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.
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 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.
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, firstname.lastname@example.org