Combining Low Pressure Liquid Affinity Chromatography and Flow Injection Analysis for the Quantitation of Immunoglobulins

Abstract

A simple, accurate method combining low pressure liquid affinity chromatography and flow injection analysis is described for the quantitation of immunoglobulins in biologicals fluids. The affinity matrix consists of Protein A covalently immobilized to a 2-fluoro-1 methylpyridinium salt activated Fractogel support. Utilizing a 1 cm³ affinity column, optimized binding and eluting buffer flow rates of 0.7 and 1.5 mL min^?1, respectively, and a sample loop size of 100 μL, IgG's can be eluted from the affinity column in about 9 min. Linear standard curves (r > 0.99) were obtained at concentrations up to at least 4.0 and 8.0 mg mL^?1 respectively for human and bovine IgG. Recovery yields are good ranging from 96–100%. The within day CV for human and bovine IgG was found to be less than 3.0% whereas the day to day CV was less than 3.4% (n=10). IgG concentrations of spiked and unspiked bovine plasma samples obtained by the low pressure affinity/flow injection method when compared to those obtained by the radial immunodiffusion agreed to within 4%.     

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional 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.

     

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.     

Comparison of protein A affinity sorbents II. Mass transfer properties

Protein A affinity chromatography is the standard purification method for isolation of therapeutic antibodies. Due to improvements in expression technology and optimization of fermentation, culture supernatants with high antibody content must be processed. Recently protein A affinity media with improved adsorption characteristics have been developed. The agarose media MabSelect Xtra and MabSelect SuRe are recent developments of the existing protein A affinity medium MabSelect. MabSelect Xtra is designed to exhibit a higher binding capacity for IgG, and MabSelect SuRe is functionalized with an alkaline stabilized protein A. ProSep-vA Ultra is a porous glass medium with a pore size of 70 nm, also developed to improve the binding capacity. Adsorption was measured in a finite and infinite bath. Mass transfer in these systems could be well described by a model including film and pore diffusion. Mass transfer parameters were used to accurately predict IgG breakthrough in packed bed mode. The dynamic binding capacity of all three media did not change when residence time was at least 4 min. All three media are suited for capture of feed stocks with high antibody content.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

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.     

Quantification of immunoglobulin G and characterization of process related impurities using coupled Protein A and size exclusion high performance liquid chromatography

The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a Protein A sensor cartridge (Protein A HPLC) and (ii) the same Protein A cartridge in combination with a size exclusion HPLC column (PSEC-HPLC). The possibility to simultaneously quantify immunoglobulin G (IgG) besides a non-binding protein such as bovine serum albumin (BSA) increases the applicability of Protein A HPLC. Its most pronounced feature is its independence of the buffer system, pH-value and salt content of the investigated sample solvent, which includes cell media. A comparison with the state-of-the-art, the photometrical Bradford method, shows that Protein A HPLC is as sensitive as Bradford, but that it comes with an extended linear range of 4 orders of magnitude, ranging from 0.15 [μg abs] to 1 [mg abs] absolute injected protein amount. The applicability of the PSEC-HPLC method is demonstrated for the analysis of real cell culture feed samples. While Protein A binds IgG, the SEC-column distributes the feed impurities by their molecular weight. The peak area ratios of IgG and the feed impurities of interest are then plotted against the collected sample fraction. These Protein A-Size-Exclusion-Chromatographic diagrams (PSEC-plot) combine the performance information of feed impurities and IgG in a single plot. Further it is shown that both methods are suitable for the performance evaluation of antibody purification media using static as well as dynamic binding experiments performed on DEAE-Fractogel and Capto Adhere. The investigated test samples were “mock” protein solutions with increasing complexity ranging from simple PBS buffer to serum free cell media and “real” cell culture feed solutions.     

Gellan beads as a transparent media for protein immobilization and affinity capture

Gellan gum beads are presented as a novel substrate for protein immobilization and immobilized protein activity measurements. The optical transparency of the gellan beads down to 200 nm provides a method for direct quantitation of the amount of protein immobilized onto the beads. The ability to utilize these beads in a non-aqueous activation step allowed for a fourfold increase in the amount of protein immobilized, and this method was used to immobilize Protein A onto gellan beads at a final yield of 1.42+/-0.07 mg of Protein A/g of beads. The optical transparency also allowed for detection of the activity of the immobilized Protein A simply by measuring the absorbance of the beads following capture of rabbit IgG. This activity measurement method was compared with a traditional method utilizing the amount of protein remaining in solution after the IgG capture step. The traditional method yielded an activity measurement of 10.9+/-0.2 mg IgG/mg of Protein A, while the absorbance method showed an activity of only 7.5+/-0.3 mg IgG/mg of Protein A. The difference can be explained by the more direct measurement used in the absorbance method. The optical transparency of the beads was also evaluated in a fluorescence based IgG capture experiment, showing that detection of fluorescent IgG captured on the beads was possible with no interference from the beads.     

Enhanced binding by dextran‐grafting to Protein A affinity chromatographic media

Dextran‐grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose‐based matrix, followed by epoxy‐activation and Protein A coupling site‐directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran‐grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction‐dried gel, increased by 24% compared with that of the non‐grafted medium. The binding capacity of dextran‐grafted medium decreased about 7% after 40 cleaning‐in‐place cycles, much lower than that of the non‐grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran‐grafted medium faster than that of non‐grafted one. Atomic force microscopy showed that this dextran‐grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non‐grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high‐performance dextran‐grafted Protein A affinity chromatographic medium has promising applications in large‐scale antibody purification.     

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu²⁺, Ni²⁺ and Zn²⁺ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu²⁺ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu²⁺ and 7–9 mg/ml with Ni²⁺ for human IgG with MMA buffer. In the case of CIM-IDA-Zn²⁺ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.     

Avid AL, a synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G.

Avid AL is an affinity gel designed for the purification of immunoglobulin G (IgG). The gel was prepared by first reacting Sepharose with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine and then with 2-mercaptoethanol. The IgG purified by Avid AL is about 95% pure. The binding parameters of Avid AL for the whole IgG, Fab and Fc fragment and the stability of gel were investigated. The IgG bound to Avid AL can be eluted with an acidic buffer or with a novel neutral buffer containing electron donors. The development of such a mild neutral elution buffer is described. Application of Avid AL in a rapid gram-scale IgG purification was demonstrated. The possible mechanism of IgG binding is discussed.     

Modified general affinity adsorbent for large-scale purification of penicillinases

N-Acetyl-D-(-)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H beta-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of D-(-)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl-D-(-)-penicillamine; (2) incorporation of 15 mumol of N-acetyl-D-(-)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.     

Screening and chromatographic assessing of a novel IgG biomimetic ligand

A novel biomimetic ligand, N-benzyloxycarbonyl-l-tyrosine (N-cbz-l-Tyr), was screened by a combination method of molecular docking and immobilized receptor technique. Then, N-cbz-l-Tyr was immobilized on Sepharose CL-4B to prepare a specific affinity adsorbent for immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on the adsorbent gave an association constant (K(a)) of 4.91 x 10(6) m(-1) and a theoretical maximum adsorption capacity of 17.3 mg IgG/mL gel. IgG with a purity of 98% was separated from human plasma by this new affinity adsorbent.     

Preparation and Characterization of PEGylated Thiophilic Nanoparticles for Rapid Antibody Separation

Magnetic nanoparticles with novel core-shell structure were prepared for immunoglobulin (IgG) separation, in which thiophilic property of sulfone groups and protein resistance of poly(ethylene glycol) (PEG) moieties were integrated. The step-wise surface reactions on the nanoparticles were characterized by ¹H nuclear magnetic resonance (NMR) and surface zeta potential measurements. With human IgG and bovine serum albumin (BSA) as model proteins, the effects of PEG chain length, conjugation group, solution pH and salt concentration on IgG selectivity were investigated using static adsorption experiments. The experiment results showed that mPEG2000-NH₂ modified magnetic nanoparticles had an adsorption capacity of 132.8 mg g^?1 and selectivity of 32.5 towards IgG under the condition of pH 7.45 and 0.15 M NaCl. In complex biological fluids, the PEG modified magnetic nanoparticles could separate IgG from fetal calf serum and Omalizumab from cell culture supernatant with purities of 96% and 99%, respectively. Moreover, the binding affinities of the proposed core-shell structure towards IgG from four animal species (human, bovine, rabbit and goat) were quantified by bio-layer interferometer (BLI). The results showed that the selectivity of this structure towards IgG varied from traditional Protein A method, suggesting its potentials in rapid separation and purification of IgG with low affinity towards Protein A.     

蛋白A高效亲和膜色谱法测定人血浆中免疫球蛋白G的含量

 研究了蛋白Ａ高效亲和膜色谱对水溶液及人血浆中人免疫球蛋白Ｇ（ＨＩｇＧ）的特异性吸附和定量测定。方法具有较高的精确度和较好的重复性：ＨＩｇＧ标样５次重复进样的相对标准偏差为１．５％，人血浆样品３次重复进样的相对标准偏差为３．６％；所测得的定量标定曲线的线性相关系数达到０．９９９３；不含己二胺间隔臂的亲和介质的非特异性吸附极低，基本检测不出来。快速实验中，一次分析可在０．５ｍｉｎ内完成。实验表明，利用所建立的方法对人血浆中的ＨＩｇＧ进行定量测定可以得到较为满意的结果。     

Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

Protein A tangential flow affinity membrane cartridge for extracorporeal immunoadsorption therapy.

Tangential flow affinity membrane cartridge (TFAMC) is a new model of immunoadsorption therapy for hemoperfusion. Recombinant Protein A was immobilized on the membrane cartridge through Schiff base formation for extracorporeal IgG and immune complex removal from blood. Flow characteristics, immunoadsorption capacity and biocompatibility of protein A TFAMC were studied. The results showed that the pressure drop increased with the increasing flow rate of water, plasma and blood, demonstrating reliable strength of membrane at high flow rate. The adsorption capacities of protein A TFAMC for IgG from human plasma and blood were measured. The cartridge with 139 mg protein A immobilized on the matrix (6 mg protein A/g dry matrix) adsorbed 553 mg IgG (23.8 mg IgG/g dry matrix) from human plasma and 499.4 mg IgG (21.5 mg IgG/g dry matrix) from human blood, respectively. The circulation time had a major influence on IgG adsorption capacity, but the flow rate had little influence. Experiments in vitro and in vivo confirmed that protein A TFAMC mainly adsorbed IgG and little of other plasma proteins, and that blood cell damage was negligible. The extracorporeal circulation system is safe and reliable.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Structural evaluation of an alternative Protein A biomimetic ligand for antibody purification

Affinity chromatography is one of the most common techniques employed at the industrial-scale for antibody purification. In particular, the purification of human immunoglobulin G (hIgG) has gained relevance with the immobilization of its natural binding counterpart—Staphylococcus aureus Protein A (SpA) or with the recent development of biomimetic affinity ligands, namely triazine-based ligands. These ligands have been developed in order to overcome economic and leaching issues associated to SpA. The most recent triazine-based ligand—TPN-BM, came up as an analogue of 2-(3-amino-phenol)-6-(4-amino-1-naphthol)-4-chloro-sym-triazine ligand also known as ligand 22/8 with improved physico-chemical properties and a greener synthetic route. This work intends to evaluate the potential of TPN-BM as an alternative affinity ligand towards antibody recognition and binding, namely IgG, at an atomic level, since it has already been tested, after immobilization onto chitosan-based monoliths and demonstrated interesting affinity behaviour for this purpose. Herein, combining automated molecular docking and molecular dynamics simulations it was predicted that TPN-BM has high propensity to bind IgG through the same binding site found in the crystallographic structure of SpA_IgG complex, as well as theoretically predicted for ligand 22/8_IgG complex. Furthermore, it was found that TPN-BM established preferential interactions with aromatic residues at the Fab domain (Trp 50, Tyr 53, Tyr 98 and Trp 100), while in the Fc domain the main interactions are based on hydrogen bonds with pH sensitive residues at operational regime for binding and elution like histidines (His 460, His 464, His 466). Moreover, the pH dependence of TPN-BM_IgG complex formation was more evident for the Fc domain, where at pH 3 the protonation state and consequently the charge alteration of histidine residues located at the IgG binding site induced ligand detachment which explains the optimal elution condition at this pH observed experimentally.