PEGylated protein separation using different hydrophobic interaction supports: Conventional and monolithic supports

Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, β‐lactoglobulin, and lysozyme. All proteins were PEGylated in the N‐terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono‐ and di‐PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of β‐lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:702–707, 2016     

Separation of hexokinase activity using different hydrophobic interaction supports

Hydrophobic interaction chromatography (HIC) has been used extensively for the separation of proteins and peptides by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this paper we compare different hydrophobic interaction chromatographic media for the separation of multiple forms of hexokinase from rabbit reticulocytes. Among the different hydrophobic chromatographic media tested (Toyopearl Phenyl 650S, Ether 650S and Butyl 650S) Toyopearl Phenyl 650S offered the best separation of multiple forms of hexokinase, probably due to its intermediate hydrophobicity. In order to establish the optimal experimental conditions, we evaluated the effects of different salts, and the results obtained demonstrated that among the antichaotropic salts, ammonium sulphate is the most suitable for the separation of hexokinase sub-types. The sample loading capacity of the three Toyopearl supports was investigated and the recovery of enzymatic activity obtained ranged from 60% to 90%, depending on the different salts and hydrophobic media used. The chromatographic profiles of hexokinase activity from various mammalian and fungal tissues also demonstrate that Toyopearl Phenyl 650S can be successfully employed for the separation of multiple forms of enzymes from different biological sources.     

Solvent modulation in hydrophobic interaction chromatography

Solvents play a critical role in hydrophobic interaction chromatography (HIC), since the separation of proteins by HIC is based on the hydrophobicity of the proteins presented to the solvents. This review first describes the solvent properties which determine the effect of cosolvents on the binding and elution of proteins in HIC; i.e., the protein solvent interactions and the surface tension of water/cosolvent mixture. Second are presented the various cosolvents which have been tested as facilitating binding or elution of the proteins. Last, some examples of solvent manipulation which resolved complex mixtures of proteins by HIC are reviewed.     

Depletion of highly abundant proteins in blood plasma by hydrophobic interaction chromatography for proteomic analysis

The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA.     

Chromatographic fractionation of yeast extract: A strategy to identify physicochemical properties of compounds promoting CHO cell culture

The study proposes to get a better knowledge of the physicochemical properties of yeast extract (YE) molecules involved in the improvement of CHO cell growth and to reduce YE complexity without losing positive effects. Various chromatographic processes were implemented for fractionation of a nanofiltrated YE (nYE). The nYE was first fractionated by one-step preparative chromatography, either with anion exchange (AEC), hydrophobic interaction (HIC) or size exclusion (SEC) methods. After analysis of its main components, each fraction was added in a control chemically defined medium to assess its impact on CHO cell growth. Results mainly underlined that AEC was the most selective separation process to purify nYE in one step without decreasing cell growth promoting effect. A three-step chromatographic process including successive AEC, HIC, and SEC was then developed to refine the physicochemical properties of nYE compounds. Among fractions that triggered similar cell growth promoting effect than nYE, one also improved IgG specific production. It mainly included cationic and hydrophilic peptides with a great proportion of lysine and arginine, low quantities of polysaccharides and no nucleic acids. Thus, this study allowed us to deepen the YE contribution to animal cell culture as well as to evaluate fractionation strategies to simplify such a complex mixture.     

Multi‐dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

A multi‐dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion‐exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size‐exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt‐gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision‐making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge‐based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation. Biotechnol. Bioeng. 2012; 109: 3070–3083. © 2012 Wiley Periodicals, Inc.     

Prediction of protein retention in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a powerful technique for protein separation. This review examines methodologies for predicting protein retention time in HIC involving elution with salt gradients. The methodologies discussed consider three-dimensional structure data of the protein and its surface hydrophobicity. Despite their limitations, the methods discussed are useful in designing purification processes for proteins and easing the tedious experimental work that is currently required for developing purification protocols.     

Quantifying process tradeoffs in the operation of chromatographic sequences

A method for the rapid representation of key process tradeoffs that need to be made during the analysis of chromatographic sequences has been proposed. It involves the construction of fractionation and maximum purification factor versus yield diagrams, which can be completed easily on the basis of chromatographic data. The output of the framework developed reflects the degree of tradeoff between levels of yield and purity and provides a fast and precise prediction of the sample fraction collection strategy needed to meet a desired process specification. The usefulness of this approach for the purposes of product purification and contaminant removal in a single chromatographic step has been successfully demonstrated in an earlier paper and it is now extended by application to a chromatographic sequence: the separation of a hypothetical three-component protein system by hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). The HIC operation has a strong impact upon the subsequent SEC step. The studies show how the analysis of performance in such a chromatographic sequence can be carried out easily and in a straightforward fashion using the fractionation diagram approach. The methodology proposed serves as a useful tool for identifying the process tradeoffs that must be made during operation of a sequence of chromatographic steps and indicates the impact on further processing of the cut-point decisions that are made.     

A Procedure for Human Pregnancy Zone Protein (and Human α2-Macroglobulin) Purification Using Hydrophobic Interaction Chromatography on Phenyl–Sepharose CL-4B Column

In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl–Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human α₂-macroglobulin (α₂-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of α₂-M from the same pregnancy plasma, based on the differential elution of PZP and α₂-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP–protease complexes, HIC on phenyl–Sepharose column could also be used for separating both conformational states of PZP.     

Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions

Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Performance was comparable to that observed using conventional HIC conditions with high salt.     

Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6®‐derived recombinant antibody

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow‐through mode for contaminant removal. As flow‐through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind‐and‐elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow‐through applications. Given these considerations, a new Sartobind Phenyl? membrane adsorber was developed for large‐scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography—virtually no diffusion limitation and shorter processing time—with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg‐MAb/cm³‐membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five‐ to ten‐fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6® cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale‐up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography. Biotechnol. Bioeng. 2010; 105: 296–305. © 2009 Wiley Periodicals, Inc.     

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein ¹. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH₄)₂SO₄). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) ². As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter ³. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs ⁴.The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light ⁵. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.Download video file.^{(63M, mov)}     

Identification and quantification of basic and acidic proteins using solution-based two-dimensional protein fractionation and label-free or 18O-labeling mass spectrometry

Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells.     

Protein instability during HIC: describing the effects of mobile phase conditions on instability and chromatographic retention

Hydrophobic interaction chromatography (HIC) is known to be potentially denaturing to proteins, but the effects of mobile phase conditions on chromatographic behavior are not well understood. In this study, we apply a model describing the effects of secondary protein unfolding equilibrium on chromatographic behavior, including the effects of salt concentration on both stability and adsorption. We use alpha-lactalbumin as a model protein that in the presence and absence of calcium, allows evaluation of adsorption parameters for folded and unfolded species independently. The HIC adsorption equilibrium under linear binding conditions and solution phase protein stability have been obtained from a combination of literature and new experiments. The effect of salt concentration on protein stability and the rate constant for unfolding on the chromatographic surface have been determined by fitting the model to isocratic chromatography data under marginally stable conditions. The model successfully describes the effects of added calcium and ammonium sulfate. The results demonstrate the importance of considering the effects on stability of mobile phase modifiers when applying HIC to marginally stable     

Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.     

<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis> gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays

Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (M_r ~ 30,000). Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of 17beta-estradiol (E₂)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed by HIC) resolved pituitary extracts into two active fractions; both induced E₂ synthesis, but one was relatively poor in eliciting DHP and testosterone production. Thus, using homologous bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E₂-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F. heteroclitus pituitaries by a variety of chromatographic procedures. This study complements previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH function in a multiple-spawning teleost.     

Protein instability during HIC: hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects

Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogen-deuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability.