Factorial screening of antibody purification processes using three chromatography steps without protein A

Protein A affinity chromatography is often employed as a capture step to meet the purity, yield, and throughput requirements for pharmaceutical antibody purification. However, a trade-off exists between step performance and price. Protein A resin removes 99.9% of feed stream impurities; however, its price is significantly greater than those of non-affinity media. With many therapeutic indications for antibodies requiring high doses and/or chronic administration, the consideration of process economics is critical. We have systematically evaluated the purification performance of cation-exchange, anion-exchange, hydroxyapatite, hydrophobic interaction, hydrophobic charge induction, and small-molecule ligand resins in each step of a three-step chromatographic purification process for a CHO-derived monoclonal antibody. Host cell proteins were removed to less-than-detectable for three processes (cation-exchange-anion-exchange-hydrophobic interaction chromatography, cation-exchange-anion-exchange-mixed cation-exchange chromatography, and cation-exchange-mixed cation-exchange-anion-exchange chromatography). The order of the process steps affected purification performance significantly.     

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.     

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.     

Adsorption Study of Immunoglobulin G Subclasses from Different Species by Pseudobioaffinity Separation on Histidyl–Bisoxirane–Sepharose

Adsorption chromatography is increasingly used for protein separations and biomedical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA, and plasma proteins must be purified before characterization and utilization. Use of immunoglobulins as immunodiagnostic and therapeutic tools has initiated many attempts to develop new adsorbents for their separation. Protein A and protein G are the affinity ligands most widely used for separation of immunoglobulins. These proteins are reliable, and have good selectivity and specificity, but are very expensive. Much attention has therefore been devoted to developing alternative methods for separation of immunoglobulins. Pseudobiospecific ligands, for example metal ions and amino acids, can be used for separation of a wide range of biological molecules. In this study, IgG₁, IgG₂, and IgG₃, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent. Adsorption of IgG from different animal species on the same chromatographic adsorbent was also tested. The high recovery and purification on histidyl–bisoxirane–Sepharose gel of IgG from all the sources tested compared well with results obtained by use of protein A–Sepharose gel.

     

Adsorption Study of Immunoglobulin G Subclasses from Different Species by Pseudobioaffinity Separation on Histidyl–Bisoxirane–Sepharose

Adsorption chromatography is increasingly used for protein separations and biomedical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA, and plasma proteins must be purified before characterization and utilization. Use of immunoglobulins as immunodiagnostic and therapeutic tools has initiated many attempts to develop new adsorbents for their separation. Protein A and protein G are the affinity ligands most widely used for separation of immunoglobulins. These proteins are reliable, and have good selectivity and specificity, but are very expensive. Much attention has therefore been devoted to developing alternative methods for separation of immunoglobulins. Pseudobiospecific ligands, for example metal ions and amino acids, can be used for separation of a wide range of biological molecules. In this study, IgG₁, IgG₂, and IgG₃, three subclasses of human IgG, were separated from human serum using the amino acid histidine grafted on to bisoxirane-activated Sepharose, as pseudobiospecific adsorbent. Adsorption of IgG from different animal species on the same chromatographic adsorbent was also tested. The high recovery and purification on histidyl–bisoxirane–Sepharose gel of IgG from all the sources tested compared well with results obtained by use of protein A–Sepharose gel.     

Evaluation and comparison of alternatives to Protein A chromatography Mimetic and hydrophobic charge induction chromatographic stationary phases

In this paper Protein A mimetic and hydrophobic charge induction chromatographic (HCIC) stationary phases are characterized in terms of their protein adsorption characteristics and their selectivity is compared with Protein A chromatography using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were employed to compare the selectivities of these resins for both non-IgG model proteins as well as antibodies and the fusion proteins. While none of the non-IgG model proteins were observed to bind to the Protein A resin, most of them did in fact bind to the alternative resins. In addition, while the elution pH was similar for the model proteins and antibodies on the HCIC resin, the mimetic resins did exhibit higher binding for the antibodies under these linear pH gradient conditions. A mixed mode preparative isotherm model previously developed for HCIC was shown to accurately describe the adsorption behavior of the mimetic materials as well. Host cell protein clearance profiles were also investigated under preparative conditions using complex biological feeds and the results indicated that while some selectivity was observed for both the HCIC and the mimetic materials, the purification factors were in general significantly less than those obtained with Protein A. It is important to note, however, that the selectivity of the mimetic and HCIC materials was also observed to be antibody specific indicating that further optimization may well result in increased selectivities for these materials.     

Analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse

Protein A resins are often reused for multiple cycles to improve process economy during mAb purification. Significant reduction in binding capacity and product recovery are typically observed due to the presence of unwanted materials (foulants) deposited on the resin upon reuse. In this paper, we have used a wide spectrum of qualitative and quantitative analytical tools (particle size analysis, HPLC, fluorescence, SEM, MS, and FTIR) to compare the strengths and shortcomings of different analytical tools in terms of their capability to detect the fouling of the resin and relate it to chromatographic cycle performance. While each tool offers an insight into this complex phenomena, fluorescence is the only one that can be used for real‐time monitoring of resin fouling. A correlation could be established between fluorescence intensity and the process performance attributes (like yield or binding capacity) impacted upon resin reuse. This demonstration of the application of fluorescence for real‐time monitoring correlated empirically with process performance attributes and the results support its use as a PAT tool as part of a process control strategy. While the focus of this paper is on fouling of protein A chromatography resin, the approach and strategy are pertinent to other modes of chromatography as well.     

Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process

Recent efforts to improve hydrophobic interaction chromatography (HIC) for use in monoclonal antibody (mAb) purification have focused on two approaches: optimization of resin pore size to facilitate mAb mass transport, and use of novel hydrophobic charge induction (HCIC) mixed mode ligands that allow capture of mAbs under low salt conditions. We evaluated standard HIC and new generation HIC and HIC-related chromatography resins for mAb purification process efficiency and product quality both as isolated chromatography steps and in purification process trains. We find that HIC resins with optimized pore size have significantly improved binding capacity which can increase HIC purification unit operation efficiency. The HCIC Mercapto-Ethyl-Pyridine (MEP) resin, which shows a different salt impact trend and impurity resolution pattern from standard HIC resin, can not only capture mAb from crude CHO fermentation supernatant but also substantially enhance mAb purification process flow efficiency when serving as a polishing role.     

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.     

A mechanistic study of Protein A chromatography resin lifetime

A mechanistic study into Protein A chromatographic resin lifetime limitations is presented. Binding and mass transport properties of two widely used agarose-based Protein A resins were studied to distinguish between the roles of resin fouling due to product/impurity build-up and ligand degradation as contributory factors towards the decline in binding capacity with use. Cycling studies were conducted with and without product loading on the columns to separate out the influence of resin fouling. Ligand degradation under the mildly alkaline conditions used for column regeneration was determined to be the primary cause for Protein A resin capacity decline with usage. The use of lower concentrations of caustic and the use of stabilizing excipients to protect the Protein A ligand during cleaning and sanitization were found to be useful techniques in maintaining column performance. The results presented in this paper provide a clearer understanding of the causative factors that limit Protein A chromatographic resin lifetime. It is anticipated that these findings will assist in the development of more robust and economical downstream manufacturing processes for monoclonal antibody and Fc fusion protein purification.     

Synergies between micropreparative high-performance liquid chromatography and an instrumental optical biosensor

The recent development of an automated surface plasmon resonance technology for the measurement of biomolecular interactions (Pharmacia BIAcore) has provided new opportunities for the detection and analysis of protein-protein interactions. In the BIAcore, detection is based on changes in surface plasmon resonance which are monitored optically. Changes in surface plasmon resonance correspond to changes in surface concentration of macromolecules and can be monitored in real time.

We have found that the detection sensitivity obtainable with this technology (ng/ml concentrations of specific ligands are readily detectable for many applications) is complementary “in a bidirectional manner” to micropreparative HPLC. Thus micropreparative HPLC may be used to purify and characterise reagents for the biosensor, whilst the biosensor may be used to define chromatographic parameters such as elution conditions for affinity chromatography or serve as an affinity detector for fractions obtained during chromatographic purification.

Examples of such applications, including the potential of the biosensor to search for and monitor the purification of unknown ligands for which the target molecule has been identified, are shown. In particular, the use of the biosensor to monitor the purification of soluble epidermal growth factor receptor from A431 cell conditioned media is demonstrated.     

Epitope affinity chromatography and biophysical studies of monoclonal antibodies and recombinant antibody fragments

Peptide epitope affinity chromatography is a powerful technique for the purification of antibodies. This study aims to demonstrate the versatility of the technique and to show how biophysical techniques such as circular dichroism (CD) and fluorescence quenching (FQ) can aid the rational design of affinity ligands and characterization of antibody-based reagents. The performance of a number of peptide ligands for the purification of a range of different antibodies and recombinant fragments is investigated by automated fast-protein liquid chromatography. Purified products are analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They are then radiolabelled and the immunoreactivity is determined. Ligands are analyzed for secondary structural characteristics by CD and for binding affinity by FQ. Finally, a study is performed to investigate the thermal stability of a recombinant antibody fragment by CD analysis. It is found that simple ligand modifications such as the introduction of a C-terminal cysteine residue can improve purification performance. The FQ studies show that the modified peptide has a higher affinity for antibody. The CD analysis shows that it has a tendency to dimerize because of the formation of disulfide bonds. The versatility of epitope affinity is demonstrated through the purification of a recombinant diabody (dbFv) and by the use of a separate peptide matrix for the purification of an unrelated antibody. All studies result in antibody preparations of high purity and immunoreactivity. The CD analysis of the dbFv shows that it is denatured at 37 degrees C and is therefore unsuitable as a targeting reagent for use in humans in its present form. It is concluded that epitope affinity chromatography coupled with biophysical analyses plays an important role in the production and characterization of antibody-based reagents for targeted diagnosis and therapy of human diseases.     

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.     

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.     

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.     

Novel Protein Binding Capacity and Selectivity of Immobilized Pyridinium Pseudo-Affinity Gels: Applications in Immunoglobulin G Purification and Quantitative Determination

Abstract

A series of novel pseudo-affinity gels using synthetic, immobilized pyridinium ligands have been prepared. These gels exhibit extraordinarily high protein binding capacity and selectivity. Initial applications of these gels were directed toward the purification and quantitative, rapid determination of immunoglobulins. The results showed that these pseudo-affinity gels functionally mimic, to a high degree of similarity, the behavior of immobilized Protein A or G for immunoglobulins purification. In contrast to the labile nature of protein-based affinity gels, these immobilized pyridinium gels can withstand harsher treatments, such as prolonged exposure of the gels to proteolytic enzymes, organic solvents, acid or base and high temperatures.     

Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography

Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50–60% range for affinity resins, and in the 80–85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

Aptamer-Based Alternatives to the Conventional Immobilized Metal Affinity Chromatography for Purification of His-Tagged Proteins

Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based affinity purification for His-tagged proteins for comparison of purification efficiency with the conventional Ni²⁺-based affinity chromatography. Thiol-functionalized aptamers able to specifically bind to His-tag were immobilized employing two crosslinking methods onto the surface of polystyrene resins. The resulting aptamer-anchored resins were successfully applied for purification of His-tagged proteins from complex E. coli and human cell lysates, respectively, and superior or at least comparable purification results to the conventional immobilized metal affinity chromatography were obtained via one-step purification.     

Poly(glycidyl methacrylate)‐grafted hydrophobic charge‐induction agarose resins with 5‐aminobenzimidazole as a functional ligand

Hydrophobic charge‐induction chromatography is a new technology for antibody purification. To improve antibody adsorption capacity of hydrophobic charge‐induction resins, new poly(glycidyl methacrylate)‐grafted hydrophobic charge‐induction resins with 5‐aminobenzimidazole as a functional ligand were prepared. Adsorption isotherms, kinetics, and dynamic binding behaviors of the poly(glycidyl methacrylate)‐grafted resins prepared were investigated using human immunoglobulin G as a model protein, and the effects of ligand density were discussed. At the moderate ligand density of 330 μmol/g, the saturated adsorption capacity and equilibrium constant reached the maximum of 140 mg/g and 25 mL/mg, respectively, which were both much higher than that of non‐grafted resin with same ligand. In addition, effective pore diffusivity and dynamic binding capacity of human immunoglobulin G onto the poly(glycidyl methacrylate)‐grafted resins also reached the maximum at the moderate ligand density of 330 μmol/g. Dynamic binding capacity at 10% breakthrough was as high as 76.3 mg/g when the linear velocity was 300 cm/h. The results indicated that the suitable polymer grafting combined with the control of ligand density would be a powerful tool to improve protein adsorption of resins, and new poly(glycidyl methacrylate)‐grafted hydrophobic charge‐induction resins have a promising potential for antibody purification applications.