Aggregation of Monoclonal Antibody Products: Formation and Removal - Aggregate formation is influenced by multiple aspects of the bioproduction process but can be mitigated by good process design and

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Aggregation of Monoclonal Antibody Products: Formation and Removal
Aggregate formation is influenced by multiple aspects of the bioproduction process but can be mitigated by good process design and control.


BioPharm International
Volume 26, Issue 3, pp. 40-45

APPROACHES FOR REDUCTION IN AGGREGATES

Perhaps the most effective way to control aggregate levels is to avoid their formation in the first place. Thus, appropriate design of the process to avoid aggregate formation at the various process unit operations mentioned above would be most desirable. If aggregate formation cannot be completely averted, appropriate controls can be put in place to ensure that aggregate levels are low and that the process performance is consistent. Based on process development of mAb products in the past decade, it has been observed that aggregate formation cannot be completely avoided and hence, means of aggregate removal are needed. The following section briefly discusses some of the most commonly used process steps for aggregate removal.

Ion-exchange chromatography

This step is the most common unit operation used for purification of mAb products. CEX is often used in a bind/elute mode as an intermediate polishing step because therapeutic mAbs are highly basic (i.e., pI >8). CEX has been optimized to remove aggregates, even though the aggregates are composed of the same polypeptide chains as the monomeric product. CEX can perform this function because the molecules in the aggregate are partially denatured and unfolded, thereby exposing different amino acids on the surface of the aggregate. The surface charge distribution may be different for denatured aggregates than for the native monomer, enabling an effective separation (27).

Charge difference between antibody products and aggregates can also be exploited by using AEX for impurity removal. The difference between the net charge of the antibody product and the aggregates enables AEX to be used in a flow-through mode (4).

Hydrophobic interaction chromatography (HIC)

This chromatography involves use of resin with immobilized hydrophobic groups for binding proteins in the feedstream on the basis of their hydrophobicity. HIC steps are often developed with the primary objective of reducing aggregate levels. High-molecular-weight aggregates of mAbs bind more tightly to the media than monomeric mAbs because of the exposed hydrophobic groups in the denatured chains of aggregate (28).

Multimodal or mixed-mode chromatography

Recently, mixed-mode anion exchange ligands with enhanced binding strength for aggregates through the addition of hydrophobic functionality have been brought to market. These ligands can increase the range of ionic strength and pH under which significant separation can occur (29). Mixed-mode resins based on hydroxyapatite, made from calcium phosphate, have both positive and negative charges and interact with proteins through a combi- nation of electrostatic interactions and coordination complex formation. Traditionally, hydroxyapatite has been used for separa- tion of protein therapeutics from host and media proteins, aggregates, DNA and Protein A, all of which tend to bind more tightly. More recently, its effectiveness for separation of aggregates has been demonstrated (4, 30).

Aqueous two-phase systems (ATPS)

ATPS has been investigated as an alternative to process chromatography for the purification of many proteins and enzymes because of its cost effectiveness, high capacity, biocompatibility, and scale-up potential (31, 32). Several ATPS have been proposed for production of mAb products, including polyethylene glycol (PEG)-phosphate, PEG-citrate, and PEG-dextran (33, 34). ATPS has been shown to be effective for reduction of low-molecular-weight and high-molecular-weight mAb aggregates (34). For example, an ATPS system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product-related impurities (i.e., aggregates and low-molecular-weight product fragments) from 40% to less than 0.5% while achieving 95% product recovery. Issues that were related to use of ATPS included handling and disposal of large quantities of raw materials needed for the process, and the limited understanding about the complex interactions between the different components in the system.

Membrane chromatography

Membrane chromatography is another emerging alternative to traditional packed-bed chromatography. HIC-based membrane adsorbers have been shown to be effective at efficient removal of dimers and higher molecular weight aggregates when used as a polishing step in a mAb purification process (35). With the high throughput that membrane adsorbers provide, this could be the technique of choice in some cases.

SUMMARY

Aggregation is a critical quality attribute of any protein-based therapeutic because of its potential impact on immunogenicity and thus, safety of the product. Appropriate process design and control can result in consistent and acceptable product quality.

Anurag S. Rathore, PhD,* (pictured) is a consultant at Biotech CMC Issues and a professor in the department of chemical engineering, Varsha Joshi is a postdoctoral associate, and Nitin Yadav is an intern, all at the Indian Institute of Technology, Dehli. Rathore is also a member of BioPharm International's Editorial Advisory Board. *To whom correspondence should be addressed,
.


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