Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase

The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu²⁺ was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH₄Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH₄Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH₄Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97–100%).     

Suitability of immobilized metal affinity chromatography for protein purification from canola

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.     

Protein A immobilized affinity cartridge for immunoglobulin purification

Recombinant Protein A was immobilized on a cellulose and acrylic composite matrix through Schiff base formation. Various factors that could affect the binding of immunoglobulin by the Protein A molecules immobilized on the solid matrix were studied to achieve optimum affinity purification. The spacer arm length and ligand concentration of Protein A were verified as factors crucial to optimized IgG purification. Liquid-phase environmental conditions such as pH and salt concentration also play important roles in adsorption capacity by affecting the molecular interaction between IgG and the immobilized Protein A. The rate of interaction between Protein A and IgG is rather fast, with minimal differences observed at 10-fold increases in the cartridge loading rate. This paper describes a cellulose/acrylic composite matrix for immobilizing Protein A, at an optimized ligand concentration, installed on a spacer arm of adequate length, to purify immunoglobulins from animal plasma. The fast-flow property of the cartridge made from such a matrix and its simplicity in operation provide effective means for purifying immunoglobulins on a relatively large scale.     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Adsorption characteristics of an immobilized metal affinity membrane.

An immobilized metal affinity (IMA) hollow-fiber membrane was prepared by radiation-induced graft polymerization of glycidyl methacrylate (GMA) onto a porous polyethylene hollow fiber, followed by chemical conversion of the produced epoxide group into an iminodiacetate (IDA) group and its chelation with copper(II) ion. The IDA hollow fiber, whose degree of GMA grafting was 120%, was found to retain 0.42 mol of Cu ion/kg of dry weight of the resulting IMA hollow fiber. The pure water flux of the affinity membrane was 0.90 m/h at a filtration pressure of 1 x 10(5) Pa. The 0.1 g/L L-histidyl-L-leucine (His-Leu) solution permeated across the IMA hollow fiber, whose inner diameter and thickness were 0.78 and 0.365 mm, respectively, at a prescribed filtration pressure ranging from 0.2 x 10(5) to 1.0 x 10(5) Pa. The adsorption of His-Leu during permeation of the solution showed that the overall adsorption rate was independent of the filtration pressure, i.e., the residence time, because of the negligible diffusional resistance of His-Leu to the pseudobioaffinity ligand located on the pore surface of the membrane. No deterioration in the adsorption capacity was observed after five cycles of His-Leu adsorption, its elution, and reimmobilization of copper. The adsorption isotherm of bovine serum albumin (BSA) on the IMA hollow fiber was measured and compared with that for the conventional agarose-based bead containing the IDA-Cu ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

蛋白A亲和层析法纯化单克隆抗体工艺的优化

治疗性单克隆抗体药物已成为生物医药领域市场最主要的产品类别。蛋白A亲和层析作为第一步捕获抗体蛋白最为有效的手段仍然在现有单克隆抗体纯化平台中占据主导地位。在本研究中,首先开发了一种基于低p H处理抗体细胞回收液的新型细胞液回收技术,该技术能有效去除宿主相关污染物(非组蛋白宿主杂质蛋白、组蛋白、DNA、蛋白聚合物等),同时保证较高的抗体回收率。通过该技术有效预处理后,蛋白A纯化效率可提高10倍左右,并且有效避免了抗体洗脱液中和后浊度的上升,大大减轻了后续蛋白纯化的压力。同时我们也对酸性处理中各种宿主杂质去除机制进行了研究。然后,预处理的洗脱液再经一步Capto adhere色谱纯化,非组蛋白宿主杂质蛋白降低至5 ppm、DNA小于1 ppb、组蛋白降低至检测限以下、蛋白聚合物小于0.01%。总过程抗体蛋白收率87%。该两步法抗体纯化技术可有效集成至当前主流抗体纯化平台,具有良好的大规模应用价值。     

Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications

Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions.     

Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 microg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M _r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.     

Evaluation of new affinity chromatography resins for polyclonal,oligoclonal and monoclonal antibody pharmaceuticals

The performance of MabSelect SuRe and IgSelect affinity chromatography resins designed for process-scale purification of antibodies was investigated. Various antibodies (4 human monoclonal, 1 human polyclonal and 1 bovine polyclonal antibody and 1 Fc-fusion protein) were used to evaluate the elution pH and dynamic binding capacity of the resins. The elution pH for each human antibody was similar on MabSelect SuRe and IgSelect (pH 3.5–3.8). No significant differences in dynamic binding capacity were observed among human antibodies on MabSelect SuRe (∼20–40 mg/mL resin) and IgSelect (∼10–30 mg/mL resin). The binding capacity order for the human antibodies was the same on MabSelect SuRe and IgSelect. Using a linear pH gradient, both resins were able to partially separate monomeric and aggregated forms of the antibodies. The results indicate that these new affinity resins are powerful tools for the purification of human polyclonal antibodies from transgenic animals and oligoclonal antibodies from CHO cell cultures.     

Nucleic acid separations utilizing immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is widely used for protein purification, e.g., in the isolation of proteins bearing the well-known hexahistidine affinity tag. We report that IMAC matrixes can also adsorb single-stranded nucleic acids through metal ion interactions with aromatic base nitrogens and propose that metal affinity technologies may find widespread application in nucleic acid technology. Oligonucleotide duplexes, plasmid, and genomic DNA show low IMAC binding affinity, while RNA and single-stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose. The affinity of yeast RNA for IDA-chelated metal ions decreases in the following order: Cu(II), Ni(II), Zn(II), and Co(II). Adsorption isotherms for 20-mer oligonucleotide homopolymers show that purines are strongly favored over pyrimidines and that double-stranded duplexes are not bound. IMAC columns have been used to purify plasmid DNA from E. coli alkaline lysates, to purify a ribozyme, to remove primers and imperfect products from PCR reactions, and to separate 20-mer oligonucleotide duplexes containing centered single-base mismatches. Potential further applications include SNP scoring, hybridization assays, and the isolation of polyadenylated messenger RNA.     

Purification of urokinase by monoclonal antibody affinity chromatography

Matrix-bound monoclonal antibodies against urokinase have been used to purify this enzyme by affinity chromatography. In a single-step procedure, urokinase can be isolated from crude preparations with high yield and high purity, and without loss of enzymatic activity.     

Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns

Human immunoglobulin G is known to contain 16 different biantennary complex-type asparagine-linked sugar chains, each of which occurs in a nonsialylated, monosialylated, or disialylated form. These oligosaccharides can be separated into 14 fractions by sequential affinity chromatography with Aleuria aurantia lectin (AAL)-Sepharose, RCA120-WG003, and E4-phytohemagglutinin-agarose columns. Twelve of them were found to contain a single oligosaccharide, while the fraction which passed through all three columns was shown to contain two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. The fraction, which bound to the AAL-Sepharose column and passed through the remaining two lectin columns, also contained two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6)GlcNAcOT. These results indicated that serial affinity chromatography with the three lectin columns can be used effectively to detect changes in the sugar chains of IgG resulting from diseases such as rheumatoid arthritis.