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We studied the aging behaviour of gelatin used to size paper. Thus far, research on the aging of paper has largely ignored the sizing agent. Degradation of the protein was characterised and the impact of paper components, such as cellulose, and aluminium potassium sulphate was evaluated. Whatman No. 1 filter papers sized with two types of gelatins (A and B) were prepared as model samples. Commercially sized modern papers (Arches) were also studied in order to compare laboratory samples with real artist papers. Both types of papers were artificially aged (80 °C, 50% relative humidity for 35 and 94 days). Historic papers were included in the study in order to compare artificially aged with naturally aged gelatin. The aqueous extracts from the papers were characterised by aqueous size-exclusion chromatography (SEC) using four PL-Aquagel-OH columns and UV photodiode array detection at 220, 254 and 280 nm. Results showed that gelatin undergoes hydrolysis upon aging, type A gelatin showing a faster degradation rate than type B. The result was an increase in the lower-molar-mass fractions, under 50 000 g mol−1, and especially in a characteristic fraction with a peak molecular mass (Mp) of 14 000 g mol−1. A significant decrease in the extraction yields of α-, β- and γ-chains occurred after aging. This was attributed to crosslinking, leading to the formation of less-soluble polypeptides with very high molar mass (>800 000 g mol−1). Less than 10% alum had no impact on the degradation rate; higher alum contents accelerated hydrolysis reactions. [Copyright &y& Elsevier]

Abstract: High throughput process development (HTPD) platforms provide time and resource effective solutions for optimization of resin as well as membrane based chromatography processes. They enable successful implementation of Quality by Design by allowing us to investigate more variables and wider ranges than would be possible using traditional lab-scale based experiments. This paper presents an investigation about non-specific protein adsorption that was found to occur on the membrane disks used in a commercially available high throughput device. Both qualitative as well as quantitative analytical tools were used in this investigation. Two biotherapeutic protein molecules, granulocyte colony stimulating factor (GCSF) and Transtuzumab were selected as model proteins in this study. Qualitative assessment was performed using Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). An empirical model based on contact angle measurement was developed for quantitative assessment of the amount of adsorbed protein. The results indicated that while significant non-specific adsorption was observed in case of highly hydrophobic GCSF molecule, adsorption was minimal in the case of Transtuzumab. The paper proposes a novel use of contact angle measurement for quantitative estimation of adsorbed protein. The study also highlights the importance of accounting for non-specific adsorption when using these HTPD devices. [Copyright &y& Elsevier]

Abstract: Many recombinant proteins (rPRTs) have a high bioactivity and some of them may eventually be classified as drugs beneficial to human health, recombinant human protein drugs (rPDs). rPDs are a high-technology product with all the associated economic benefits, therefore the liquid chromatography (LC) of rPRT is different from that of proteins isolated in laboratory scale for purely research purposes. The design of a purification scheme for an rPRT depends on the intended function of the purified rPRT, as a pure sample for research in small scale, or as a product for industrial production. This review paper mainly deals with the latter instance, producing rPD at a large scale. Pharmaceutical economics is considered not only for each step of purification, but also the whole production process. This strategy restricts the content of this review paper to the factors affecting the optimization source, the character of rPRT in up-stream technology and the purification of the rPRT in down-stream production. In the latter instance, the purification step is required to be as efficient as possible and LC is the core of the refined purification method, which is either a single LC method or combination of LC methods, sometimes, it may be a combination of LC and other non-LC separation methods comprising an optimized purification technology. Here some typical examples of rPRT purification at the large scale, recent developments, such as protein folding liquid chromatography, short column chromatography, and new packing material and column techniques are introduced. [Copyright &y& Elsevier]

Abstract: This paper describes approaches to optimize the chromatographic performance for our recently developed LC–MS platform, extended range proteomic analysis (ERPA), for comprehensive protein characterization at the ultratrace level. Large digested peptide fragments up to 10kDa (e.g., from lysyl endopeptidase digestion) with or without modifications were well separated with high resolution using narrow bore (20 and 50μm I.D.) poly(styrene-divinylbenzene) (PS-DVB) monolithic columns constructed by in situ solution polymerization. Importantly, the macroporous structure of the monolithic columns facilitated mass transport of large peptides with improved recovery relative to small pore size reversed-phase packings. High sequence coverage (>95%), including identification of phosphorylated and glycosylated particles was achieved for β-casein and epidermal growth factor receptor (EGFR) at the 4 and 20fmol levels per injection, respectively, using the 20μm I.D. PS-DVB monolithic column. For peptides with greater ionization efficiency, the detection limit could be lowered to ∼400zmol. Typically, the separation system produced a peak capacity of ∼200 for a 10cm column. This paper demonstrates that narrow-bore monolithic columns are suitable for high sensitivity and high-resolution separation of large peptide fragments by LC–MS analysis. [Copyright &y& Elsevier]

Abstract: This paper examines chemically selective displacement chromatography using affinity ranking plots, batch displacer screening experiments, column displacements, multi-component adsorption isotherms and spectroscopy. The affinity ranking plot indicated that the displacers, sucrose octasulfate (SOS) and tatrazine, should possess sufficient affinity to displace the proteins amyloglucosidase and apoferritin over a wide range of operating conditions. In addition, the plots indicated that the separation of these proteins by displacement chromatography would be extremely difficult. Further, the two proteins were shown to have very similar retention times under shallow linear gradient conditions. When batch displacement experiments were carried out, both tartrazine and SOS exhibited significant selectivity differences with respect to their ability to displace these two proteins, in contrast to the affinity ranking plot results. Column displacement experiments carried out with sucrose octasulfate agreed with the predictions of the affinity ranking plots, with both proteins being displaced but poorly resolved under several column displacement conditions. On the other hand, column displacement with tartrazine as the displacer resulted in the selective displacement and partial purification of apoferritin. Single- and multi-component isotherms of the proteins with or without the presence of displacers were determined and were used to help explain the selectivity reversals observed in the column and batch displacement experiments. In addition, fluorescence and CD spectra suggested that the displacers did not induce any structural changes to either of the proteins. The results in this paper indicate that multi-component adsorption behavior can be exploited for creating chemically selective displacement separations. [Copyright &y& Elsevier]

Mixed mode or multimodal chromatography has been developed for rational use of multiple interactions in a controlled manner, in contrast to non-specific interactions. Indeed, as the term “mixed mode” suggests, these resins allow different types of interactions within a single chromatographic medium. In this paper, HEA HyperCel™, PPA HyperCel™ mixed-mode chromatographic media have been studied. These mixed-mode sorbents typically involve hydrophobic pseudo-affinity interactions for binding and essentially ionic interactions (charge repulsion) for elution. We identified and characterized these different interactions in chromatographic experiments by exploiting specific properties of proteins using protein standards and complex mixtures. We highlighted the major intervention of at least two types of interactions in these media: hydrophobic and electrostatic interactions. We observed the behaviour of these resins at different pH, ionic strength, with different salts and buffers types and in the presence of different organic compounds. [ABSTRACT FROM AUTHOR]

Protein A affinity chromatography, which is the standard method for purifying monoclonal antibodies (mAbs) is expensive and involves elution at acidic pH, which could degrade the mAb. Moreover, it cannot be used to fractionate charge variants or to remove mAb aggregates. In this study, we examine the purification of Trastuzumab by cation-exchange z2 laterally-fed membrane chromatography (or z2LFMC). It has been shown that z2LFMC is suitable for carrying out high-speed, high-resolution protein purification. The results discussed in this paper demonstrate that the purity of Trastuzumab obtained by the z2LFMC process was comparable to that obtained by protein A chromatography (i.e., >95% with both methods), while the recovery was significantly greater with z2LFMC (90.4% as opposed to 81.7%). Also, the z2LFMC process was faster, did not require acidic pH conditions for elution, and the mAb was eluted in 2.24 bed volumes as opposed to 2.74 bed volumes with protein A chromatography. The mAb productivity obtained with the z2LFMC process was more than 3 times higher than that with the column-based purification process. The z2LFMC device is significantly cheaper than its equivalent protein A affinity column, and earlier studies have shown it to be suitable for separating mAb charge variants and aggregates. Therefore, the z2LFMC process could be evaluated as a cheaper and more efficient alternative to protein A affinity chromatography. • An economic and efficient alternative to protein A affinity chromatography. • Monoclonal antibody purified under mild processing conditions. • Faster separation than other methods including protein A affinity chromatography. • Comparable purity to that obtained by protein A affinity chromatography. • Higher recovery than Protein A affinity chromatography. [ABSTRACT FROM AUTHOR]

This paper presents a mixed integer linear programming (MILP) model for the optimal synthesis of chromatographic protein purification processes including the time line in which our target protein product is collected. The model is linearised using piecewise linear approximation strategies and tested on three example protein mixtures, containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The results are also compared with previous literature models attempting to solve the same problem and show that the proposed approach offers significant gains in computational efficiency without compromising the quality of the solution. [ABSTRACT FROM AUTHOR]

Abstract: Protein glycosylation has significant effects on the structure and function of proteins. The efficient separation and enrichment of glycoproteins from complex biological samples is one key aspect and represents a major bottleneck of glycoproteome research. In this paper, we have explored pH multimodal hydrophobic interaction chromatography to separate glycosylated from non-glycosylated forms of proteins. Three different proteins, ribonuclease, invertase and IgG, have been examined and different glycoforms have been identified. The media itself shows strong responsiveness to small variations in pH, which makes it possible to fine-tune the chromatographic conditions according to the properties of the protein isolated. Optimal glycoprotein separation has been obtained at pH 4. The pH responsive multimodal HIC medium in contrast to conventional HIC media is able to resolve contaminating DNA. [ABSTRACT FROM AUTHOR]

Abstract: The osmotic second virial coefficient is a key parameter in light scattering, protein crystallisation, self-interaction chromatography, and osmometry. The interpretation of the osmotic second virial coefficient depends on the set of independent variables. This commonly includes the independent variables associated with the Kirkwood–Buff, the McMillan–Mayer, and the Lewis–Randall solution theories. In this paper we analyse the osmotic second virial coefficient using a Gibbs–McMillan–Mayer framework which is similar to the McMillan–Mayer framework with the exception that pressure rather than volume is an independent variable. A Taylor expansion is applied to the osmotic pressure of a solution where one of the solutes is a small molecule, a salt for instance, that equilibrates between the two phases. Other solutes are retained. Solvents are small molecules that equilibrate between the two phases. The independent variables of the solvents are temperature, pressure, and chemical potentials. The derivatives in the Gibbs–McMillan–Mayer framework are transformed into derivatives in the Gibbs framework. This offers the possibility for an interpretation and correlation of the osmotic second virial coefficient using activity coefficient models. [Copyright &y& Elsevier]

Abstract: Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme. [Copyright &y& Elsevier]

Abstract: We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of β-lactoglobulin A and β-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method. [Copyright &y& Elsevier]

Abstract: The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD600 =40) culture of recombinant E. coli, and an SK protein concentration of 1080mgl−1 was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18×105 in cell lysate to 0.53×105 IUmg−1 resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96×105 IUmg−1) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps. [Copyright &y& Elsevier]

Abstract: This paper gives a summary of different aspects for predicting protein behaviour in hydrophobic interaction chromatography (HIC). First, a brief description of HIC, hydrophobic interactions, amino acid and protein hydrophobicity is presented. After that, several factors affecting protein chromatographic behaviour in HIC are described. Finally, different approaches for predicting protein retention time in HIC are shown. Using all this information, it could be possible to carry out computational experiments by varying the different operating conditions for the purification of a target protein; and then selecting the best conditions in silico and designing a rational protein purification process involving an HIC step. [Copyright &y& Elsevier]

Abstract: There is significant interest in establishing appropriate bioprocessing conditions for protein adsorption in hydrophobic interaction chromatographic (HIC) systems without the need for high salt concentrations. In this paper, the adsorption and recovery of proteins under low salt conditions in HIC systems was investigated using a variety of experimental and computational techniques. Parallel batch screening was employed to determine protein adsorption and recovery. Experiments were carried out with twenty six proteins using five resins with different ligand chemistry, ligand density and backbone chemistry. Proteins were classified based on various combinations of adsorption and recovery behavior. In order to gain insight into the effect of protein properties on this behavior, molecular descriptors were computed based on protein crystal structure and primary sequence information as well as a set of hydrophobicity descriptors based on the solvent accessible surface area of the proteins. Finally, classification software CART was employed to determine the key molecular descriptors associated with various types of adsorption behavior. [Copyright &y& Elsevier]

Abstract: In this paper Protein A mimetic and hydrophobic charge induction chromatographic (HCIC) stationary phases are characterized in terms of their protein adsorption characteristics and their selectivity is compared with Protein A chromatography using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were employed to compare the selectivities of these resins for both non-IgG model proteins as well as antibodies and the fusion proteins. While none of the non-IgG model proteins were observed to bind to the Protein A resin, most of them did in fact bind to the alternative resins. In addition, while the elution pH was similar for the model proteins and antibodies on the HCIC resin, the mimetic resins did exhibit higher binding for the antibodies under these linear pH gradient conditions. A mixed mode preparative isotherm model previously developed for HCIC was shown to accurately describe the adsorption behavior of the mimetic materials as well. Host cell protein clearance profiles were also investigated under preparative conditions using complex biological feeds and the results indicated that while some selectivity was observed for both the HCIC and the mimetic materials, the purification factors were in general significantly less than those obtained with Protein A. It is important to note, however, that the selectivity of the mimetic and HCIC materials was also observed to be antibody specific indicating that further optimization may well result in increased selectivities for these materials. [Copyright &y& Elsevier]

Abstract: Strong and weak cation-exchangers were compared for a number of chromatographic parameters, i.e. pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and scanning electron microscopy (SEM) pictures. Chromatographic resins investigated were Fractogel EMD SO3 − (M), Fractogel EMD SE Hicap (M), Fractogel EMD COO− (M), MacroPrep 25S, MacroPrep High S, MacroPrep CM, CM HyperZ, and Matrex Cellufine C-500. Testing was done with three proteins: Anti-FVII Mab (IgG), aprotinin, and lysozyme. For lysozyme and aprotinin with pI above experimental pH, dependence of pH on retention was generally low, though some pronounced decrease of retention with increasing pH was observed for CM HyperZ. For Anti-FVII Mab with pI <7.5, binding was observed on several resins at pH 7.5. Efficiency results present the expected trend of increasing dependence of plate height as a function of increasing flow rate, and the highest flow dependence was observed for Fractogel EMD COO−. Particle size distribution was determined by two independent methods, coulter counting and SEM pictures, with fair agreement. Binding strength data of cation-exchange resins as a function of ionic strength depends on the protein, but binding and elution at high salt concentration may in general be performed with MacroPrep resins. Comparison of dynamic capacity data at 10% break-through and static capacity measurements shows that a very diverse utilization of approximately 25–90% of the total available capacity is employed during chromatographic operation. The effect of competitive binding from yeast fermentation components on dynamic binding capacity of aprotinin was studied showing a significant decrease in binding capacity. Sepharose FF, Toyopearl 650M, and Ceramic HyperD F strong and weak cation-exchange resins were included in this study. Resins with good pure aprotinin capacity also performed well for aprotinin in fermentation broth, but the highest relative capacity was obtained with MacroPrep High S having a fairly low pure component dynamic capacity. Results of this paper may be used in the selection of resins for further testing in biopharmaceutical protein purification process development. [Copyright &y& Elsevier]

Abstract: We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14–26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A 330 nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 104–106 response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude that the majority of the major modified amino acids present in early stage brunescent Indian cataract lens proteins appear to arise as a result of ascorbic acid modification, and are presumably advanced glycation end-products. [Copyright &y& Elsevier]

Abstract: A novel continuous supermacroporous monolithic cryogel embedded with nanometer-size particles was prepared by the radical cryogenic co-polymerization of acrylamide (AAm), N,N′-methylene-bis-acrylamide (MBAAm), allyl glycidyl ether (AGE) and the dispersed surfactant-stabilized Fe3O4 nanoparticles under the freezing-temperature variation condition in a glass column. This special separation matrix has interconnected supermacropores with pore size of 10–50μm, which permit the free-passage of microbial cells or cell debris in the culture fluids and then is interest in downstream processes. The axial liquid dispersion coefficients of the new continuous supermacroporous monolithic bed at different liquid flow rates were obtained by measuring residence time distributions (RTDs) using tracer pulse-response method. The experimental results showed that the axial liquid dispersion within the bed was weak in a wide water flow rate of 0.5–15cm/min. The axial dispersion coefficient was found to be increased exponentially with the increase of liquid flow rate. Chromatographic process of bovine serum albumin (BSA) in the cryogel monolithic bed was carried out to reveal the protein breakthrough and elution characteristics. Compared with other reported cryogel beds in literature, the protein adsorption capacity of the present cryogel bed was improved due to the embedded nano-sized solid adsorbents in the gel matrix. Microstructure morphology of the embedded nanoparticles in the cryogel and the gel matrix structure were also analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in this paper. [Copyright &y& Elsevier]

Abstract: The extraction of high-value dairy proteins such as lactoferrin and lactoperoxidase normally requires extensive pre-treatments of milk to remove fat and caseins by centrifugation, precipitation, Ca2+ chelation and/or filtration. Similarly, fat and caseins are normally removed prior to capture of recombinant proteins from the milk of transgenic animals. Such pre-treatments can result in significant loss of protein yield and/or activity. In this paper, we demonstrate that it is possible to pass significant quantities of raw, untreated milk through a 5cm high XK16 chromatography column (volume 10mL) packed with SP Sepharose Big Beads™ (GE Healthcare, Uppsala, Sweden) without exceeding the maximum allowable backpressure, provided that the processing temperature is kept nominally around milking temperature (35–37°C). Results show that more than 100 column volumes of raw milk could be loaded at 300cm/h before breakthrough of lactoperoxidase occurred. The dynamic capacity for adsorbing lactoferrin and lactoperoxidase simultaneously under these conditions was approximately 48.6mg/mL of packed resin. Minor leakage (4.6% of the feed concentration) of lactoferrin occurred throughout the loading process but major breakthrough occurred only after approximately 100 column volumes were loaded. [Copyright &y& Elsevier]

Abstract: This paper focuses on the prediction of the dimensionless retention time (DRT) of proteins in hydrophobic interaction chromatography (HIC) by means of mathematical models based on the statistical description of the amino acid surface distribution. Previous models characterises the protein surface as a whole. However, most of the time it is not the whole protein but some of its specific regions that interact with the environment. It seems much more natural to use local measurements of the characteristics of the surface. Therefore, the statistical characterisation of the distribution of an amino acid property on the protein surface was carried out from the systematic calculation of the local average of this property in a neighbourhood placed sequentially on each of the amino acids on the protein surface. This process allowed us to characterise the distribution of this property quantitatively using three main statistics: average, standard deviation and maximum. In particular, if the property considered is a hydrophobicity scale, these statistics allowed us to characterise the average hydrophobicity and the hydrophobic content of the most hydrophobic cluster or hotspot, as well as the heterogeneity of the hydrophobicity distribution on the protein surface. We tested the performance of the DRT predictive models based on these statistics on a set of 15 proteins. We obtained better predictive results with respect to the models previously reported. The best predictive model was a linear model based on the maximum. This statistic was calculated using an index of the mobilities of amino acids in chromatography. The predictive performance of this model (measured as the Jack Knife MSE) was 26.9% better than those obtained by the best model which does not consider the amino acid distribution and 19.5% better than the model based on the hydrophobic imbalance (HI). In addition, the best performance was obtained by a linear multivariable model based on the HI and the maximum. The difference between the experimental data and the prediction carried out by this model was smaller than those observed previously. In fact, this model obtained better predictive capacities than a previous linear multivariable model decreasing the Jack Knife MSE in 8.7%. In addition, this model allowed us to diminish the number of variables required, increasing, in this way, the degrees of freedom of the model. [Copyright &y& Elsevier]

Abstract: This paper focuses on the prediction of the dimensionless retention time of proteins (DRT) in hydrophobic interaction chromatography (HIC) by means of mathematical models based on characteristics of the surface hydrophobicity distribution. We introduce a new parameter, called hydrophobic imbalance (HI), obtained from the three-dimensional structure of proteins. This parameter quantifies the displacement of the superficial geometric centre of the protein when the effect of the hydrophobicity of each amino acid is considered. This parameter is simpler and less expensive than those reported previously. We use HI as a way to incorporate information about the surface hydrophobicity distribution in order to improve the prediction of DRT. We tested the performance of our DRT predictive models in a set of 15 proteins. This set includes four proteins whose DRTs are known as very difficult to predict. By means of the variable HI, it was possible to improve the predictive characteristics obtained by models based on the average surface hydrophobicity (ASH) by 9.1%. Also, we studied linear multivariable models based on characteristics determined from the HI. By using this multivariable model, a correlation coefficient of 0.899 was obtained. With this model, we managed to improve the predictive characteristics shown by previous models based on ASH by 31.8%. [Copyright &y& Elsevier]

Abstract: Poly(ethylene glycol)-conjugated (or PEGylated) proteins are an increasingly important class of therapeutic proteins that offer improved in vivo circulation half lives over their corresponding native forms. Their production involves covalent attachment of one or more poly(ethylene glycol) molecules to a native protein, followed by purification. Because of the extremely high costs involved in producing native therapeutic proteins it is important that subsequent PEGylation processes are as efficient as possible. In this paper, reaction engineering and purification issues for PEGylated proteins are reviewed. Paramount considerations for PEGylation reactions are specificity with respect to the conjugation site and overall yield. Batch PEGylation reaction methods are discussed, along with innovative methods using packed bed or “on-column” approaches to improve specificity and yield. Purification methods are currently dominated by ion exchange and size exclusion chromatography. Other methods in common use for protein separations, including hydrophobic interaction chromatography, affinity chromatography and membrane separations, are rarely used in PEGylated protein purification schemes. A better understanding of the effects of PEGylation on the physicochemical properties of proteins (isoelectric point, surface charge density and distribution, molecular size and relative hydrophobicity) and interactions between PEGylated proteins and surfaces is needed for the future development of optimal purification processes and media. [Copyright &y& Elsevier]

Abstract: This paper focuses on the prediction of the dimensionless retention time of proteins (DRT) in hydrophobic interaction chromatography (HIC) by means of mathematical models based, essentially, only on aminoacidic composition. The results show that such prediction is indeed possible. Our main contribution was the design of models that predict the DRT using the minimal information concerning a protein: its aminoacidic composition. The performance is similar to that observed in models that use much more sophisticated information such as the three-dimensional structure of proteins. Three models that, in addition to the amino acid composition, use different assumptions about the amino acids tendency to be exposed to the solvent, were evaluated in 12 proteins with known experimental DRT. In all the cases analyzed, the model that obtained the best results was the one based on a linear estimation of the aminoacidic surface composition. The models were adjusted using a collection of 74 vectors of aminoacidic properties plus a set of 6388 vectors derived from these using two mathematical tools: k-means and self-organizing maps (SOM) algorithms. The best vector was generated by the SOM algorithm and was interpreted as a hydrophobicity scale based partly on the tendency of the amino acids to be hidden in proteins. The prediction error (MSEJK) obtained by this model was almost 35% smaller than that obtained by the model that supposes that all the amino acids are completely exposed and 40% smaller than that obtained by the model that uses a simple correction factor considering the general tendency of each amino acid to be exposed to the solvent. In fact, the performance of the best model based on the aminoacidic composition was 5% better than that observed in the model based on the three-dimensional structure of proteins. [Copyright &y& Elsevier]

Abstract: This paper presents the development and application of a solid phase microextraction method for the full investigation of chlorhexidine interaction with saliva during a pharmacokinetic study: chemical stability, binding to proteins, free concentration, total concentration and kinetics of elimination after oral administration. Only 0.1mL sample were needed for each time point and the concentration of salivary proteins was determined as well. It was shown that chlorhexidine remained stable in the oral cavity for at least 9h and high concentrations of the drug (2μg/mL total) were still present even 8h after mouthrinsing. Supplementary facts were uncovered: while the total concentration followed first-order elimination kinetics, the free concentration remained almost constant for several hours; this showed that the oral cavity acted like a reservoir that slowly released the drug. It was also revealed that following oral administration of chlorhexidine, the normal composition of saliva changed for a few hours, probably as a physiological response to the bitter taste of the medicine. The method had a wide linear range (0.1–40μg/mL free chlorhexidine) that was perfectly suitable for the study of chlorhexidine retention in the oral cavity. Separation and quantitation were achieved by liquid chromatography coupled to mass spectrometry; no interference from endogenous compounds was observed. This selective and sensitive solid-phase microextraction (SPME) approach for monitoring the free and total concentration of a drug, as well as the concentration of proteins that bind that drug, should prove to be more useful for pharmacokinetic studies than classic methods that only provide the total concentration as a final result. [Copyright &y& Elsevier]

Abstract: There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 × 10−13 and 2.4 × 10−13 m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1–3μm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow. [Copyright &y& Elsevier]

Abstract: This paper presents a methodology to gain process knowledge and assist in the robustness analysis of an ion-exchange step in a protein purification process using a model-based approach. Factorial experimental design is common practice in industry today to obtain robustness characterization of unit operations with respect to variations in process parameters. This work aims at providing a better insight into what process variations affect quality and to further reduce the experimental work to the regions of process variation that are of most interest. This methodology also greatly increases the ability to predict process performance and promotes process understanding. The model calibration part of the methodology involves three consecutive steps to calibrate a steric mass action (SMA) ion-exchange chromatography model. Firstly, a number of gradient elution experiments are performed. Secondly, experimental breakthrough curves have to be generated for the proteins if the adsorption capacity of the medium for each component is not known. Thirdly, a multi-component loading experiment is performed to calibrate the multi-component effects that cannot be determined from the single-component experiments. The separation process studied in this work is the separation of polyclonal IgG from a mixture containing IgG, myoglobin and BSA. The calibrated model is used to simulate six process variations in a full factorial experiment. The results of the simulations provide information about the importance of the different process variations and the simulations are also used to determine the crucial points for the process parameter variations. The methodology can be used to assist in the robustness analysis normally performed in the pharmaceutical industry today as it is able to predict the impact on process performance resulting from variations in salt concentration, column load, protein concentration and flow rate. [Copyright &y& Elsevier]

Monolithic columns for capillary electrochromatography are receiving quite remarkable attention. This review summarizes results excerpted from numerous papers concerning this rapidly growing area with a focus on monoliths prepared from synthetic polymers. Both the simplicity of the in situ preparation and the large number of readily available chemistries make the monolithic separation media a vital alternative to capillary columns packed with particulate materials. Therefore, they are now a well-established stationary phase format in the field of capillary electrochromatography. A wide variety of synthetic approaches as well as materials used for the preparation of the monolithic stationary phases are presented in detail. The analytical potential of these columns is demonstrated with separations involving various families of compounds and different chromatographic modes. [Copyright &y& Elsevier]

The flexibility and selectivity of size exclusion chromatography (SEC) for protein purification can be modified by adding non-ionic micelle-forming surfactants to the mobile phase. The micelles exclude proteins from a liquid phase similar to the exclusion effect of the polymer fibers of the size exclusion resin. This surfactant-aided size exclusion chromatography technology (SASEC) is demonstrated on the separation of two model proteins; bovine serum albumin (BSA) and myoglobin (Myo). The effect of the added surfactants on the distribution behavior of the proteins is predicted adequately by a size exclusion model presented in this paper. [Copyright &y& Elsevier]

This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate–polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22 000 and 25 000. [Copyright &y& Elsevier]

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and β-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity. [Copyright &y& Elsevier]

A method to suppress the interference of pigments in the analysis of proteinaceous media used in paintings is presented in this paper. This method is based on the formation of metallic ion–ethylenediaminetetraacetic acid (EDTA) complexes previous to the derivatisation process, using ethyl chloroformate (ECF), to transform the amino acids in N(O,S)-ethoxycarbonyl (EOC) ethyl esters. Test specimens, containing different proteinaceous media such as albumin, porcine gelatine and casein mixed with lead white, chalk, verdigris and raw Sienna have been prepared for carrying out this study. Different pH conditions have been probed for the different pigments studied. Values of peak area ratio of amino acids relative to the alanine, obtained using the proposed method on a series of protein–pigment test specimens, have been compared to those from specimens of pure protein in which direct method of derivatisation was applied. Finally, the method has been successfully applied to the analysis of 18th century wall paintings in which animal glue was used as binding medium. [Copyright &y& Elsevier]

Hydrophobic interaction chromatography (HIC) is widely used in the downstream processing of proteins. Resolution of HIC is very good, but sometimes not as high as expected. Resolution values could be increased if good operating conditions were selected. In this paper we present a methodology for selecting good operating conditions. First, it is necessary to predict the dimensionless retention time (DRT) of each protein in the mixture. We use a correlation such as DRT=A+Bφ+Cφ2, where φ is the superficial hydrophobicity of the protein, which is calculated considering the hydrophobicity of the superficial amino acids using the Miyazawa–Jernigan scale. Considering that there was little interaction amongst proteins in a mixture at the concentrations investigated (2 g/l of each protein), the behaviour of the proteins in the mixture was considered to be similar to that of the individual proteins. Using simulations it was possible to test different operating conditions for the purification of a target protein from a mixture of proteins and it was possible to select ideal conditions. The methodology developed was also tested for the purification of a recombinant protein from a fermentation extract of yeast producing human superoxide dismutase and the results have been satisfactory. [Copyright &y& Elsevier]

A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [α1(V)]2α2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods. [Copyright &y& Elsevier]

In process-scale antibody purification, protein-A affinity chromatography is commonly used as the initial purification step. In this paper, two different protein-A media were evaluated. These adsorbents have a porous glass backbone with different pore sizes: 700 A˚ and 1000 A˚. Adsorption equilibrium data of human immunoglobulins on these media were measured via a batch technique and correlated using the Langmuir isotherm model. A larger static capacity was found for the smaller pore size material, which is probably a result of the larger specific surface area and associated higher ligand concentration. The protein uptake kinetics were also obtained via a stirred tank experiment using different initial protein concentrations. A surface layer model was used to represent the protein uptake by the media and to estimate values of a concentration-independent effective diffusivity within the particle. Experimental breakthrough curves were also obtained from packed beds operated under different conditions. Calculated breakthrough profiles were found to be in good agreement with the experimental results. Experimental breakthrough data were used to determine the dependence of the dynamic capacity of the media as a function of the fluid residence time. A larger dynamic capacity was also found for the smaller pore size media. The permeability of large scale packed beds was also reported and used in conjunction with the dynamic capacity to calculate the process production rate. [Copyright &y& Elsevier]

This paper reports a system for three-dimensional electrochromatography in a chip format. The steps involved included trypsin digestion, copper(II)-immobilized metal affinity chromatography [Cu(II)-IMAC] selection of histidine-containing peptides, and reversed-phase capillary electrochromatography of the selected peptides. Trypsin digestion and affinity chromatography were achieved in particle-based columns with a microfabricated frit whereas reversed-phase separations were executed on a column of collocated monolithic support structures. Column frits were designed to maintain constant cross sectional area and path length in all channels and to retain particles down to a size of 3 μm. Cu(II)-IMAC selection of histidine-containing peptides from standard peptide mixtures and protein digests followed by reversed-phase chromatography of the selected peptides was demonstrated in the electrochromatography mode. The possibility to run a comprehensive proteomic analysis by combining trypsin digestion, affinity selection, and a reversed-phase separation on chips was shown using fluorescein isothiocyanate-labeled bovine serum albumin as an example. [Copyright &y& Elsevier]

This paper reports how a novel method for determining the surface hydrophobicity of a protein can be used to predict the dimensionless retention time (DRT) in hydrophobic interaction chromatography. The methodology has three steps. First, it is necessary to know the three-dimensional structure of the target protein. Then, it is necessary to calculate the surface hydrophobicity of the protein, considering that each amino acid has a relative contribution to the surface properties, using an appropriate equation. This will depend largely on the relative scale used to evaluate the hydrophobicity of each amino acid. Forty-two scales were investigated and two gave the best correlation, the Miyazawa–Jernigan and the Cowan–Whittaker scales. Finally, a “quadratic model” (DRT=a+bΦsurface+cΦsurface2) is used to predict the retention time of the target protein. The methodology was able to correlate adequately the retention data for monomeric proteins not included in the generation of the model. [Copyright &y& Elsevier]

Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(Hc)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is ∼3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(Hc) proteins. [Copyright &y& Elsevier]

Some of the problems associated with packed bed chromatography can be overcome by using synthetic macroporous and microporous membranes as chromatographic media. This paper reviews the current state of development in the area of membrane chromatographic separation of proteins. The transport phenomenon of membrane chromatography is briefly discussed and work done in this area is reviewed. The various separation chemistries which have been utilised for protein separation, along with different applications, are also reviewed. The technical challenges facing membrane chromatography are highlighted and the scope for future work is discussed. [Copyright &y& Elsevier]

High-performance monolithic disk chromatography (HPMDC), including its affinity mode, is a very efficient method for fast separations of biological molecules of different sizes and shapes. In this paper, protein and peptide ligands, immobilized on the inner surface of thin, monolithic supports (Convective Interaction Media or CIM® disks), have been used to develop methods for fast, quantitative affinity fractionation of pools of polyclonal antibodies from blood sera of rabbits, immunized with complex protein–peptide conjugates. The combination of several disks with different affinity functionalities in the same cartridge enables the separation of different antibodies to be achieved within a few minutes. The apparent dissociation constants of affinity complexes were determined by frontal analysis. Variation of elution flow rate over a broad range does not affect the affinity separation characteristics. Indifferent synthetic peptides used as biocompatible spacers do not change the affinity properties of the ligands. The highly reproducible results of immunoaffinity HPMDC are compared with data obtained by widely used enzyme-linked immunosorbent assay. [Copyright &y& Elsevier]

• Supercritical fluid chromatography and enhanced fluidity liquid chromatography. • Free amino acids, peptides, proteins and nucleic acid bricks. • Mobile phases of CO 2 and co-solvent, comprising water and additives. • Detection issues and hyphenation to mass spectrometry. • Applications in foodomics, metabolomics, clinical and pharmaceutical areas. Biomolecules like proteins, peptides and nucleic acids widely emerge in pharmaceutical applications, either as synthetic active pharmaceutical ingredients, or from natural products as in traditional Chinese medicine. Liquid-phase chromatographic methods (LC) are widely employed for the analysis and/or purification of such molecules. On another hand, to answer the ever-increasing requests from scientists involved in biomolecules projects, other chromatographic methods emerge as useful complements to LC. In particular, there is a growing interest for chromatography with a mobile phase comprising pressurized carbon dioxide, which can be named either (i) supercritical (or subcritical) fluid chromatography (SFC) when CO 2 is the major constituent of the mobile phase, or (ii) enhanced fluidity liquid chromatography (EFLC) when hydro-organic or purely organic solvents are the major constituents of the mobile phase. Despite the low polarity of CO 2 , supposedly inadequate to solubilize such biomolecules, SFC and EFLC were both employed in many occasions for this purpose. This paper specifically reviews the literature related to the SFC/EFLC analysis of free amino acids, peptides, proteins, nucleobases, nucleosides and nucleotides. The analytical conditions employed for specific molecular families are presented, with a focus on the nature of the stationary phase and the mobile phase composition. We also discuss the potential benefits of combining SFC/EFLC to LC in a single gradient elution, a method sometimes designated as unified chromatography (UC). Finally, detection issues are presented, and more particularly hyphenation to mass spectrometry. [ABSTRACT FROM AUTHOR]