Purification of humanized monoclonal antibody by hydrophobic interaction membrane chromatography

Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained.     

Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies.

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.     

Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein.

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.     

Study of the separation of mouse monoclonal antibodies by pseudobioaffinity chromatography using matrix-linked histidine and histamine.

The selective retention of proteins on matrix-linked histidine has been shown to depend on chromatographic conditions: pH, temperature and ionic strength. An extension of this study to separate mouse monoclonal antibodies on histidyl-Sepharose is presented here; the roles of different functional groups such as imidazole, primary amine and carboxyl groups are elucidated by using histamine-Sepharose and histidine linked via the carboxyl group of the alpha-amino acid. We separated two monoclonal antibodies, immunoglobulin G1 (IgG1) from a culture supernatant and IgG2b from ascites fluid precipitated with 50% ammonium sulphate. The pseudoselective retention of monoclonal IgG1 on the three different matrices and IgG2b on histidyl-aminohexyl-Sepharose was achieved at pH 7.4. The purity of the final monoclonal antibody preparation determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions proved the separation of the monoclonal antibodies (IgG1, IgG2b) from other contaminating proteins such as albumin and transferrin. Quantitation of the mouse monoclonal antibodies was carried out using enzyme-linked immunosorbent assay.     

Protein Purification on a New Preparative Ion Exchanger

Abstract

The ACCELL chromatographic media is a new packing designed specifically for the isolation and purification of proteins. The anionic and cationic functional groups are bonded to an encapsulated 40μ silica particle which can be readily packed into any size column. These columns can be operated on both high performance and medium performance liquid chromatographic equipment. The optimization of the separation conditions on the anion exchange media for the preparative purification of a monoclonal antibody (Mab) from ascites will be discussed as well as a multi-step purification of the enzyme prostatic acid phosphatase (PAP) from seminal fluids on the anion and cation exchangers.     

pH-based cation exchange chromatography in the capture and elution of monoclonal antibodies

Cation exchange chromatography separates adsorbed proteins by controlling the salt concentration or the mobile phase pH. This study examines the pH‐based method for binding and elution of monoclonal antibodies (MAbs). Five different clones with isoelectric points from 6 to 9 were evaluated. We performed our studies using a new chromatography resin (Nuvia? S), which has high binding capacity. A three‐column process incorporating Nuvia S as a capture step was also demonstrated for the purification of MAb from tissue culture fluid. Chromatography performance of Nuvia S was demonstrated in a 50‐cycle study.     

Monoclonal Antibody-Based Immunolocalization of Bound Triazine Residues in Two Aquatic Macrophytes (Elodea Canadensis and Myriophyllum Spicatum)

Abstract

Immunohistochemical localization offers a fast and reliable method of obtaining information about the distribution of bound pesticide residues in plants. In the present study aquatic macrophytes (Elodea canadensis, Myriophyllum spicatum) were grown in laboratory model ecosystems spiked with 50 μg/l atrazine. To label cryosections, monoclonal antibodies from cell culture supernatants and ascites fluid with differing specificities for s-triazines were used in combination with the fluorescent dye Phycoerythrin and biotin-streptavidin amplification. The best results were observed with antibodies gained from mice immunized with an ametryn sulfoxide-BSA conjugate, regardless of the method employed for antibody production. The consequences with respect to herbicide metabolism and binding are discussed.     

A high-throughput microchip-based glycan screening assay for antibody cell culture samples

A high-throughput screening assay was developed to quantify major glycan species in the crude mammalian cell culture samples for monoclonal antibodies (mAbs). This method utilizes high-speed microchip electrophoresis separation following a fast sample preparation procedure. Using a 96-well ultra-filtration membrane, interfering species in the cell culture media were efficiently removed as the samples were concentrated. A commercial microchip electrophoresis instrument was used for high-speed separation, allowing each sample to be analyzed in less than 1 min. This method is well suited for the purpose of high-throughput antibody glycan profiling during cell culture expression, including clone selection and cell culture process optimization. The relative levels of high mannose (HM), fucosylated and galactosylated glycan species in the Fc domain can be determined for hundreds of crude cell culture samples in a few hours.     

Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.     

Separation of post-translational modifications in monoclonal antibodies by exploiting subtle conformational changes under mildly acidic conditions

Chromatographic separation plays a key role in the identification, quantification, and characterization of protein variants. Here we describe separation of species containing two post-translational modifications (glycosylation and methionine oxidation) in the Fc fragment of a monoclonal antibody. The method is based on cation-exchange chromatography under mildly acidic conditions that destabilize mainly the CH2 domain. Our data suggest that the separation is not mediated by the chemical modification itself, but rather by subtle structural changes induced by the chemical modification in the domain-decoupled conformation that monoclonal antibodies adopt around pH 4. Compared to other procedures already described in the literature, this method demonstrates an improved separation and allows purification of species in the native fold for additional functional characterization. This approach of separation under conditions where the protein assumes an alternative conformation could find a more general utility for the separation of chemical modifications in proteins.     

Research and development activities on radioimmunoassay at CIAE

Research and development activities on the field of radioimmunoassay has been reviewed in the past decade at China Institute of Atomic Energy (CIAE), including polyclonal and monoclonal antibodies, recombinant antigens, radioiodination method and tracer purification, separation systems, and nonisotopic label immunoassay.     

Pseudoaffinity Chromatography Using a Convective Interaction Media®-Disk Monolithic Column

Monolithic convective interaction media-disk (CIM-disk) chromatography is one of the fastest liquid chromatographic methods for the separation and purification of biomolecules due to its high mass transfer rate. In this way, all separated molecules are transported by convection into the pores of the matrix, resulting in a very fast separation due to the low mass transfer resistance of the CIM-disk. Due to the advantages of monolith technique, in recent years, CIM-disk affinity chromatography has been developed and investigated for purification of peptides, restriction enzymes, antibodies, etc. In this review, applications of monolithic affinity chromatography are discussed. The purification of restriction enzymes, polyclonal and monoclonal antibodies using a new monolithic CIM-disk system with immobilized histidine affinity chromatography are presented.     

Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography

We developed a simple high-performance liquid chromatography assay to monitor high-mannose glycans in monoclonal antibodies by monitoring terminal alpha-mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2-aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha-mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase-treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal-mannose binding to the column and comparable data trending with high mannose levels by 2-aminobenzamide assay confirmed that terminal-mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high-mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high-mannose content in samples compared with traditional glycan profiling by 2-aminobenzamide or mass spectrometry-based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two-dimensional analysis, high-mannose data could be relayed to the manufacturing team in less than 30 min, allowing near-real-time monitoring of high-mannose levels in the cell culture process.     

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。     

The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process

Increased upstream productivity and the continuous pressure to deliver high quality drug product have resulted in the development of new separation technologies and platform strategies for downstream purification processes of monoclonal antibodies (mAb). In this study, the separation attributes of three mixed-mode resins, Mercapto-Ethyl-Pyridine (MEP) hydrophobic charge induction resin, Capto adhere multi-modal anion exchange resin, and ceramic hydroxyapatite/fluoroapatite (CHT/CFT) resins, were investigated to define their roles in monoclonal antibody purification processes. We demonstrated that the multi-modal nature of ligands on mixed-mode resins allows the separation resolution to be honed, either through a single dominant mechanism or through mix-modal balanced purification strategies. In addition, the three mixed-mode resins present different purification powers for different types of impurities. We also demonstrated that besides enhancing chromatography separation and improve product quality, especially for high molecular weight (HMW) aggregate reduction, mixed-mode resins can also help to improve process efficiency in industrial-scale mAb drug manufacturing. Our results underscore the importance of selecting appropriate chromatography resins during DSP design to obtain the best overall process outcome.     

Comparison of protein A affinity sorbents III. Life time study

Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.     

Cation exchange media provide a multi-stage,orthogonal catch and release method for MIDA boronates bearing basic centers

An operationally simple and rapid purification of MIDA boronates is described. This method allows separation of MIDA boronates containing basic centers from those that are neutral as well as separation from species that do not contain the MIDA moiety using a single “catch and release” purification medium. Application of this method to the purification of reductive amination products is described. It is hoped that this facile, rapid and conceptually new isolation will stimulate further investigation of other functionalized silica gel media for the isolation of MIDA boronate building blocks.     

Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia based cation-exchange support

A method suitable for the isolation of monoclonal antibodies (Mabs) is described. The protocol utilizes a zirconia based column modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid to create a novel cation-exchange chromatographic support. Initial experiments using a linear salt gradient demonstrate the ability of this support to efficiently separate Mab from transferrin and bovine serum albumin in a model matrix. Results of the purification of Mab from an actual cell culture supernatant over a range in protein concentrations are also shown. Analyses by enzyme-linked immunosorbent assay and gel electrophoresis demonstrate that Mabs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (> 95%) in a single chromatographic step.     

An enzyme-linked immunosorbent assay system for quantitative determination of calphobindin I, a new placental anticoagulant protein, and its application to various specimens

We developed a sandwich enzyme-linked immunosorbent assay (ELISA) system for calphobindin I (CPB-I), a new placental coagulation inhibitor, using two monoclonal antibodies. This ELISA system can detect CPB-I at concentrations of between 0.4 and 25 ng/ml in buffer and allow almost quantitative determination of it in human plasma. Using this ELISA system, CPB-I levels in many kinds of specimens were measured. Levels in the plasma and urine of women were as low as 10 ng/ml, and no significant differences were observed throughout the trimesters of pregnancy and during different stages of the menstrual cycle. Toxemic patients were slightly higher in CPB-I levels than normal pregnant women, and levels in body fluids such as the amniotic fluid, saliva, milk, ascites, and semen were higher than those in the plasma. The high levels of CPB-I were found, being in the order of micrograms/ml, in the ascites of carcinomatous peritonitis as well as seminal plasma. Measurements of the levels in ovarian follicular fluid samples at different stages of the menstrual cycle showed that those in the immature and atretic stages were higher than those in mature stages. CPB-I levels in many types of cultured human cells ranged from 0.023 to 10.30 micrograms/mg protein, and levels in cultured human lymphocytes were less than those in other types of cells measured. Little of this inhibitor was secreted into media from cultured human lymphocytes, and it was found in all measured tissues of Macacus irus at levels ranging from 0.232 to 1.557 micrograms/mg protein. From these results, it was suggested that CPB-I might be a ubiquitous protein in the body that has an important physiological role.     

Isolation of Der pI, the Dermatophagoides pteronyssinus major mite allergen, from a crude mite culture extract, purification by ion-chromatography, and comparison between the material obtained and a cDNA-coded Der pI.

A high degree of purity is a prerequisite for an allergen preparation to be suitable for clinical diagnosis and therapy. A pure allergen can easily be obtained from a crude mite culture extract by using an immunosorbent prepared with highly specific monoclonal antibodies or from a cDNA-coded material. However, up to now none of these methods has been performed on a process scale. Here large-scale purification is defined as a process in which a crude Dermatophagoides pteronyssinus mite culture extract is essentially fractionated by acetone and ammonium sulphate precipitations followed by anion-exchange high-performance liquid chromatography. A high yield of a very pure Der pI allergen is obtained during the first isocratic run, as shown by sodium dodecylsulphate-polyacrylamide gel electrophoresis, capillary electrophoresis, chromatofocusing and a two site monoclonal antibody enzyme-linked immunosorbent assay. Microsequencing revealed that the 25-residue sequence obtained is entirely in agreement with the sequence derived from the cDNA of Der pI.