Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography

Chromatographic resins of a family of linear Fc-binding hexamer peptides (HWRGWV, HYFKFD, and HFRRHL) exhibited the ability to selectively adsorb and isolate human IgG (hIgG) from complete mammalian cell culture medium (cMEM). Among them, the HWRGWV resin with a peptide density of 0.08 mequiv./g of resin was able to purify hIgG from cMEM with both purity and yield as high as 95%, comparable to Protein A and A2P agarose gels. The influences of N-terminal acetylation of the HWRGWV resin, ligand density on the resin, initial hIgG concentration, and temperature on IgG isolation were also investigated. The results indicate that these small peptide ligands, especially HWRGWV, offer a potential alternative to the use of Protein A or Protein G for large scale affinity chromatography.     

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.     

Oriented immobilized anti‐hIgG via Fc fragment‐imprinted PHEMA cryogel for IgG purification

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F_c fragment‐imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti‐hIgG for IgG purification from human plasma. Non‐imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti‐hIgG to compare the adsorption capacities of oriented (MIP/anti‐hIgG) and random (NIP/anti‐hIgG) cryogel columns. The amount of immobilized anti‐hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti‐hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti‐hIgG column (29.7 mg/g) for the MIP/anti‐hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti‐hIgG cryogel column. Adsorbed IgG was eluted using 1.0 m NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti‐hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Effect of the counter-anion type and concentration on the liquid chromatography retention of beta-blockers

Zirconia beads (25-38 microm in diameter) were modified with N,N,N',N'-ethylenediaminetetramethylenephosphonic acid to generate a zirconia based pseudoaffinity support, further referred to as r_PEZ. The influence of pH, salt concentration and temperature on the binding of human immunoglobulin G (hIgG) to r_PEZ was studied. Temperature had no significant impact on the maximum binding capacity (Qmax), and the equilibrium-binding constant (Kd), whereas pH and the salt concentration had a noticeable impact on both Qmax and Kd. The Qmax value of 55 mg hIgG/ml of bead was obtained at a pH of 5.5 and found to decrease with an increase of pH. The modified zirconia support allowed the separation of immunoglobulins (IgG, IgA and IgM) from untreated human serum. Elution was possible under mild conditions with a step salt gradient. Overall protein recoveries in the range of 109-125% were obtained with human serum. Human IgG, human IgA, and human IgM yields of 29.50+/-6.3, 3.22+/-0.7, and 6.84+/-0.7%, respectively, were obtained at a linear velocity of 4.32 cm/min. Purity of products, obtained from a single chromatographic step was estimated to be greater than 89.0+/-2.6%. The utility of r_PEZ in the selective removal of immunoglobulins, as in immunoadsorption was discussed.     

Accelerated purification process development of monoclonal antibodies for shortening time to clinic. Design and case study of chromatography processes

For accelerating the purification process development of human monoclonal antibodies (hmAbs) for pharmaceutical drugs, we designed a standardized method for setting the conditions of the purification process, which could be applied to hmAbs for the early phase of pharmaceutical development. The process includes three sequential chromatography steps: Protein A affinity chromatography (AFC), anion-exchange chromatography (AIEC) and cation-exchange chromatography (CIEC), and also includes a low pH virus inactivation step after the AFC step. We predicted the elution pH in the AFC and elution salt concentration in the CIEC from the amino acid sequences of hmAbs, as described in our previous paper. The mobile phase pH in AIEC and the pH for virus inactivation were also predicted based on the amino acid sequence of hmAb. As a case study, six hmAbs (two of IgG(1), two of IgG(2) and two of IgG(4)) were purified with the standardized method. The recovery, purity and clearance of impurities (DNA, host cell proteins (HCP), and Protein A) were examined. All the six hmAbs were purified with high recovery and high clearance of the impurities. Factors affecting the impurities level in the purified products are also discussed.     

Evaluation of Amino Acid O-Phosphoserine as Ligand for the Capture of Immunoglubulin G from Human Serum

The amino acid ortho-phosphoserine (OPS) immobilized on agarose gel was evaluated as a ligand for adsorption of polyclonal human immunoglobulin G (IgG) from human serum in the presence of low ionic strength buffers. Screening of buffer systems showed sodium phosphate as the buffer that exhibited higher IgG purity values. Through breakthrough curve analysis for agarose-OPS (feeding of 31.93?mg of total protein per mL of gel), a purification factor of 5.4 with an IgG purity of 89?% was obtained (based on IgG, IgM, IgA, HSA, and Trf nephelometric analysis). IgG adsorption equilibrium studies showed that these data followed the Langmuir-Freundlich model, with cooperativity parameter (n) equal to 1.74, indicating the presence of positive cooperativity, probably due to multipoint interactions. The maximum IgG binding capacity was 24.2?mg?mL^?1, near the value for the bioaffinity ligand protein A. The agarose-OPS adsorbent provides an attractive alternative for capturing of IgG from human serum.     

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)‐based anion‐exchange cryogel

Bovine milk whey contains several bioactive proteins such as α‐lactalbumin, β‐lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2‐hydroxyethyl methacrylate)‐based anion‐exchange cryogel. The monolithic cryogel was prepared by grafting 2‐(dimethylamino) ethyl methacrylate onto the poly(2‐hydroxyethyl methacrylate)‐based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high‐purity IgG from bovine milk whey.     

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

A one-step purification process using flow-through mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min^?1. Ft-IEC using Tris?CHCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris?CHCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.     

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.     

Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.     

Comparison of affinity membranes for the purification of immunoglobulins

The purification of immunoglobulins was studied by comparing 10 different affinity membranes, prepared by coupling various affinity ligands to different microfiltration membranes. Membranes carrying the synthetic peptide TG19318, histidine, the thiophilic ligand and iminodiacetic acid complexed with Zn(II) showed a weak affinity for human IgG, as expressed by apparent association constants (K_A) in the order of 10⁵ M⁻¹. Human IgM and rat IgG bound with high affinity to TG19318 membranes, thus, demonstrating the potential of this sorbent for the purification of immunoglobulins other than human IgG. When carrying Protein-A ligands, membranes based on Nylon 66 coated with low-molar-mass dextran or poly(vinylalcohol), as well as commercial pre-activated polysulfone (Ultrabind^®) and regenerated cellulose (Sartobind^®) membranes, showed high affinity for human IgG (K_A≈10⁶ M⁻¹). In contrast, a nylon membrane coated with high-molar-mass dextran yielded only K_A≈10⁵ M⁻¹, which was attributed to a low accessibility of the immobilized ligand. Besides the high association constants, Protein-A adsorbers based on polysulfone and regenerated cellulose membranes showed several other advantages, such as enhanced charge-to-charge consistency, simpler preparation procedure, membrane sterilisability, good selectivity for IgG purification from cell culture supernatant and good stability throughout repeated adsorption–elution cycles.     

AbSep--an amino acid based pseudobioaffinity adsorbent for the purification of immunoglobulin G

The present work deals with the development and characterization of a tryptophan based pseudobioaffinity adsorbent for the purification of monoclonal and polyclonal antibodies. Tryptophan as a ligand was selected based on molecular docking and experimental screening studies of the amino acids involved in IgG-Protein A interaction. The ligand was coupled to a polymethacrylate based rigid, porous SEPABEADS beaded matrix to obtain the desired affinity adsorbent, which was named AbSep. Characterization studies with regards to the effect of matrix properties (pore size, particle size, nature of matrix, spacer arm) and the medium properties (pH, conductivity, additives) were performed to elucidate the nature of IgG-AbSep interactions and to determine the optimal conditions for obtaining high binding and purity of IgG. The equilibrium binding capacity of AbSep and dissociation constant was found to be 78 mg/ml and 5.31×10(-6)M respectively. AbSep was able to successfully purify polyclonal human IgG from plasma and monoclonal antibody (chimeric IgG1) from CHO cell culture supernatant. Both binding and elution steps were performed at near neutral pH resulting in a purity and recovery of more than 90% and 85% respectively. Additionally, AbSep was shown to be stable to 0.5M NaOH solutions, the preferred agent for cleaning and sanitization of chromatographic media.     

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.     

Isolation of human serum immunoglobulins with a new salt-promoted adsorbent

In this work a highly acetylated-ethylenediamine-Novarose (HA-EDA-Novarose) gel was synthesized and used as a new agarose-based salt-promoted adsorption chromatography (SPAC) matrix to effectively isolate serum immunoglobulins without the need of denaturing conditions. Samples of human serum in 0.5 M Na2SO4, 10 mM 3-(N-morpholino)-propane-sulfonic acid (MOPS), pH 7.6 were applied to a chromatographic column packed with the SPAC gel. Immunoglobulins (Igs) with affinity for the HA-EDA ligands were specifically adsorbed to the matrix, non-bound serum proteins were readily removed by washing the column with the same feed solution buffer. Bound Igs were effectively and very gently eluted by simply removing the salt from the feed solution buffer. The elution buffer consisted thus of only 10 mM MOPS, at pH 7.6 and no salt. The salt-dependent adsorption capacity of this system was estimated to be 7.3 mg/ml with protein recovery of about 93%. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis analysis, radial immunodiffusion and enzyme-linked immunosorbent assays showed that immunoglobulins G, M and A (IgG, IgM and IgA) were the main components present in the elution fraction. The new SPAC adsorbent was used to purify Igs from human serum and IgG and IgA from non-pure commercially available Igs preparations in a very gentle single step.     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared. They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid. The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells. For the latter enzyme, a 11,000-fold purification was achieved in one step.     

亲和色谱-氢化物发生原子荧光分光光度法纯化检测人血浆中的硒蛋白-P

开发了两步亲和色谱法:肝素-琼脂糖凝胶、Ni-琼脂糖凝胶色谱纯化人血浆中硒蛋白-P的方法,并采用氢化物发生-原子荧光分光光度法(HG-AFS)检测,成功搭建了硒蛋白-P的纯化检测平台。确定了亲和色谱纯化的最佳梯度洗脱条件,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)定性检测,得到了一定纯度的硒蛋白-P,其回收率达43.2%。HG-AFS方法的线性相关系数为0.999 1,检出限为0.09μg/L,日内精密度(RSD)为0.12%,日间精密度(RSD)为0.27%,加标回收率为95%～104%。该亲和色谱纯化方法简单易控、回收率高,HG-AFS检测灵敏度高,结果准确可靠。     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

嗜硫类磁性聚合物微球高效分离抗体

用微悬浮聚合技术制备微米级顺磁性的聚合物微球,并通过水解反应在其表面生成丰富的羟基,进而通过二乙烯基砜活化,在其表面修饰2-巯基嘧啶,实现磁性微球对人体免疫球蛋白G(IgG)的特异性识别.进一步探讨了不同解吸附环境,如pH值、温度、离子种类、离子强度等条件,对这种特异性吸附行为的影响.采用此磁性分离技术后,分离抗体的纯度超过92%,抗体活性高于99%.通过连续分离工艺,IgG的分离效率达到86.8%.