Chromatographic Methods for the Purification of Monoclonal Antibodies and their Alternatives:A Review

Transcription

1 Chromatographic Methods for the Purification of Monoclonal Antibodies and their Alternatives:A Review Ishan Arora 1 1 Department of Chemical Engineering, Indian Institute of Technology Delhi Abstract Monoclonal Antibodies (mabs) have emerged as a unique and exciting group of biological products. They represent a rapidly growing biopharmaceutical market segment with interesting and extremely useful medical applications in the treatment of numerous diseases such as cancer and immunological disorders. The monoclonal antibody purification process that is adopted should be reliable, robust and must efficiently remove various impurities such as aggregates, host cell protein, DNA, some cell culture media additives, clipped/low molecular weight species, viruses etc. The currently established purification platform typically employs three chromatography steps with Protein A chromatography as the initial capture step followed by two polishing steps which may include cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography etc. But conventional packed bed chromatography suffers from certain serious drawbacks. As a result, alternatives to chromatography are also becoming important. In this paper, various techniques used for the purification of monoclonal antibodies are discussed and the merits and demerits of each technique are critically analyzed besides discussing about the typical applications of each of these methods. Keywords Aqueous two phase systems,, Impurities, Monoclonal antibodies, Resin, Yield I. INTRODUCTION Monoclonal antibodies (mabs) have emerged as an extremely important and valuable class of therapeutic products. They have been successfully introduced as therapies to myriad diseases such as rheumatoid arthritis, auto-inflammatory disease, colorectal cancer, allergic asthma and multiple sclerosis [1],[2]. This is primarily due to their high specificity and amazing versatility [3]. As a result, monoclonal antibodies represent a rapidly growing biotechnology field. A pertinent concern is the development of a reliable purification process that can efficiently remove the different types of impurities in order to produce products suitable for human use. Also, the loss of yield of the product during purification should be minimum. The purification process must be capable of removing both product related impurities which include high molecular weight aggregates and several isoforms that may be formed as a consequence of deamidation, oxidation or shuffling of disulfide bonds and process related impurities which include leached protein A, DNA, certain cell culture media additives and host cell protein [3]-[5]. It is essential to remove these impurities since they may seriously hamper the biological activity of the biopharmaceutical. The cost effectiveness and ease of scale up of the purification process are also crucial considerations. Currently, packed bed chromatography is the backbone of downstream processing. We typically use three chromatography steps the first being Protein A chromatography followed by two polishing chromatography steps which may be anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography etc. [1],[6]. The choice of the appropriate polishing steps is mainly governed by the nature of the impurities that need to be removed. Generally, one of the polishing chromatography steps involves the use of ion exchange chromatography. Protein A chromatography is extremely effective and selective in removing host cell proteins, virus particles, DNA and other impurities and at the same time gives a very good yield [5],[7]. But it does have a few drawbacks such as the possibility of formation of aggregates due to the elution at low ph and inability to remove aggregates formed during earlier processing steps [6],[7]. The polishing chromatography steps are typically used to remove the remaining aggregates, viruses, host cell protein and leached protein A [8]. However, the use of chromatographic separations has certain demerits such as the requirement of large liquid volumes, high cost and comparatively long cycle times. Hence, there is significant interest in alternatives to chromatography such as aqueous two phase extraction, precipitation and charged ultrafiltration membranes [6],[9],[10]. 475

2 Fig. 1: Commonly used chromatographic methods for monoclonal antibody purification and some novel alternatives In Fig.1, HIC refers to Hydrophobic Interaction, IEC refers to Ion Exchange and ATPS refers to Aqueous Two Phase Systems II. DISCUSSION OF DIFFERENT CHROMATOGRAPHY TECHNIQUES A. Protein A It is a mode of affinity chromatography that makes use of the specific interactions which take place between the Fc region of mabs and immobilized protein A [3]. Protein A chromatography is an unparalleled and versatile technique since it allows the removal of more than 95% impurities in a single step [8]. As a result, it is almost unanimously selected as the first step (capture step) in the monoclonal antibody purification process. Another remarkable feature of this technique is its simplicity and ease of operation. However, as mentioned earlier, the low ph elution conditions used in Protein A chromatography are a serious cause of concern since they can lead to aggregation and loss of biological activity [11]. The leaching out of Protein A in small amounts from its support matrix is also a major problem since the leachate and its fragments are immunotoxic [7]. There has been a lot of research to address the important problem of aggregate formation during Protein A elution and it has been demonstrated that the addition of stabilizers such as arginine to the Protein A elution buffer can help reduce aggregation [3]. B. Ion Exchange (IEC) It is the most common polishing step used in the purification of monoclonal antibodies to remove the trace amounts of impurities remaining after the Protein A chromatography step. As the name suggests, Ion Exchange implies separation on the basis of charge. The separation is on account of differential electrostatic affinities of the biomolecules for a charged stationary phase material [12]. It is a very common practice to classify ion exchange stationary phases as strong and weak exchangers. This is on the basis of the ph range over which these ion exchangers retain their charge. Those ion exchangers which have the ability to retain their charge over a wide ph range are classified as strong exchangers while those which can do so over a narrow ph range are known as weak exchangers [11],[12]. Ion Exchange is classified into two categories: Cation Exchange and Anion Exchange. 476

3 TABLE I PROPERTIES OF SOME COMMON CATION EXCHANGE CHROMATOGRAPHY RESINS Resin Type Vendor Functional Group Backbone Particle Size (in micron) Fractogel EMD SO 3 - (M) Strong Merck Sulfoisobutyl Methacrylate Fractogel EMD SO 3 - (S) Strong Merck Sulfoisobutyl Methacrylate Fractogel EMD COO - (M) Weak Merck Carboxyethyl Methacrylate ESHMUNO S Strong Merck Sulfoisobutyl Surface grafted rigid polyvinyl ether hydrophilic polymer CM Sepharose Weak GE Healthcare Carboxymethyl Cross linked agarose Capto S Strong GE Healthcare Sulfoethyl Cross linked agarose with dextran surface extender 90 (average) Capto SP ImpRes Strong GE Healthcare Sulfopropyl High flow agarose S Ceramic HyperD Strong Pall Sulfopropyl Polystyrene shell and hydrogel 50 (average) POROS XS Strong Applied Biosystems Sulfopropyl Cross-linked poly(styrenedivinylbenzene) 50 (average) 1) Cation Exchange : In Cation Exchange, negatively charged functional groups such as carboxymethyl, sulfopropyl and sulfoisobutyl are immobilized to the resin so that it has affinity for positively charged ions (cations) [5]. It is an extremely useful technique for removing certain impurities such as aggregates, deamidated (acidic material), oxidized species which would be present even after Protein A chromatography [4]. Table I highlights the properties of some commonly used cation exchange chromatography resins. 2) Anion Exchange : It retains negatively charged ions (anions) because the stationary phase contains positively charged functional groups such as Diethylaminoethyl (DEAE), Trimethylammoniumethyl (TMAE) etc. It is conventionally operated in flow through mode or bind and elute mode but recently a new mode of anion exchange chromatography known as weak partitioning mode has become popular which can provide an enhanced clearance of impurities since the ph and conductivity are selected in such a way that weaker binding impurities can be removed more efficiently as compared to the flow through mode [1]. Some commonly used anion exchangers are Fractogel EMD DEAE(M), Q Hyper D and Q Sepharose Fast Flow [12],[13]. The properties of some common anion exchangers are depicted in Table II. 477

4 TABLE II PROPERTIES OF SOME COMMON ANION EXCHANGE CHROMATOGRAPHY RESINS Resin Type Vendor Functional Group Backbone Particle Size (in micron) Capto Q ImpRes Strong GE Healthcare Quaternary amine High-flow agarose DEAE Sepharose Fast Flow Weak GE Healthcare Diethylaminoethyl 6% cross-linked agarose Q Sepharose Fast Flow Strong GE Healthcare Quaternary amine 6% cross-linked agarose Fractogel EMD DEAE(M) Weak Merck Diethylaminoethyl Methacrylate Fractogel EMD TMAE(M) Strong Merck Trimethylammoniumethyl Methacrylate C. Hydrophobic Interaction (HIC) The distinct advantage offered by hydrophobic interaction chromatography over other techniques is its ability to efficiently remove aggregates. It can also remove other impurities such as host cell protein, DNA, leached Protein A etc. making it a good candidate for being used after ion exchange chromatography as a polishing step [5] The basic principle is that proteins and other molecules which have hydrophobic surface properties get preferentially attached (bound) to HIC resins containing ligands such as phenyl and butyl under aqueous conditions with a high salt concentration in the buffer but get eluted when a gradient of decreasing salt concentration is used [14]. But there are some limitations associated with using HIC in bind and elute mode which hinder its widespread application. It has relatively lower yield compared to other chromatography steps and the elution product pool may contain fair amount of salt since high concentrations of binding salts are typically required in order to achieve good capacities [4], [15]. Recently, there has been a lot of work on resin pore size optimization which can lead to a considerable improvement in the binding capacity of HIC resins [15]. D. Multimodal (Mixed Mode ) As the name suggests, Multimodal possesses the unique ability to combine various types of interactions such as hydrophobic interaction, hydrogen bonding and ionic interaction within one single resin [5],[16]. It can lead to dramatic improvements in the monoclonal antibody purification process by providing improved selectivity, new separation mechanisms, salt tolerant adsorption and a remarkably high loading capacity [17],[18],[19]. It can prove to be an extremely important technique for removing aggregates and enhancing the process efficiency for downstream processing of mabs [20]. In certain cases, using multimodal chromatography as a purification step after Protein A chromatography may help to reduce the number of chromatography steps from three to two, thus increasing the productivity of the process [21]. Examples of multimodal chromatographic resins are Capto adhere, Capto MMC, HEA HyperCel, MEP HyperCel and PPA HyperCel [19]. It is interesting that if we can combine an externally controlled ph gradient with such resins, we may be successful in carrying out certain separations that cannot be achieved by conventional methods [16]. But there are certain issues that need careful study such as the complexity of interactions involved and resin lifetime. 478

5 TABLE III SUMMARY OF THE VARIOUS CHROMATOGRAPHIC METHODS USED FOR MONOCLONAL ANTIBODY PURIFICATION Technique Importance Distinct Advantages Available Resins Limitations Protein A chromatography Primarily used as capture step to remove DNA, host cell protein, cell culture media components and various other impurities Extremely efficient, highly selective, removal of most of the impurities achieved in a single step, very robust MabSelect SuRe, ProSep A High Capacity, POROS Mabcapture A, nprotein A Sepharose 4 Fast Flow Elution at low ph leads to aggregate formation, high cost of resins, leached Protein A ligand added as impurity Cation Exchange Effective in removing both process and product related impurities such as leached Protein A, host cell protein, aggregates High step yield, relatively less expensive resins, very significant reduction of host cell protein level, high capacity S Ceramic HyperD, CM Ceramic HyperD, SP Sepharose Fast Flow, POROS XS, Capto SP ImpRes Sample preparation needs buffer exchange if conductivity is not low, partial insolubility of some mabs under low ph and low conductivity conditions used for achieving high binding capacity Anion Exchange Generally employed as the last chromatography step in order to remove residual host cell proteins, DNA, endotoxin, leached Protein A and viruses Excellent removal of endotoxin, high loading capacity in flow through mode, inexpensive resins Capto Q ImpRes, Fractogel EMD TMAE(S), DEAE Ceramic HyperD, DEAE Sepharose Fast Flow Requirement for low loading buffer conductivity, large column volume needed for fast flow Hydrophobic Interaction Used as a polishing step for removing high molecular weight aggregates, host cell protein Very effective for aggregate removal, easily removes those contaminants which are most difficult for other chromatography techniques Octyl Sepharose 4 Fast Flow, Fractogel EMD Phenyl(S), SOURCE 15ETH, Capto Butyl High salt concentration in elution product pool, relatively lower binding capacity in bind and elute mode, not very effective for removal of DNA and leached Protein A in flow through mode Multimodal Mostly used after the Protein A chromatography step to selectively remove DNA, aggregates, host cell proteins and viruses and can convert a conventional three step purification process to an extremely productive two step process Resin can bind through hydrogen bonding, hydrophobic and electrostatic interactions, unique selectivity, stronger binding, savings in operational costs by using only two chromatography steps MEP HyperCel, Capto MMC, ESHMUNO HCX, Capto adhere Interactions involved are complex, lack of understanding about nature of binding, extensive optimization required 479

6 III. NOVEL ALTERNATIVES TO COLUMN CHROMATOGRAPHY E. Aqueous Two Phase Systems (ATPS) Aqueous two phase systems are spontaneously formed when aqueous solutions of two mutually incompatible components are mixed such as two polymers which are structurally different or a polymer (typically PEG) and a salt (generally citrate, phosphate or sulphate) above a certain critical concentration [2],[22],[23]. Aqueous two phase extraction can serve as an important and novel technique for the purification of monoclonal antibodies owing to its cost effectiveness, high capacity, biocompatibility and scale up potential [22], [23]. The selective partitioning between the two phases in ATPS is governed by numerous factors which include intrinsic properties such as surface hydrophobicity, charge, conformational characteristics and system properties such as ph, ionic strength, polymer type, salt type, concentration of salt etc. [2],[24]. An amazing feature of this technique is that it has the potential to integrate clarification, concentration and partial purification of mabs into just one single step [25]. But certain challenges need to be overcome before its full potential can be tapped such as the problems associated with the handling and disposal of large quantities of raw materials needed for the process and the limited understanding we have about the complex interactions between the different components in the system [2],[10]. F. Membrane This is an innovative and special technique in which we typically use microporous membranes consisting of a polymeric substrate to which certain functional ligands are coupled [5]. Membrane chromatography has a remarkable potential and is rapidly emerging as an alternative to conventional column chromatography. This is primarily because membrane chromatography offers certain distinct benefits. These include reduction of buffer consumption, quick operation and low space requirement [26]. Here, convection is the main mechanism of transport of molecules to their binding sites with negligible pore diffusion and hence, the total mass transfer resistance in such a case is substantially lower than traditional chromatography columns [27]. It eliminates the need for column packing which is strenuous and unreliable [28]. This method helps us overcome the problem of high pressure drop which is prevalent in conventional column chromatography [27]. Also, another big advantage is that membrane chromatography allows us to use much higher flow rates than column chromatography. Hydrophobic Interaction Membrane has been demonstrated to be useful in simultaneously removing leached protein A and aggregates from monoclonal antibodies, a remarkable breakthrough indeed! [7]. However, this is just one side of the coin. Membrane chromatography does suffer from certain limitations which need to be overcome before the potential of this incredible technique can be fully tapped. These include membrane fouling, lower throughput on account of lower unit surface areas, lower binding capacity, uneven flow distribution etc. [5],[6],[29],[30]. We need to study more about the various aspects of membrane chromatography and come up with ingenious solutions to combat these challenging problems. IV. CONCLUSION Although a wide range of techniques are available for the purification of monoclonal antibodies, most of them rely on the use of Protein A chromatography as the capture step followed by one or two polishing steps which are generally selected based on the particular impurity clearance challenge [1]. has been the workhorse of downstream processing primarily due to its simplicity and high resolving power [2]. Protein A chromatography is almost unanimously selected as the initial capture step but it has certain serious drawbacks which have attracted significant interest and lot of research has been done to overcome these shortcomings as far as possible. Cation exchange chromatography, anion exchange chromatography and hydrophobic interaction chromatography are generally used as polishing steps for the removal of product and process related impurities and viruses [5]. Hydrophobic Interaction (HIC) is very useful for the removal of aggregates. Anion exchange chromatography provides excellent removal of endotoxin while Cation exchange chromatography is particularly good for removal of host cell protein besides some other impurities. It has been demonstrated that Multimodal chromatography, when used after Protein A chromatography, can help reduce the number of chromatographic steps for monoclonal antibody purification from three to two, thus improving the yield and shortening the process time [21]. However, extensive optimization and proper understanding of the nature of interactions are required before we can make the best use of the amazing potential of Multimodal chromatography. 480

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