Integrated isolation of antibody fragments from microbial cell culture fluids using supermacroporous cryogels

The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10-100 microm) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+-iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)6-tagged single chain (sc) Fv antibody fragments, (His)6-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+-IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84-96% recovery of (His)6-scFv fragments with a purification factor of 13-15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques.     

Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent

A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N'-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10-100 microm size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 degrees C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.     

A novel adsorbent for protein chromatography: supermacroporous monolithic cryogel embedded with Cu2+-attached sporopollenin particles

The aim of this study is to prepare supermacroporous cryogels embedded with Cu(2+)-attached sporopollenin particles (Cu(2+)-ASP) having large surface area for high protein adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Cu(2+)-ASP was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for affinity purification of human serum albumin (HSA). Firstly, Cu(2+) ions were attached to sporopollenin particles (SP), then the supermacroporous PHEMA cryogel with embedded Cu(2+)-ASP was produced by free radical polymerization using N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Embedded particles (10 mg) in PHEMA-based cryogel column were used in the adsorption/desorption of HSA from aqueous solutions. Optimum conditions of adsorption experiments were performed at pH 8.0 phosphate buffer, with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of HSA adsorption from aqueous solution was very high (677.4 mg/g SP) with initial concentration 6 mg/mL. It was observed that HSA could be repeatedly adsorbed and desorbed to the embedded Cu(2+)-ASP in PHEMA cryogel without significant loss of adsorption capacity.     

Affinity Chromatography of Mushroom Tyrosinase

Abstract

The purification by column chromatography of a phenol-oxidizing enzyme, mushroom tyrosinase, was investigated using solid phase adsorbents designed to have specific affinity for the enzyme. Sepharose 4B, aminophenyl-bearing porous glass, and p-aminobenzylcellulose were chemically modified to introduce phenolic, catecholic, or benzoic groups on the polymer surface. The resulting preparations were tested for their effectiveness in separating tyrosinase from an impure protein mixture. The phenolic and benzoic polymers displayed no specific affinity for tyrosinase. Aminophenyl glass, with or without an attached phenolic group, adsorbed appreciable quantities of protein nonspecif-ically, thus complicating studies of its tyrosinase affinity properties. Dopamine, a dihydroxyphenyl derivative, was bound to Sepharose and was found to be effective in retaining tyrosinase at pH 5.5; elution of the enzyme by washing at pH 8.8 resulted in its purification by a factor of 10 to 14. Enzymatic oxidation of the adsorbent limited the number of purification cycles which could be carried out on a single column.     

分离尿激酶的亲和色谱填料的制备

 合成了分别以 Sepharose和聚甲基丙烯酸环氧丙酯为基质、对氨基苯甲脒为配基的分离尿激酶的两种亲和色谱填料 ,并用于尿激酶粗品的直接纯化 ,活性回收率分别为 1 0 8.3 %和 43 .4% ,比活提高倍数分别为 9.0 6倍和 3 6.9倍。     

Synthesis and characterization of supermacroporous cryogel with immobilized p-aminobenzenesulfonamide as affinity ligand for the purification of lactoperoxidase from whey

This study proposed the development of a monolithic supermacroporous affinity column for direct capture of lactoperoxidase, a glycoprotein present in milk, whey, and colostrum, with several applications due to its wide antimicrobial activity. A poly(acrylamide)-based cryogel was produced by radical co-polymerization of monomers in frozen aqueous solution and activated with p-aminobenzenesulfonamide as a ligand for specific interaction with the lactoperoxidase. The axial liquid dispersion coefficients at different liquid flow rates were determined by measuring residence time distributions using the tracer pulse-response method. The axial dispersion coefficient was low and the height equivalent to theoretical plate was not dependent on the flow velocity. The adsorptive capacity of affinity cryogel was studied as a function of flow velocity and the best condition was 0.9 cm/min. The response surface methodology was applied to optimize the capture of the enzyme, as a function of pH and salt concentration. Higher purification factor value was found at a salt concentration of 80 mmol/L and pH of 8.0 (p < 0.05). There was no influence of the variables under study on the yield (p > 0.05). The results indicated that affinity cryogel is a promising chromatography support for the use in high-throughput one-step purification of lactoperoxidase from whey.     

Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells

Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm x 7.1 mm ?) with immobilized concanavalin A was used. Chromatographic columns (113 mm x 7.1 mm ?) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl alpha-D-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.     

Preparation,characterization, and binding profile of imprinted semi-IPN cryogel composite for aluminum

Human body is greatly exposed to aluminum due to its high abundance in the environment. This nonessential metal is a threat to the patients of chronic renal disorders, as it is easily retained in their plasma and quickly accumulates in different tissues. Thus, there is great need to remove it from the aqueous environment. In this study, Al³⁺ imprinted semiinterpenetrating polymer network (semi-IPN)-based cryogel composite was prepared and applied for the purification of environmental and drinking water samples from aluminum. Poly (2-hydroxyethyl methacrylate) (pHEMA) discs were produced via cryogenic treatment and imprinted semi-IPN was introduced to the 3-(trimethoxysilyl) propyl acrylatemodified macroporous cryogel discs. The adsorption properties and selectivity of the aluminum (III) imprinted semi-IPN cryogel composite were studied in detail. The imprinted semi-IPN cryogel composite showed good selectivity towards aluminum (III) ions with the imprinting factor (IF) of 76.4 in the presence of competing copper (II), nickle (II), and iron (III) ions. The maximum adsorption capacity of 271 μmol g^-1 was obtained for aluminum (III) at pH 7.0 within 10 min using imprinted semi-IPN cryogel composite. The good selectivity and reusability of aluminum (III)-imprinted semi-IPN cryogel composite makes this material an eligible candidate for the purification of drinking water from aluminum (III) leaving important minerals remained in the water.     

Comparison among three anion exchange chromatographic supports to capture erythropoietin from cell culture supernatant Lourdes

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin (EPO) from mammalian cell culture supernatant. Currently, chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work, three anion exchange chromatographic supports (gel, membrane and monolithic) were evaluated in the capture step of the recombinant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis, as a decision tool for larger scale implementation, was done. As a conclusion, the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity. However, the monolithic column admits high operating velocity, showing the highest adsorption capacity and productivity.     

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonas aeruginosa on immobilized metal chelates

The aim of this work was to devise a one‐step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild‐type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)–IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 ± 0.015 and 3.214 ± 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K_D of 4.53 × 10⁻⁷ m was obtained from batch isotherm measurements. The combination of tailor‐made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one‐step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial‐Zn(II) and EPI‐30–IDA–Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A‐Sepharose CL‐4B. This MAb preparation revealed on SDS–PAGE two protein bands with M_r of 50 and 22 kDa corresponding to the heavy and light chains, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format

Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples.     

Adsorption Study of Immunoglobulin G Subclasses from Different Species by Pseudobioaffinity Separation on Histidyl–Bisoxirane–Sepharose

Adsorption chromatography is increasingly used for protein separations and biomedical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA, and plasma proteins must be purified before characterization and utilization. Use of immunoglobulins as immunodiagnostic and therapeutic tools has initiated many attempts to develop new adsorbents for their separation. Protein A and protein G are the affinity ligands most widely used for separation of immunoglobulins. These proteins are reliable, and have good selectivity and specificity, but are very expensive. Much attention has therefore been devoted to developing alternative methods for separation of immunoglobulins. Pseudobiospecific ligands, for example metal ions and amino acids, can be used for separation of a wide range of biological molecules. In this study, IgG₁, IgG₂, and IgG₃, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent. Adsorption of IgG from different animal species on the same chromatographic adsorbent was also tested. The high recovery and purification on histidyl–bisoxirane–Sepharose gel of IgG from all the sources tested compared well with results obtained by use of protein A–Sepharose gel.     

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.     

固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶

以Ｃｕ~（２＋）－Ｓｅｐｈａｒｏｓｅ４Ｂ固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶,考察了不同的洗脱缓冲溶液及其ｐＨ对纯化效率的影响,显示了方法的有效性。     

Protein digestion and phosphopeptide enrichment on a glass microchip

This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using β-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.     

Purification of Papain Using Reactive Green 5 Attached Supermacroporous Monolithic Cryogel

Supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] monolithic cryogel was prepared by radical cryocopolymerization of HEMA with N,N??-methylene bisacrylamide as crosslinker. Reactive Green 5 dye was immobilized to the cryogel with nucleophilic substitution reaction, and this dye attached cryogel column was used for affinity purification of papain from Carica papaya latex. Reactive Green 5-immobilized poly(HEMA) cryogel was characterized by swelling studies, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray analysis. Maximum papain adsorption capacity was found to be 68.5?mg/g polymer while nonspecific papain adsorption onto plain cryogel was negligible (3.07?mg/g polymer). Papain from C. papaya was purified 42-fold in single step with dye attached cryogel, and purity of papain was shown by silver-stained sodium dodecyl sulfate?Cpolyacrylamide gel electrophoresis.     

Oriented Covalent Immobilization of Engineered ZZ-Cys onto Maleimide-Sepharose: An Affinity Platform for IgG Purification

Protein A affinity chromatography is an important technique that is widely used in purifying polyclonal and monoclonal antibodies. However, improving the IgG loading capacity of protein A affinity materials remains crucial. In this study, a smaller divalent IgG binding molecule derived from the B domain of protein A, i.e., ZZ-domain, was used to develop an affinity adsorbent with high IgG loading capacity by improving the unit area yield of the site-specific immobilization affinity ligand. The engineered ZZ-Cys was tightly immobilized onto Sepharose support via the covalent incorporation of a cysteine handle and a maleimide group, with oriented manner and divalent IgG binding capacity, thereby resulting in homogenous conjugates, namely, Sepharose–ZZ^SA. Approximately 1.19 mg of ZZ-Cys was coupled onto wet Sepharose g⁻¹ and the maximum saturation binding capacity of Sepharose–ZZ^SA g⁻¹ was approximately 23.80 mg of IgG. The smaller engineered ZZ-Cys can be produced at a lower cost than protein A and covalently conjugated onto matrix surface with high density and full IgG binding capacity. Thus, the proposed platform may be of general use for IgG purification in an efficient and economical manner.