William Evansis technical service specialist at Tosoh Bioscience, 800.366.4875, techservice.tbl@ tosoh.com.

William Evans

 	Monoclonal antibodies (mAbs) are a successful class of therapeutic products used increasingly in the past 15-20 years, but the manufacture of safe and effective mAb drug products provides many challenges to downstream molecule purification. One of these challenges is the tendency of mAb molecules to aggregate during processing. Aggregates in the final drug product are undesirable for two major reasons. First, aggregates may cause a decrease in product efficacy due to lowering the effective concentration of the drug product. Second, aggregates increase the risk of an immunogenic response in patients, including anaphylaxis. For these reasons, the removal of aggregates is a focus of downstream processing. 	 	The mAb purification procedure must effectively reduce aggregate concentration in the drug product, and processes are usually optimized to target an aggregate concentration of less than 1% for the final mAb drug product. Additionally, processes must be optimized to prevent the additional aggregation of the drug molecule during processing.	
	


	Removal of aggregates, especially soluble aggregates, presents a challenge due to the physical and chemical similarity of the aggregates to the drug product itself, which is usually a monomer. Chromatography steps can effectively remove aggregates, and, typically, one or more chromatography steps in a process will be optimized for aggregate removal. The need for aggregate removal, however, must be balanced by the productivity of the process, the step yield, and the overall purity of the product through the removal of host-cell proteins and other contaminants.

	For mAb products, nearly all processes begin with an initial Protein A affinity chromatography step to remove the bulk of impurities present in the clarified harvest. This initial step provides a product that is typically >90% pure, and the subsequent processing steps focus on the removal of the remaining minor impurities. Due to the chemical similarity of aggregates to the monomer molecule, however, Protein A chromatography does not effectively remove aggregates already present in the feedstock. Additionally, Protein A elution conditions must be optimized to prevent the further aggregation of molecules due to denaturation at the acidic pHs typically used. Finally, Protein A eluate is usually maintained at a low pH for 30-60 minutes as a viral inactivation measure. This hold has the potential to exacerbate aggregate formation.

	Secondary purification steps are often used to remove aggregates following Protein A chromatography and low pH treatment. Because aggregate molecules are, chemically, multiples of the monomer, aggregate molecules will have proportionately greater surface charge or surface hydrophobicity. Ion (anion or cation) exchange and hydrophobic interaction chromatography modes may be employed to take advantage of this increased charge and hydrophobicity of the aggregates to separate them from the monomer molecule. 

 shows the strategy for aggregate removal with ion exchange (IEX), hydrophobic interaction (HIC), and mixed-mode chromatography. IEX methods will separate based on molecule charge, while HIC methods separate based on hydrophobicity. Mixed-mode methods separate based on both charge and hydrophobicity. Merits of the various chromatography modes are discussed as follows.

 In cation exchange chromatography (CEX), a positively-charged molecule is adsorbed by the column resin and then displaced with a high concentration of a positively-charged ion such as Na+. As they require a positively-charged molecule, CEX steps are performed at a pH below the isoelectric point of the target molecule; for mAbs this is often in the range of 7.5-9. To determine elution conditions during process development, separations of aggregate-containing product may be done in which material is bound at low conductivity and eluted with a conductivity (salt) gradient (

	The strength of the binding of the mAb to a CEX resin is determined by the pH and the type of CEX ligand used. Alteration of the pH will affect the charge of the molecule and increase or decrease the binding to a given resin. The chemical properties of the ligand will also affect the binding affinity of the molecule. For instance, carboxylic-type (CM) resins often show a stronger binding then sulfonic acid-type, thus requiring a higher solution conductivity to cause elution. For this reason, the CM-type resins may be said to be “salt-tolerant”. During process development, it is common to screen multiple resins at various pH points to optimize the resolution between monomer and aggregate peaks. In the final processes, elution conditions are set to collect primarily monomer. Species that bind more weakly than the mAb are washed off the column prior to elution, while those that bind more strongly, including aggregates, remain bound and are cleaned off prior to the processing of the next batch. Theoretically, it is also possible to adjust solution conductivity to allow the monomer to flow through and the aggregate to be retained, although this method is not typically employed.

 Anion exchange (AEX) chromatography may also be employed to remove aggregates. Often, anion exchange is employed in mAb processes in a product flow-through mode to remove DNA and some viruses that are highly negatively charged at neutral pHs. Salt-tolerant anion exchange resins, though, may be used in bind-and-elute mode, because significant antibody binding can be achieved at neutral to slightly-basic pHs. In this mode, the salt-tolerant AEX resin would be used in a manner similar to CEX described previously.


	HIC may be used in much the same manner as IEX to remove aggregates. In this case, the aggregates will show increased surface hydrophobicity relative to the monomer. HIC steps present more variables for optimization during process development, as the hydrophobicity of the protein, the type and concentration of salt in the feedstock, and the hydrophobicity of the resin come into play. Protein hydrophobicity may be modulated by change in pH to increase or decrease the net charge of the molecule. Different types of salts may be used to modulate the strength of the hydrophobic interaction of the mAb with the resin. Different resin ligands will also have varying levels of hydrophobicity. For instance, a butyl ligand will be more hydrophobic than a phenyl ligand, which will, in turn, be more hydrophobic than an ether ligand.

	Usually, HIC is performed in bind-and-elute mode. The product is bound under high-salt conditions to strengthen hydrophobic effect with the resin. To determine elution conditions, a reverse-salt gradient is then used to elute the protein (

). Analogous to cation exchange, the aggregate will remain bound following monomer elution due to greater surface hydrophobicity. During process development, various salt/resin combinations may be screened to provide the greatest aggregate removal. Use of HIC in bind-and-elute mode, however, provides additional challenges, especially at a manufacturing scale. The HIC eluate may contain substantial amounts of salt, which then must be removed by diafiltration, or reduced in concentration by the dilution of the eluate prior to further processing. This has led to the development of low-salt or salt-free flow-through HIC steps in some processes. For these, a highly hydrophobic ligand (such as hexyl) is used, and the hydrophobicity of the mAb molecule is modulated by pH change to allow the monomer to flow through the column, while aggregate and other contaminants remain bound.

 Mixed chromatographic modes allow separation based on both charge and hydrophobicity in a single step. Mixed-mode chromatography (MMC) can provide a different selectivity than either IEX or HIC alone. One drawback of mixed-mode chromatography, however, is that because of the increased binding due to hydrophobic interactions, a higher salt concentration is usually needed to elute molecules bound to an MMC resin than a simple IEX; in effect, mixed-mode resins may be employed as highly salt-tolerant IEX resins (

). An alternate strategy for separation by MMC is to use both a conductivity and pH change for elution; this may provide the needed selectivity for separation, as well as reducing the salt concentration of the eluate. Optimum elution conditions may be determined by using combined salt and pH gradients for elution, as shown in 

 A special mention should be given to size-exclusion chromatography (SEC) for the purposes of aggregate removal. As aggregates would be proportionally sized relative to the monomer (e.g., a dimer would occupy twice the volume of the monomer), molecule size should provide a basis to separate out aggregates. In analytical chromatography (HPLC, UHPLC), SEC is often used to determine the amount of aggregate in a sample, as monomer will be more highly retained due to pore access, and elute later than aggregates (

). For purification, however, it is difficult to conduct SEC on the scale necessary to perform industrial mAb manufacture, and, therefore, it is not used in practice. SEC is very useful, though, on a laboratory scale, especially as a final purification step for material that must be very pure (e.g., for crystallography).


	The removal of aggregates present in a mAb purification process presents a challenge to process development. However, several chromatographic tools are available to reduce the concentration of aggregates to maintain drug product efficacy and safety. Ion exchange, HIC, and MMC may be used for aggregate removal.


	William Evans is technical service specialist at Tosoh Bioscience, 800.366.4875, techservice.tbl@tosoh.com.

 When referring to this article, please cite it as W. Evans, “Removing Aggregates in Monoclonal Antibody Purification,” 

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Several chromatographic resins are available for downstream purification.