Aggregate Removal
Aggregates are important product-related impurities in monoclonal antibody manufacturing and require effective control during downstream purification. In this case, the antibody feed material exhibited a relatively high aggregate level, while the existing cation-exchange chromatography step provided limited aggregate removal and was associated with self-association during purification.
A customized hydrophobic interaction chromatography (HIC) process operated in flow-through mode was therefore evaluated as an alternative approach for aggregate removal. The optimized process improved aggregate clearance while achieving higher chromatography recovery.
Challenge
The initial purification process presented several challenges:
- High aggregate content in the antibody feed material
- Limited aggregate removal by cation-exchange chromatography
- Self-association during the existing chromatography step
- Insufficient product purity
- Relatively low chromatography recovery of approximately 70%
These limitations created a need for an alternative purification strategy capable of improving aggregate clearance while maintaining high product recovery.
Purification Strategy
- Hydrophobic Resin Screening
Hydrophobic interaction chromatography was investigated as an alternative separation mechanism based on its potential to differentiate antibody monomers from higher-order aggregates.
Suitable hydrophobic chromatography media were screened to identify a resin with effective aggregate removal performance.
- Flow-Through Chromatography Optimization
Following resin selection, the HIC conditions were optimized in flow-through mode. Process conditions were adjusted to promote aggregate retention while allowing the target antibody to pass through the column.
The loading conditions were also evaluated to determine whether effective aggregate removal could be maintained at a higher loading capacity than the original cation-exchange step.
The optimized process was assessed using key purification performance indicators, including:
- Aggregate clearance
- SEC purity
- Chromatography recovery
- Loading capacity
- Process practicality
Results
The optimized flow-through HIC process provided effective aggregate removal while improving chromatography recovery.
≥99%
SEC Purity
The optimized process effectively reduced aggregates, resulting in a purified antibody with SEC purity above 99%.
~70%
→
90%
Chromatography Recovery
Chromatography recovery increased from approximately 70% to up to 90% following process optimization.
Figure 1. Aggregate removal performance following optimization of the flow-through hydrophobic chromatography process.
Effective aggregate removal was maintained under loading conditions higher than those used for the original cation-exchange chromatography process, demonstrating improved process efficiency.
Key Results
| Parameter |
Initial Process |
Optimized Process |
| Aggregate Removal |
Limited |
Effective |
| SEC Purity |
Below target |
>99% |
| Chromatography Recovery |
~70% |
Up to 90% |
| Loading Capacity |
Lower |
Higher |
| Chromatography Mode |
Cation Exchange |
Flow-Through HIC |
Project Outcome
The case demonstrates that flow-through hydrophobic interaction chromatography can provide an effective alternative for aggregate control in monoclonal antibody purification when conventional cation-exchange chromatography does not provide sufficient separation.
Through hydrophobic resin screening and process condition optimization, the resulting purification process achieved SEC purity above 99% and chromatography recovery of up to 90%. The process also demonstrated effective aggregate removal at higher loading conditions, providing practical and economic advantages for manufacturing.
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