligomeric assembly of GPR still remains. We present here the first tag-free purification of pentameric GPR. The combination of ion exchange and size exclusion chromatography yields homogeneous and highly pure untagged pentamers from GPR overexpressing Escherichia coli. The presented purification procedure provides native-like protein and excludes the need for affinity purification tags. Importantly, three-dimensional protein crystals of GPR were successfully grown and analyzed by X-ray crystallography. These results together with data from single particle cryo-electron microscopy provide direct evidence for the pentameric stoichiometry of purified GPR.

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process‐ and product‐related impurities. IFN‐α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product‐related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed‐phase chromatography of the fractions collected were used to generate a retention database for IFN‐α2b and its variants. These data, in combination with a previously constructed process‐related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product‐related impurities. Top‐ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind‐elute steps and the other having two bind‐elute steps and a flowthrough operation. This approach suggests a new platform‐like approach for rapidly designing purification processes for a range of proteins where separations of both process‐ and product‐related impurities are needed. [ABSTRACT FROM AUTHOR]

• Deformation of protein peaks in extra-column volume (ECV) was indicated. • ECV effect stems from radial velocity distribution and slow protein diffusivity. • ECV effect influences on the protein retention on a small column. • Mathematical model was used to predict the ECV effect. 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. The phenomenon enhanced with increasing molecular weight of the model compound, when radial diffusion dominated the mechanism of band spreading. The band shape was also affected by the geometry of the injection system used, i.e., an injection loop capillary or a superloop. The phenomenon vanished for a small molecule compound, for which plug flow conditions could be established. The difference in flow behaviour of the macromolecule and small-molecule compounds caused them to migrate with different velocities in ECV, which resulted in partial separation of their bands. The ECV effect influenced the retention behaviour of macromolecules in a small column; it caused tailing of peaks and asymmetry of breakthrough curves. To describe the elution profiles in ECV and in the column, a mathematical model was used which accounted for non-ideality of the flow pattern. The model reproduced accurately band profiles of macromolecules within a range of relatively low velocities, typical however for protein chromatography. [ABSTRACT FROM AUTHOR]

Cyanobacterial phycobilisomes are made up from phycobiliproteins held together by linker proteins. Incident photons are absorbed by the phycobiliproteins that carry linear tetrapyrrole chromophores with extended conjugated bond systems. The chemical structure of the chromophores also bestows the phycobiliproteins with excellent radical scavenging and antioxidant properties and phycobiliproteins have pharmaceutical value as such. We purified and characterized the phycobilisome and its major protein constituent C-phycocyanin from the freshwater filamentous cyanobacterium Aphanizomenon gracile. Isolated phycobilisomes were shown to be intact and contained the phycobiliproteins allophycocyanin and C-phycocyanin, but not phycoerythrin, together with the classic set of linker proteins. C-phycocyanin could be easily purified and was of analytical grade with spectral characteristics similar to that of other cyanobacterial species. To our best knowledge, this is the first study to report the purification and characterization of the Aphanizomenon gracile phycobilisome and C-phycocyanin. [ABSTRACT FROM AUTHOR]

The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea. Current treatments are based on nitro drugs, especially metronidazole. Nitro compounds are activated by reduction, yielding toxic intermediates. The enzymatic systems responsible for this activation are not completely understood. By fractionating cell free crude extracts by size exclusion chromatography followed by mass spectrometry, enzymes with nitroreductase (NR) activities are identified. The protein encoded by ORF 17150 found in two pools with NR activities is overexpressed and characterized. In pools of fractions with main NR activities, previously-known NRs are identified, as well as a previously uncharacterized protein encoded by ORF 17150. Recombinant protein 17150 is a flavoprotein with NADPH-dependent quinone reductase and NR activities. Besides a set of previously identified NRs, we have identified a novel enzyme with NR activity.

Highlights • Polymer-grafted cation exchangers of 6 chain lengths/ionic capacities were prepared. • Adsorption capacity reached over 300 mg/mL at ionic capacities of 480 and 590 mmol/L. • Protein uptake rates on the two resins enhanced with increasing salt concentration. • Dynamic capacities of the two resins kept at 200–270 mg/mL below 100 mmol/L NaCl. Abstract This work was designed to acquire novel high-capacity cation exchangers by grafting glycidyl methacrylate (GMA)-iminodiacetic acid (IDA) conjugate (GMA-IDA) to Sepharose FF. Six GMA-IDA-grafted resins of the same grafting density but different chain lengths (ionic capacities, ICs) were synthesized and denoted as FF-Br2-pG-I n ( n denotes for IC value). Lysozyme adsorption and chromatography were studied to evaluate the cation exchangers. It was found that resins of moderate IC values (FF-Br2-pG-I480 and FF-Br2-pG-I590) afforded lysozyme adsorption capacities higher than 300 mg/mL at 50 and 100 mmol/L NaCl, over three times higher than the commercial resin CM Sepharose FF. Beside, the uptake rate ( D e / D 0 ) of lysozyme decreased with increasing IC at 50 mmol/L NaCl because of the increased polymer chain length that limited surface diffusion of the bound protein. Protein adsorption capacities of FF-Br2-pG-I480 and FF-Br2-pG-I590 kept higher than that of CM Sepharose FF in the salt concentration range of 0–150 mmol/L, and the uptake rates on the two resins increased with increasing salt concentration. Therefore, the dynamic binding capacities of them were remarkably higher than that of CM Sepharose FF and kept over 70 mg/mL at the salt concentration up to 150 mmol/L. The results demonstrated that GMA-IDA-grafted resins are promising for use as high-capacity protein chromatography. [ABSTRACT FROM AUTHOR]

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. The so-called transport dispersive model was eventually employed for the process simulations. The model was modified to account for the protein dispersion in void volumes of chromatographic systems. The manner of the model calibration was suggested, which was based on a few chromatographic runs and verified over a wide space of the operating parameters, including composition and flow rate of the mobile phase, column dimensions, residence time, and mass loading. The model system for the study was ion-exchange chromatography. The analysis was performed based on the elution profiles of basic fibroblast growth factor 2 and lysozyme, on two different IEX media. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by ultra-high resolution ESI-MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated β-domain, the smallest peptide with 9 cysteines in the sequence, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length (20 cysteines in the sequence) and α-domain (11 cysteiens in the sequence) peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da, and can infer structural properties from the LC-MS data, analogous to more complicated and less ubiquitous ion-mobility experiments. The data suggest a compact globular structure for each of the apo-MTs, but where the β-domain is more easily unfolded. This differential folding stability may have biological implications in terms of domain-specific participation of MT in cellular redox chemistry and resulting metal release. [ABSTRACT FROM AUTHOR]

This study introduces an improved approach for determining the most suitable mobile phase maximizing repulsion between biomass and adsorbent to reduce biomass fouling in expanded bed adsorption (EBA) chromatography. Extended DLVO theory (xDLVO) has been utilized to comprehensively calculate optimal mobile phase compositions, thus increasing efficiency, improving hydrodynamics as well as product recovery. Biomass-adsorbent interaction energy surfaces were generated as a function of pH, ionic strength, and salt type, investigating the interactions of CHO cells with MabDirect® Protein-A beads. We have shown that mobile phases consisting of Na 2 SO 4 were best suited at low ionic strength, whereas NaCl was more favorable at higher ionic strength. This was found to increase product recovery while maintaining a constant level of purity of monoclonal antibodies. Our method provides a tool for rapid method development of EBA purifications that can be dynamically adapted to operational requirements. In our case, the resulting optimized process was able to outperform the current protocol by up to 10% in terms of product recovery. [ABSTRACT FROM AUTHOR]

Macroporous cellulose monolith as chromatographic support was successfully fabricated from an ionic liquid dissolved cellulose solution by an emulsification method and followed by the cross-linking reaction and DEAE modification. With the physical characterization, the cellulose monolith featured by both the interconnected macropores in range of 0.5–2.5 μm and the diffusion pores centered at about 10 nm. Given the bimodal pore system, the monolith possessed the specific surface area of 36.4 m 2 g −1 and the column permeability of about 7.45 × 10 −14 m 2 . After the DEAE modification, the anion cellulose monolith was evaluated for its chromatography performances. It demonstrated that the static and dynamic adsorption capacity of BSA reached about 66.7 mg mL −1 and 43.9 mg mL −1 at 10% breakthrough point, respectively. The results were comparable to other chromatographic adsorbent. In addition, the proteins mixture with different p I was well separated at high flow velocity (611.0 cm h −1 ) and high protein recovery (over 97%), proving the macroporous cellulose monolith had excellent separation performance. In this way, the prepared cellulose monolith with bimodal pores system is expected for the potential application in high-speed chromatography. [ABSTRACT FROM AUTHOR]

• Protein elution in a lab chromatographic system was described by the CFD model. • Elution of low-viscosity (LV) and high-viscosity (HV) samples was examined. • For LV band broadening was pronounced in the injector and the column hardware. • For HV band broadening was pronounced in packed bed due to viscosity effects. • CFD model accurately predicted band broadening and viscous fingering effect. A fluid dynamics model has been developed to describe flow behavior in a lab-scale chromatographic system dedicated for protein processing. The case study included a detailed analysis of elution pattern of a protein, which was a monoclonal antibody, glycerol, and their mixtures in aqueous solutions. Glycerol solutions mimicked viscous environment of the concentrated protein solutions. The model accounted for concentration dependences of solution viscosity and density, and dispersion anisotropy in the packed bed. It was implemented into a commercial computational fluid dynamics software using user-defined functions. The prediction efficiency was successfully verified by comparing the model simulations in the form of the concentration profiles and their variances with the corresponding experimental data. The contribution of the individual elements of the chromatographic system to protein band broadening was evaluated for different configurations: for the extra-column volumes in the absence of the chromatographic column, for the zero-length column without the packed bed and for the column containing the packed bed. The influence of the operating variables, including: the mobile phase flowrate, the type of the injection system, i.e., the injection loop capillary or the superloop, the injection volume and the length of the packed bed, on band broadening of the protein was determined under nonadsorbing conditions. For protein solutions having viscosity comparable with the mobile phase, the flow behavior either in the column hardware or in the injection system made major contributions to band broadening, which depended on the type of the injection system. For highly viscous protein solution, the flow behavior in the packed bed exerted a dominant influence on band broadening. [ABSTRACT FROM AUTHOR]

• Multi-size optimization of macroporous cellulose microspheres. • Larger macropores are beneficial to the mass transfer of proteins. • Smaller macropores can also improve uptake kinetic by surface diffusion. • Increasing the ligand length significantly improves the adsorption performance. Multi-size optimization of ion exchangers based on protein characteristics and understanding of underlying mechanism is crucial to achieve maximum separation performance in terms of adsorption capacity and uptake kinetic. Herein, we characterize the effects of three different sizes, macropore size, protein size, and ligand length, on the protein adsorption capacity and uptake kinetic of macroporous cellulose beads, and provide insights into the underlying mechanism. In detail, (1) for smaller bovine serum albumin, macropore size has a negligible effect on the adsorption capacity, while for larger γ-globulin, larger macropores improve the adsorption capacity due to the high accessibility of binding sites; (2) there is a critical pore size (CPZ), at which the adsorption uptake kinetic is minimum. When pore sizes are higher than the CPZ, uptake kinetics are enhanced by pore diffusion. When pore sizes are lower than CPZ, uptake kinetics are enhanced by surface diffusion; (3) increasing ligand length improves the adsorption capacity by three-dimensionally extended polymer chains in pores and enhances uptake kinetic by improved surface diffusion. This study offers an integrated perspective to qualitatively assess the effects of multiple sizes, providing guidance for designing advanced ion exchangers for protein chromatography. [ABSTRACT FROM AUTHOR]

[Display omitted] • Effect of pore size on the performance of grafting-ligand ion exchangers was studied. • Four media with the average pore sizes ranging from 164.57 to 281.96 nm were prepared. • A critical pore size with maximum adsorption capacity and fastest uptake kinetic exists. • The critical pore sizes of BSA and γ-globulin are 216.64 and 266.93 nm, respectively. To study the details of the effect of pore size on the performance of grafting-ligand ion exchangers, four diethylaminoethyl-dextran grafted macroporous cellulose microspheres with different pore sizes ranging from 164.57 to 281.96 nm were selected to investigate their adsorption equilibria and kinetics for bovine serum albumin (BSA) and γ-globulin. It was found that for whether adsorption equilibria or uptake kinetics, both the proteins showed increasing-then-decreasing trends with the decreasing pore size. More importantly, for a specific protein, the maximum adsorption capacity and fastest uptake rate were achieved on the same media, indicating the existence of a critical pore size (CPZ), at which the adsorption capacity and uptake kinetic are significantly enhanced synchronously. The CPZs of BSA and γ-globulin were identified to be 216.64 and 266.93 nm, respectively. Experimental results and analyses revealed that the high adsorption capacities at CPZ are attributed to the high utilization of binding sites in pores, and the uptake kinetics are enhanced by the combination of fast pore diffusion and chain delivery effect. [ABSTRACT FROM AUTHOR]

Gloydius venom is a rich natural source of proteins possessing platelet aggregation inhibition activity. These proteins or their recombinant analogs are universal instruments for the investigation of proteins and receptors interaction in hemostatic system. At the same time previously studied and potentially new pharmacological properties of platelet aggregation inhibitors from snake venoms create a background for the development of the manufacturing technologies. This study presents a part of technological development of several target proteins parallel obtaining from G. blomhoffii brevicaudus snake venom, in particular deve­lop­ment of manufacturing steps for platelet aggregation inhibitor.The target protein was purified using three different chromatographic approaches. It was shown that it is a single chain protein with molecular weight 12001.55 Da, isoelectrical point 8.03, which inhibited platelet aggregation in the rabbit platelet rich plasma with ID50 = 3.26×10-6 g or 542×10-9 М and revealing no enzyme activity.

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations. [ABSTRACT FROM AUTHOR]

We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding. [ABSTRACT FROM AUTHOR]

Various liquid chromatographic techniques are considered standard analytical methods in proteins characterization. These methods provide essential information for drug approval, for biological and life sciences. On the other hand, there are some issues and challenges which have to be taken into account when analyzing these biopharmaceuticals. The aim of this review to summarize the most recent knowledge relating to the following topics: i) sample stability and complexity ii) adsorption problems: instrument inertness iii) adsorption problems: recovery from the stationary phase and iv) challenges in method development. This information is supposed to help practicing chromatographers in the emerging field of therapeutic protein chromatography. [ABSTRACT FROM AUTHOR]

Capillary-channeled polymer (C-CP) fiber stationary phases are finding utility in the realms of protein analytics as well as downstream processing. We have recently described the modification of poly(ethylene terephthalate) (PET) C-CP fibers to affect amine-rich phases for the weak anion-exchange (WAX) separation of proteins. Polyethylenimine (PEI) is covalently coupled to the PET surface, with subsequent cross-linking imparted by treatment with 1,4-butanediol diglycidyl ether (BUDGE). These modifications yield vastly improved dynamic binding capacities over the unmodified fibers. We have also previously employed native (unmodified) nylon 6 C-CP fibers as weak anion/cation-exchange (mixed-mode) and hydrophobic interaction chromatography (HIC) phases for protein separations. Polyamide, nylon 6, consists of amide groups along the polymer backbone, with primary amines and carboxylic acid end groups. The analytical separation characteristics of these three amine-based C-CP fiber phases are compared here. Each of the C-CP fiber columns in this study was shown to be able to separate a bovine serum albumin/hemoglobin/lysozyme mixture at high mobile phase linear velocity (∼70 mm s) but with different elution characteristics. These differences reflect the types of protein-surface interactions that are occurring, based on the active group composition of the fiber surfaces. This study provides important fundamental understanding for the development of surface-modified C-CP fiber columns for protein separation. [ABSTRACT FROM AUTHOR]

Measurements of the nanoscale structure of chromatographic adsorbents and the associated distribution of sorbed protein within the media can facilitate improvements in such media. We demonstrate a new technique for this purpose using small-angle neutron scattering (SANS) to characterize the nano- to microscale structure of the chromatographic media and sorbed protein under conditions relevant for preparative chromatographic separations. The adsorption of lysozyme on cellulosic S HyperCel™ (Pall Corporation), a strong cation exchanger, was investigated by SANS. The scattering spectrum is reduced to three contributions arising from (1) the chromatographic medium, (2) discrete protein molecules, and (3) the distribution of sorbed protein within the medium. These contributions are quantified for a range of protein loadings. The total concentration of protein in the chromatographic media can be quantified from the SANS spectrum and the protein is observed to retain its tertiary structure upon adsorption, within the resolution of the method. Further analysis of the SANS spectra shows that protein adsorption is uniform in the media. These measurement techniques provide new and valuable nanoscale information about protein sorption in chromatographic media. [ABSTRACT FROM AUTHOR]

Chromatography is the key technology in protein purification as well as in protein refolding. Taking the scientific development and technological innovation of protein chromatography as the objective, this article is devoted to an overview of protein behavior at chromatographic surfaces, including protein orientation, conformational transitions (unfolding and refolding), and protein transport. Recent advances achieved by using molecular simulations as well as theoretical and experimental investigations are elaborated and discussed with emphasis on their implications to the rational design of novel chromatographic surfaces or materials and mobile phase conditions for the development of high-performance protein chromatography. [ABSTRACT FROM AUTHOR]

Protein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial-and-error method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to optimize the operating conditions. In this article, we suggest a design procedure for matrix-assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion-exchange chromatography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α-lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix-assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used. [ABSTRACT FROM AUTHOR]

A high-speed thermoresponsive medium was developed by grafting poly( N -isopropylacrylamide- co -butyl methacrylate) (P(NIPAM- co -BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS–P(NIPAM- co -BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM- co -BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS–P(NIPAM- co -BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h −1 . In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in ‘green’ high-speed preparative protein chromatography. [ABSTRACT FROM AUTHOR]

A multiscale model is presented to elucidate protein adsorption and transport behaviors in ion-exchange chromatography (IEC) adsorbent particles that have either an open pore structure or charged dextran polymers grafted into the pores. Molecular dynamics simulation is used to determine protein diffusion and partitioning in different regions of the adsorbent pore, and these outputs are used in numerical simulations of mass transfer to determine the intraparticle protein concentration profile and the mass-transfer rate. Modeling results indicate that, consistent with experimental observations, protein transport can be faster in the polymer-grafted material compared to the open pore case. This occurs when favorable partitioning of protein into the polymer-filled pore space is combined with relatively high protein mobility within this region. The modeling approach presented here should be applicable to proteins and adsorbents with different properties, and could help elucidate the factors that control adsorption and transport in various IEC systems. © 2014 American Institute of Chemical Engineers AIChE J, 60: 3888-3901, 2014 [ABSTRACT FROM AUTHOR]

A novel composite cryogel monolith was developed by coating poly(glycidyl methacrylate) nanoparticles (NPs) onto the pore wall surface of poly(acrylamide) cryogel. The NPs-coated column was double-modified with poly(ethylenimine) (PEI) and diethylaminoethyl in sequence. Scanning electron microscopy revealed the dense coating of the NPs on the cryogel surface, but the NPs-coating did not result in distinct changes of the column porosity and permeability. The rough pore wall surface and extended polymer chains offered more binding sites, so the dynamic binding capacity of the composite cryogel bed for bovine serum albumin reached 11.7 mg/mL bed volume at a flow rate of 6 cm/min, which was 4.2 times higher than that of the cryogel bed modified with PEI without coating NPs (2.8 mg/mL). The binding capacity as well as column efficiency decreased only slightly with increasing flow rate from 0.6 to 12 cm/min. The results indicated that the strategy of NPs-coating incorporating with double ion-exchanger modifications is promising for enhancing cryogel capacities, and the novel material would be useful for high-speed protein chromatography. [ABSTRACT FROM AUTHOR]

Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100µm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties. [ABSTRACT FROM AUTHOR]

Highlights: [•] Steric exclusion chromatography was evaluated on cryogel monolith column. [•] The retention capacity of the cryogel column for γ-globulin reached 20mg/mL. [•] Compact packing of protein precipitates on the cryogel surface was revealed by SEM. [•] Primary separation of serum proteins was achieved with concentrated γ-globulin. [Copyright &y& Elsevier]