Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification (original) (raw)
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Affinity puricication of polyclonal antibodies using immobilized multimeric peptides
Journal of Chromatography B: Biomedical Sciences and Applications, 1995
The possibility of using multiple antigenic peptides (MAP) not only for the production and characterisation of antibodies but also for their purification by affinity chromatography, has been explored with two different tetrameric MAPs synthesised starting from a tetradentate lysine core. Recognition selectivity and specificity of the multimeric antigens were retained after immobilization on preactivated affinity supports, allowing convenient antibody purification directly from crude sera in a single chromatographic step. Since antibodies raised against MAPs recognise very frequently the N-terminal portion of the peptide antigen, results suggest that only a limited number of peptide chains remains covalently linked to the solid phase, leaving the others uncoupled and free to interact fully with the antibody. Recovery of antibody immunoreactivity from affinity purifications on MAPcolumns was much higher than that obtained from columns prepared by immobilizing at the same density the corresponding linear peptide antigen. The purity of thus obtained antibodies is also far superior, as detected by SDS-PAGE analysis. Retention of the multimeric peptide recognition properties for the corresponding antibodies after immobilization on solid supports suggests that production, characterization, and even the affinity purification of anti-peptide antibodies, could be carried out simply and conveniently via the synthesis of a single multimeric antigen, without additional steps.
Novel peptide ligand with high binding capacity for antibody purification
Journal of Chromatography A, 2012
Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1 M NaOH. Here, we present a novel synthetic peptide ligand for purification of human IgG. Immobilized on WorkBeads, an agarose-based base matrix from Bio-Works, the ligand has a dynamic binding capacity of up to 48 mg/mL and purifies IgG from harvest cell culture fluid with purities and recovery of >93%. The binding affinity is ∼10 5 M −1 and the interaction is favorable and entropy-driven with an enthalpy penalty. Our results show that the binding of the Fc fragment of IgG is mediated by hydrophobic interactions and that elution at low pH is most likely due to electrostatic repulsion. Furthermore, we have separated aggregated IgG from non-aggregated IgG, indicating that the ligand could be used both as a primary purification step of IgG as well as a subsequent polishing step.
Affinity chromatography is likely to play an everincreasing important role in protein purification as it is the most effective method for the direct isolation and purification of biomolecules from complex mixtures. Successful separation by affinity chromatography requires the availability of a selective ligand. Short peptides are excellent ligands for affinity separations as they have higher selectivity than dyes and metals, they are more stable than antibodies to elution and cleaning conditions, and they are not likely to cause an immune response in case of leakage into the product. Furthermore, the combinatorial synthesis of peptide libraries allows obtaining millions of peptides, thus greatly facilitating the discovery of suitable affinity ligands for any given protein of interest. After screening of the library the peptides with affinity for the target protein can be identified, typically by Edman microsequencing or mass spectrometry in the case of synthetic libraries, or by DNA...
Focused peptide library screening as a route to a superior affinity ligand for antibody purification
Scientific Reports
Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio ...
Industrial & Engineering Chemistry Research, 2013
High binding capacity and selectivity are key features for the successful application of affinity adsorbents for antibody purification. This study presents the development of affinity resins based on hexapeptide ligand HWRGWV for recovering monoclonal antibodies from cell culture fluids. Methods are presented for the immobilization of the peptide ligand and its variants on polymethacrylate and agarose based chromatographic supports using two main coupling strategies. The first one involves the formation of a peptide bond between the amino groups on the substrate and the peptide C-terminus activated with the uronium coupling agent HATU. The second approach involves resin activation with iodoacetic acid, followed by coupling of a cysteine-terminated variant of the ligand to form a thioether bond. The reaction conditions of peptide coupling were optimized to maximize the binding capacity of the resulting adsorbents. The peptide resins were characterized by measuring their static IgG binding capacities. The measured static binding capacity ranged from 35 to 48 mg/mL. The dynamic binding capacities (DBC) of four selected adsorbents were also determined, and they ranged from of 35 to 42 mg/mL with a 5-min residence time. All the resins exhibited high selectivity toward the Fc fragment of IgG. The affinity resins were used to purify two MAbs, a chimeric IgG 1 and a humanized IgG 4 , from commercial CHO cell culture fluids. The resulting yields and purities for both MAbs were found to be in the range of 87−93% and >94%, respectively, which compare well with the purity and yield values obtained using commercially available Protein A media. Finally, the peptide resin with the highest IgG binding capacity, HWRGWVC-WorkBeads, was tested for 20 DBC cycles which included cleaning in place with 0.1 M NaOH after every cycle. The resin showed a high degree of reusability and alkaline stability, as it maintained 90% of its initial capacity.
Journal of Immunological Methods, 2002
This study investigates the applicability of the protein A Mimetic (PAM) affinity ligand, obtained from the screening of a multimeric combinatorial peptide library, in immunoglobulin isolation from serum. To avoid protease degradation, the ligand has been substituted by its inverso form, named D-PAM, synthesized by replacing all amino acids with the corresponding D derivatives. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphazek, were able to capture IgG directly from the serum in a single chromatographic step, with a recovery yield ranging from 60% to 90%, a purity degree higher than 90%, and with a full recovery of antibody activity. Column capacity, determined by applying a large excess of purified IgG to 1 ml bed volume column, was close to 52 mg/ml for bovine IgG, 58 mg/ml for goat IgG, 66 mg/ml for horse IgG, 50 mg/ml for human IgG, 52 mg/ml for mouse IgG, 36 mg/ml for rabbit IgG and 48 mg/ml for sheep IgG. D-PAM peptide was found to be very stable to protease activity, and after prolonged incubation with mouse serum. Similarly, the corresponding derivatized matrix tested before and after various treatments, including sanitization and autoclaving procedures maintained its IgG binding properties, thus indicating a very high stability in terms of ligand leakage and degradation.
Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies
Journal of Separation Science, 2019
Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large‐scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.
Analytical Chemistry, 2012
The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a K d ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBS IBA). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBS IBA column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBS IBA column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBS IBA column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBS IBA platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.
Separation & Purification Technology, 2017
Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therapeu-tics in the biotechnological and biopharmaceutical industry with more than 250 therapeutic mAbs currently undergoing clinical trials. High titer producing cultures and complex mixtures containing high cell densities, together with an increasing growing demand for highly pure mAbs is making recovery and purification processes hot targets for improvement and opens important technological challenges in mAbs manufacturing platforms. This work explores the use of an affinity dual ligand based on a choline binding polypeptide tag (C-LytA) fused to the synthetic antibody binding Z domain (LYTAG-Z) in aqueous two-phase systems (ATPS) composed of phase forming polymers able to bind to the choline binding site of C-LytA (polyethy-lene glycol-PEG-and thermosensitive polymers-EOPO) for mAbs selective extraction. Integration of harvesting and ATPS affinity extraction steps were evaluated with ATPS proving to be an alternative strategy for integrating the clarification and the primary recovery of mAbs. An extraction yield of 89% and a clarification higher than 95% were achieved using a system composed of 7% PEG 3350 and 6% dextran 500,000.
Efficient purification of unique antibodies using peptide affinity-matrix columns
Journal of Immunological Methods, 2004
Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85 -99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.1, was selected and linked to beaded agarose and demonstrated excellent performance as a peptide affinity chromatography matrix. This epitope matrix was efficient in the purification of MK16 Fab fragments and had no affinity for other antibodies. Using this peptide matrix MK16 IgG could be purified from cell culture supernatants thereby separating MK16 IgG from bovine IgG normally present in the enriched growth media used for such cells.