Capturing of cell culture‐derived modified Vaccinia Ankara virus by ion exchange and pseudo‐affinity membrane adsorbers

Smallpox is an acute, highly infectious viral disease unique to humans, and responsible for an estimated 300–500 million deaths in the 20th century. Following successful vaccination campaigns through the 19th and 20th centuries, smallpox was declared eradicated by the World Health Organization in 1980. However, the threat of using smallpox as a biological weapon prompted efforts of some governments to produce smallpox vaccines for emergency preparedness. An additional aspect for the interest in smallpox virus is its potential use as a platform technology for vector vaccines. In particular, the latter requires a high safety level for routine applications. IMVAMUNE®, a third generation smallpox vaccine based on the attenuated Modified Vaccinia Ankara (MVA) virus, demonstrates superior safety compared to earlier generations and represents therefore an interesting choice as viral vector. Current downstream production processes of Vaccinia virus and MVA are mainly based on labor‐intensive centrifugation and filtration methods, requiring expensive nuclease treatment in order to achieve sufficient low host‐cell DNA levels for human vaccines. This study compares different ion exchange and pseudo‐affinity membrane adsorbers (MA) to capture chicken embryo fibroblast cell‐derived MVA‐BN® after cell homogenization and clarification. In parallel, the overall performance of classical bead‐based resin chromatography (Cellufine® sulfate and Toyopearl® AF‐Heparin) was investigated. The two tested pseudo‐affinity MA (i.e., sulfated cellulose and heparin) were superior over the applied ion exchange MA in terms of virus yield and contaminant depletion. Furthermore, studies confirmed an expected increase in productivity resulting from the increased volume throughput of MA compared to classical bead‐based column chromatography methods. Overall virus recovery was ～60% for both pseudo‐affinity MA and the Cellufine® sulfate resin. Depletion of total protein ranged between 86% and 102% for all tested matrices. Remaining dsDNA in the product fraction varied between 24% and 7% for the pseudo‐affinity chromatography materials. Cellufine® sulfate and the reinforced sulfated cellulose MA achieved the lowest dsDNA product contamination. Finally, by a combination of pseudo‐affinity with anion exchange MA a further reduction of host‐cell DNA was achieved. Biotechnol. Bioeng. 2010. 105: 761–769. © 2009 Wiley Periodicals, Inc.     

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.     

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography

Abstract

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400?SEC, we obtained highly pure, i.e.,?>?90%, rHBsAg virus-like particles (VLP).     

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.     

Sulfated membrane adsorbers for economic pseudo‐affinity capture of influenza virus particles

Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture‐based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu®) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine® sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture‐derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell‐derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM‐adsorbers were compared directly to column‐based Cellufine® sulfate and commercially available cation‐exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM‐capture step resulted in a higher reduction of dsDNA compared to the tested cation‐exchange membrane adsorbers. The productivity of the SCM‐based unit operation could be significantly improved by a 30‐fold increase in volumetric flow rate during adsorption compared to the bead‐based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell‐derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM‐adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine® sulfate. Biotechnol. Bioeng. 2009;103: 1144–1154. © 2009 Wiley Periodicals, Inc.     

Norovirus‐binding proteins recovered from activated sludge micro‐organisms with an affinity to a noroviral capsid peptide

Aims: Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro‐organisms could be totally different from that of those particles moving freely, micro‐organisms with natural affinity ligands such as virus‐binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus‐binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. Methods and Results: NoVBPs were recovered from activated sludge micro‐organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus‐like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two‐dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N‐terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. Conclusions: NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro‐organisms. Significance and Impact of the Study: NoVBPs could be natural viral ligands and play an important role in the NoV transmission.     

Optimization of cell culture‐derived influenza A virus particles purification using sulfated cellulose membrane adsorbers

Downstream processing remains one of the biggest challenges in manufacturing of biologicals and vaccines. This work focuses on a Design of Experiments approach to understand factors influencing the performance of sulfated cellulose membrane adsorbers for the chromatographic purification of a cell culture‐derived H1N1 influenza virus strain (A/Puerto Rico/8/34). Membranes with a medium ligand density together with low conductivity and a high virus titer in the feed stream resulted in optimum virus yields and low protein and DNA content in the product fraction. Flow rate and salt concentration in the buffer used for elution were of secondary importance while membrane permeability had no significant impact on separation performance. A virus loss of 2.1% in the flow through, a yield of 57.4% together with a contamination level of 5.1 pg_DNA HAU^?1 and 1.2 ng_prot HAU^?1 were experimentally confirmed for the optimal operating point predicted. The critical process parameters identified and their optimal settings should support the optimization of sulfated cellulose membrane adsorbers based purification trains for other influenza virus strains, streamlining cell culture‐derived vaccine manufacturing.     

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.     

Protein instability during HIC: Evidence of unfolding reversibility,and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

A two‐conformation, four‐state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four‐state model to the effect of salt concentration on the adsorption and unfolding of the model protein α‐lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild‐type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of α‐lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild‐type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (～6–8 kJ/mol) due to increased instability of the protein. Finally, the four‐state model is used to accurately predict the apparent adsorption strength of a disulfide bond‐reduced variant. Biotechnol. Bioeng. 2009;102: 1416–1427. © 2008 Wiley Periodicals, Inc.     

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.     

Impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses

The study aims on affinity matrix selection for a cell culture derived influenza virus capture step in downstream processing. Euonymus europaeus lectin (EEL) was used as an affinity ligand. Human influenza A/Puerto Rico/8/34 (H1N1) virus produced in MDCK cells was chosen as a model strain. The chromatographic separation characteristics of reinforced cellulose membranes and different matrices such as agarose, cellulose, polymer and glass particles with immobilized EEL have been determined. Results obtained were compared to affinity matrices, which are currently used in large-scale vaccine manufacturing. Mass balances for the viral membrane protein hemagglutinin showed that EEL affinity chromatography results in higher recoveries than conventional processes using Cellufine sulphate and heparinized agarose. The most efficient media, a polymer and a cellulose membrane, have been further characterized by protein and host cell DNA measurements. Separations based on the polymer matrix and the cellulose membrane removed contaminating DNA to 0.2 and 1%, respectively. Total protein contents were decreased to 50 and 31%, respectively. The EEL-membrane showed the highest influenza virus binding capacity. These characteristics demonstrate that EEL affinity chromatography is a promising candidate for capturing influenza viruses from MDCK cell culture broths in addition to currently applied chromatographic media.     

蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议     

Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid‐envelope virus‐like particles

This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid‐envelope virus‐like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving ～70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of ～80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions (～400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low‐shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two‐fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A rapid and universal tandem-purification strategy for recombinant proteins

A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.     

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single‐chain variable fragment

Single‐chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L‐based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed‐mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel?), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere?). The DoE designs of the polishing step included both binding and flow‐through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere? in binding‐mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere? below its theoretical pI, while the majority of HCPs were in the flow‐through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.     

乳腺生物反应器表达的重组人乳清白蛋白的高效分离纯化

利用乳腺生物反应器高效地表达重组人乳清白蛋白,但是目标产物分离纯化的难度较大。通过分子模拟计算比较待分离原料中主要蛋白组分的物化性质,包括表面电势和表面疏水性,在此基础上设计了高分辨率、快速的分离纯化工艺。通过硫酸铵沉淀的正交试验条件优化,有效地去除了干扰层析精制过程的IgG杂质,提高了后续疏水层析的稳定性,从而成功地分离开目标蛋白及与其同源的牛乳清白蛋白杂质,得到纯度>95%的rHLA纯品,工艺回收率48.6%。乳糖合成活性和圆二色谱检测结果表明,纯化rHLA具有调节β-1,4-半乳糖苷转移酶活性和天然的空间结构。     

Process and economic evaluation for monoclonal antibody purification using a membrane-only process

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high-value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra-high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane-only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane-only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.     

Influence of tryptophan tags on the purification of cutinase, secreted by a recombinant Saccharomyces cerevisiae, using cationic expanded bed adsorption and hydrophobic interaction chromatography

During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.     

Downstream process design for Gag HIV-1 based virus-like particles

Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 10¹⁰ virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.     

Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.