Purification of Monoclonal Antibodies Using High Performance Liquid Chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Purification of monoclonal antibodies using high performance liquid chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Identifying analytics for high throughput bioprocess development studies

In recent years, high throughput screening (HTS) studies have been increasingly employed as an integral element of bioprocess development activities. These studies are often limited by an analytical bottleneck; they generate multiple samples for analysis and the available analytical methods cannot always cope with the added analytical burden. A potential solution to this challenge is offered by the deployment of appropriate analytics. This article outlines features of analytical methods that affect their fit to high throughput (HT) applications. These are discussed for a range of analytics frequently used in bioprocess development studies of monoclonal antibodies. It then outlines how these features need to be considered in order to classify analytical methods in terms of their particular application in high throughput scenarios. Biotechnol. Bioeng. 2013; 110: 1924–1935. © 2013 Wiley Periodicals, Inc.     

Novel ligands for the affinity-chromatographic purification of antibodies.

Affinity chromatography represents one of the most powerful fractionation techniques for the large-scale purification of biotechnological products. Despite its potential, the use of this methodology is limited by the availability of specific ligands for each target. Combinatorial chemistry and molecular modeling, often combined, have become interesting and innovative methods for generating novel ligands, tailored to specific biotechnological needs. One of the greatest area of application has been the discovery of novel ligands for the purification of antibodies, which represent an emerging but very important class of innovative therapeutic agents for the treatment of a vast array of diseases. Naturally available affinity ligands, such as Protein A or G for IgG purification or lectins for IgA and IgM purification, which are obtained from microorganisms or genetically modified bacteria through complex and expensive procedures, are not well suited for large-scale purification and require moreover time-consuming analytical controls to check for the presence of contaminants which may affect the safety of the purified antibody for clinical purposes. Recent results suggest that the application of combinatorial technologies and molecular modeling for the discovery of synthetic ligands may open new avenues for the development of more efficient, less expensive and--more importantly--safer procedures for antibody purification at the industrial level.     

A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.

The genomics revolution has created a need for increased speed and generality for recombinant protein production systems as well as general methods for conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety of biochemical assays. Here we present a standardized system for purifying proteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein purification have been generated and characterized. To purify these antibodies from serum-containing medium without carrying through contaminating immunoglobulin G, a peptide-based purification process was developed. A fluorescence polarization binding assay was developed to characterize the antigen-antibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution parameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of recombinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields. The combination of the antibodies described here and 9E10 allow for almost any biochemical application to be utilized with a single simple peptide tag.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.     

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture

Oligosaccharides attached to Asn297 in each of the C_H2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.     

Principles and methods for the analysis and purification of synthetic deoxyribonucleotides by high-performance liquid chromatography

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques, A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purfication of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.Key words: monoclonal antibody, recovery, purification, chromatography, membrane, filtration, platform process     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

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

Purification of a corpuscular influenza vaccine by means of immunoaffinity chromatography

Two immunoaffinity chromatographic methods for the purification of corpuscular influenza vaccine from the admixture of chick embryo components have been examined. The isolation of the virus on immobilized antiviral antibodies has proved to be unsuitable for preparative purposes. The method for the purification of the vaccine from ovalbumin with the use of immobilized anti-ovalbumin antibodies has proved to be highly effective. When introduced into guinea pigs in 3 injections, the vaccine purified by immunosorption has been found to produce no anaphylactic reactions.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, M_w and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset M_w criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their M_w using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (M_w increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.     

Host cell protein analysis in therapeutic protein bioprocessing – methods and applications

The analysis of host cell proteins (HCPs) is one of the most important analytical requirements during bioprocess development of therapeutic moieties. In this review, we focus on the comparison of different methods for the analysis of HCPs and how cell lines, fermentation conditions, and unit operations influence HCP distribution during the process chain. Current guidelines typically require reduction of HCPs to the ppm level, depending on the intended use, the route of administration of the product, and the production system. A range of immunospecific and non-specific methods are available that have been globally accepted by regulatory bodies. Immunospecific methods, such as ELISA, are simple to use in routine analysis and can quantify low levels of HCPs when specific antibodies are available. Non-specific methods are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Different methods for the comparison of bioprocessing strategies during scale-up and purification development are compared herein. The methods include immunospecific methods, such as ELISA, western blot, and threshold, and non-specific methods, such as 2D-DIGE and 2D-HPLC combined with MS.     

嗅鞘细胞的不同纯化方法及结果

培养的嗅鞘细胞的最终纯度受到多种因素的影响,如嗅鞘细胞的取材来源、分离方法等等;对培养的嗅鞘细胞进行纯化可获得高纯度的嗅鞘细胞。纯化嗅鞘细胞的方法有许多种,主要有单纯差速贴壁法、免疫吸附法、化学药物抑制法、无血清饥饿法等,现在的实验研究更趋向于以上2-3种方法联合应用对嗅鞘细胞进行纯化,这些联合纯化方案主要是在采用单纯差速贴壁方法的基础上再次运用其他一种或几种方法进行嗅鞘细胞的纯化。就获取的嗅鞘细胞的最终纯度而言,许多方法取得了可观的效果。但不同的纯化方法各有利弊,除了价格不同外,不同的纯化方法对嗅鞘细胞的生物活性造成不同程度的影响。因此在选择纯化方法时,应综合考虑各方面因素,根据研究目的和实际需要选择合理的方案进行纯化。本文通过查阅各数据库中与嗅鞘细胞的分离培养及纯化有关的文献和其他相关书籍,来探讨纯化嗅鞘细胞的不同方法以及这些纯化方法对嗅鞘细胞最终纯度的影响。     

Comparative evaluation of two purification methods of anti-CD19-c-myc-His6-Cys scFv

Different chromatographic methods have been used to purify bacterially expressed single chain antibodies in soluble or insoluble form. Here, we compared two methods for purification of anti-CD19-c-myc-His6-Cys scFv expressed in Escherichia coli as soluble protein. The protein-L-agarose purification method is a one step purification method that yielded significant amounts of pure protein compared to the two-step Ni-NTA-agarose plus Resource 15S purification method. However, the protein-L purification method exhibited an additional lower molecular weight protein contaminant. Based on results from in vitro gel digestion, mass spectrometry and database search results, we confirmed that the lower molecular weight protein contaminant, which could not be purified by Ni-NTA-agarose and 15S column method, is a degraded product of the full length scFv construct.     

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.     

Flow cytometry‐based methods for assessing soluble scFv activities and detecting antigens in solution

Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast‐display libraries. Yeast‐display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from non‐immune libraries is the conversion of highly active yeast‐displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast‐display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast‐displayed and ‐secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast‐displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast‐displayed scFv that bind non‐overlapping or non‐competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast‐displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast‐displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast‐display libraries. Biotechnol. Bioeng. 2010;105: 973–981. © 2009 Wiley Periodicals, Inc.     

Improved process analytical technology for protein a chromatography using predictive principal component analysis tools

Protein A chromatography is widely employed for the capture and purification of antibodies and Fc‐fusion proteins. Due to the high cost of protein A resins, there is a significant economic driving force for using these chromatographic materials for a large number of cycles. The maintenance of column performance over the resin lifetime is also a significant concern in large‐scale manufacturing. In this work, several statistical methods are employed to develop a novel principal component analysis (PCA)‐based tool for predicting protein A chromatographic column performance over time. A method is developed to carry out detection of column integrity failures before their occurrence without the need for a separate integrity test. In addition, analysis of various transitions in the chromatograms was also employed to develop PCA‐based models to predict both subtle and general trends in real‐time protein A column yield decay. The developed approach has significant potential for facilitating timely and improved decisions in large‐scale chromatographic operations in line with the process analytical technology (PAT) guidance from the Food and Drug Administration (FDA). Biotechnol. Bioeng. 2011; 108:59–68. © 2010 Wiley Periodicals, Inc.     

Use of a Western blotting technique in the purification of a cysteine proteinase inhibitor

In Western blotting procedures, proteins are resolved in sodium dodecyl sulfate-polyacrylamide gels with subsequent electrophoretic transfer onto nitrocellulose membranes. Although this procedure is generally employed as an analytical technique for assessing interactions of proteins with antibodies, the present report describes the use of Western blotting as a preparative procedure in the purification of a biologically active proteinase inhibitor from the cellular slime mold, Dictyostelium discoideum. The feasibility of using Western blotting for inhibitor purification depended upon the unique stability properties of the inhibitor under denaturing conditions.