疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.     

Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.     

Synthesis and properties of chitosan hydrogels modified with heterocycles

Preparation and properties of chitosan modified with heterocycles in absence or presence of gluteraldehyde as a cross linker is described. New modified chitosan–heterocyclic hydrogels were prepared from chitosan and heterocyclic compounds such as N,N′-biisomaleimide, N,N′-biisophthalimide, and N,N′-phthalimidomaleimide via a crosslinking reaction. The new hydrogels chemical structure was characterized by spectral analysis (IR), X-ray diffraction, thermal gravimetric analysis (TGA), solubility, and swellability in water and different organic solvents. Evaluation of the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems was carried out and promising results were obtained.     

On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins

The behavior of a series of pure proteins partitioned in aqueous two-phase systems is compared with their behavior during mild hydrophobic interaction chromatography (HIC). A simple theoretical rationale for this comparison is presented based upon solvophobic theory. Similarities were found in the behavior of the model proteins in the two forms of partition chromatography. This indicates that HIC may be employed as a rapid instrumental technique for the broad characterization of protein behavior, which may be of benefit in the development of liquid-liquid partitioning strategies. However, it has proved difficult to completely account for this behavior on the basis of the known physical and structural properties of the proteins used. The variety in the detailed partitioning behavior of this small sample of protein types suggests that partition in aqueous two-phase systems is uniquely sensitive to subtle differences in surface properties of complex macromolecules. (c) 1994 John Wiley & Sons, Inc.     

Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography

Plants have attracted interest as hosts for protein expression because of the promise of a large production capacity and a low production cost. However, recovery costs remain a challenge as illustrated for recovery of recombinant aprotinin, a trypsin inhibitor, with removal of native corn trypsin inhibitor from transgenic corn (Azzoni et al. in Biotechnol Bioeng 80:268–276, 2002). When expression is targeted to corn grain fractions, dry milling can separate germ and endosperm fractions. Hence, only the product-containing fraction needs to be extracted, reducing the cost of extraction and the impurity level of the extract. Selective extraction conditions can reduce impurity levels to the point that low-cost adsorbents can result in relatively high purity levels. In this work, we attempted to achieve comparable purity with these lower cost methods. We replaced whole grain extraction and purification of recombinant aprotinin with sequential trypsin affinity and IMAC steps with an alternative of germ fraction extraction and purification with ion exchange and hydrophobic interaction chromatography (HIC). Using germ extraction at acidic pH supplemented with heat precipitation to remove additional host proteins resulted in a higher specific activity feed to the chromatographic steps. The cation exchange step provided 7.6× purification with 76.4% yield and no sodium dodecyl sulfate–polyacrylamide gel electrophoresis detectable native corn trypsin inhibitor. After the HIC step (2.7× step purification with 44.0% yield), the final product had a specific activity that was 75.3% of that of the affinity-purified aprotinin.     

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.     

Studies on the <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> CCT-4518 laccase purified by hydrophobic interaction chromatography

A laccase from Pycnoporus sanguineus was purified by two steps using phenyl-Sepharose columm. A typical procedure provided 54.1-fold purification, with a yield of 8.37%, using syringaldazine as substrate. The molecular weight of the purified laccase was 69 and 68 kDa as estimated by 12% (w/v) SDS-PAGE gel and by gel filtration, respectively. The K _m values for the substrates ABTS, syringaldazine, and guaiacol were 58, 8.3, and 370 μM, respectively. The enzyme’s pH optimum for syringaldazine was 4.2 and optimal activity was 50°C. The enzyme showed to be thermostable because when kept at 50°C for 24 and 48 h it retained 93 and 76% activity. This laccase was inhibited by l-cysteine, β-mercaptoethanol, NaN₃, NaF, and HgCl₂.     

Scale-up of hydrophobic interaction chromatography for the purification of a DNA vaccine against rabies

The purification of a DNA vaccine against rabies by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatised with 1,4-butanediol diglycidyl ether was scaled up 60 times. The purification profile was not affected by increased loadings (up to 15 mg DNA) and a product with a consistent quality was obtained. Fourteen mg of plasmid with an HPLC purity of 100% were obtained in one run, corresponding to a 95% yield.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

疏水层析用于大规模纯化重组HBsAg的工艺研究

应用疏水层析法从CHO细胞培养液中纯化HBsAg,每根制备柱每次可处理细胞收液350L,在适宜的上样流速和层析温度条件下,层析后可去除96％的杂蛋白,再经超速离心和凝胶过滤层析,可获HBsAg纯品。经检定,HPLC纯度高于95％,其余各项检定指标均符合《中国生物制品规程》要求。结果表明,此方法纯化效率高、处理样品量大、成本低,适于大规模生产。     

Glutaraldehyde cross-linked chitosan microspheres for controlled release of centchroman

Glutaraldehyde cross-linked chitosan microspheres were prepared for controlled release of centchroman, a nonsteroidal contraceptive. The cross-linked microspheres with low-molecular-weight (LMW) chitosan (260 kg mol(-1)) have shown maximum degree of swelling (287 wt%) but were found to be poor in loading and release behavior for centchroman. The microspheres with medium-molecular-weight (MMW) chitosan (1134 kg mol(-1)) have shown 250 wt% degree of swelling and 37.5 wt% loading of centchroman, but microspheres with high-molecular-weight (HMW) chitosan (2224 kg mol(-1)) have shown a low degree of swelling (150 wt%) and centchroman loading (30 wt%). The microspheres with MMW chitosan have released 82 wt% of loaded centchroman in a controlled release manner within a period of 70 h in comparison to low- (260 kg mol(-1)) and high-MW (2224 kg mol(-1)) chitosan microspheres. The chitosan microspheres with 62 wt% degree of deacetylation (DDA) were more efficient in the controlled release of centchroman in comparison to chitosan microspheres with low (48 wt%) and high-DDA (75 wt%). The fractional release of centchroman (M(t)/M(infinity)) from chitosan microspheres was used to predict the mechanism of drug release and to determine the diffusion constant (D) of centchroman.     

Characterization of denatured proteins by hydrophobic interaction chromatography: a preliminary study

In this preliminary study hydrophobic interaction chromatography (HIC) is proposed as a good tool in order to detect conformational changes induced by chemical denaturants in two globular proteins, cytochrome C (Cyt C) and myoglobin (MYO). Alterations in protein structure were manifested chromatographically by reproducible changes in peak heights, retention time, and appearance of multiple peaks. The HIC behavior of the two model proteins denatured by guanidinium thyocyanate (GdmSCN) was investigated, keeping constant various concentrations of urea in the mobile phase in a TSK-Gel Phenyl-5PW column (TosoBiosep). Suitable elution conditions provide evidence of the simultaneous presence of two denatured forms in the case of MYO, and sequential different denatured states of Cyt C.     

Purification of cell culture‐derived modified vaccinia ankara virus by pseudo‐affinity membrane adsorbers and hydrophobic interaction chromatography

A purification scheme for cell culture‐derived smallpox vaccines based on an orthogonal downstream process of pseudo‐affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo‐affinity chromatography, based on reinforced sulfated cellulose and heparin‐MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo‐affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated.The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312–320. © 2010 Wiley Periodicals, Inc.     

Mechanistic modeling based process analytical technology implementation for pooling in hydrophobic interaction chromatography

A major challenge in chromatography purification of therapeutic proteins is batch-to-batch variability with respect to impurity levels and product concentration in the feed. Mechanistic model can enable process analytical technology (PAT) implementation by predicting impact of such variations and thereby improving the robustness of the resulting process and controls. This article presents one such application of mechanistic model of hydrophobic interaction chromatography (HIC) as a PAT tool for making robust pooling decisions to enable clearance of aggregates for a monoclonal antibody (mAb) therapeutic. Model predictions were performed before the actual chromatography experiments to facilitate feedforward control. The approach has been successfully demonstrated for four different feeds with varying aggregate levels (3.84%–5.54%) and feed concentration (0.6 mg/mL–1 mg/mL). The resulting pool consistently yielded a product with 1.32 ± 0.03% aggregate vs. a target of 1.5%. A comparison of the traditional approach involving column fractionation with the proposed approach indicates that the proposed approach results in achievement of satisfactory product purity (98.68 ± 0.03% for mechanistic model based PAT controlled pooling vs. 98.64 ± 0.16% for offline column fractionation based pooling). © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2758, 2019.     

Environment friendly modified chitosan hydrogels as a matrix for adsorption of metal ions, synthesis and characterization

Environment friendly modified chitosan hydrogels for metal ions absorption from aqueous systems were designed using simple technology. Chitosan was modified with [N,N′-bi-α-azidosuccinimide and N-phthalimido-α-azide succinimide] under different reaction conditions to prepare new hydrogels with high metal ion absorption efficiency. The hydrogels were characterized by FTIR, thermal stability, crystallography, solubility and swelling capacity. Promising results were obtained from this preliminary study to evaluate the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems.     

Influence of mixed electrolytes on the adsorption of lysozyme,PEG, and PEGylated lysozyme on a hydrophobic interaction chromatography resin

In a recent work (Werner A and Hasse H in J Chromatogr A 2013;1315:135) the influence of mixed electrolytes on the adsorption of the macromolecules lysozyme, PEG and di‐PEGylated lysozyme on a hydrophobic resin has been studied, but only at one overall ionic strength (3000 mM). The present work, therefore, extends these studies to other ionic strengths (2400 and 2700 mM), and explores the application of a model to predict the entire data set. The adsorbent is Toyopearl PPG‐600M. The solvent is a 25 mM aqueous sodium phosphate buffer at pH 7.0. The studied salts are sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. Pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied at 25 °C. The loading of the adsorbent increases with increasing salt concentration for all macromolecules. Synergetic effects of the mixed electrolytes are observed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1104–1115, 2017     

Fungal mycelium--the source of chitosan for chromatography

Mycelium of the mold Aspergillus niger was used as a raw material for the preparation of microbial chitosan. Aspergillus niger, the mold used for the production of citric acid, contains approx. 15% of chitin, which can be separated, transformed into chitosan, and used as a sorbent for chromatography. The main advantage of this material in comparison with krill chitosan is the uniformity of particle size leading to the low back-pressure in the column. The other advantage is the fact, that original fibrous structure of mycelial pellets could be stabilized before chitosan preparation by cross-linking with glutaraldehyde. The product prepared by this way -- crosslinked chitosan of uniform particle size, is highly porous, with high water regain and, as a result, low sedimentation velocity. Low sedimentation velocity is not disadvantage in chromatographic application, but may form some problems in batchwise operation. Chitosan as a polymer of glucosamine is anion exchanger in nature and the chromatographic properties of this anion exchanger was demonstrated by the chromatography of bovine blood plasma, glucose oxidase, and chicken pepsinogen. In all cases, the course of chromatography on crosslinked chitosan was compared with the chromatography on MONO Q (bovine blood plasma) or DEAE-cellulose (glucose oxidase, chicken pepsinogen) under the same protocol.     

An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.     

An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.