Accelerated Purification Process Development for a Complex Antibody Program
Downstream process development for bispecific antibodies can be particularly challenging when the expressed material contains elevated levels of protein fragments and aggregates. These impurities may require carefully designed purification strategies to achieve the desired product quality while maintaining acceptable recovery.
In this case, Protheragen supported the downstream process development of a bispecific antibody program under an aggressive project timeline. The expressed material presented relatively high levels of protein fragments and aggregates, creating additional challenges for impurity clearance and product recovery.
A three-step chromatography process incorporating a mixed-mode chromatography resin was developed to improve impurity removal. Process confirmation demonstrated that the developed downstream process could achieve SEC purity above 98% and nrCE purity above 96%, while maintaining an overall downstream recovery of approximately 73%. The effective recovery of the target molecule during the filtration step could reach 80%.
The Challenge
The project presented two major challenges.
First, the development timeline was highly compressed, requiring an efficient approach to downstream process development and optimization.
Second, the material obtained after expression contained relatively high levels of protein fragments and aggregates. These product-related impurities can be particularly challenging to control during purification because effective impurity removal must be balanced against target molecule recovery.
The downstream process therefore needed to achieve three objectives:
- Effectively remove protein fragments and aggregates;
- Achieve high product purity;
- Maintain an acceptable overall process recovery.
This required a purification strategy that could provide sufficient impurity clearance without introducing excessive product loss.
Our Strategy
- Three-step Chromatography Process Development
To address the complex impurity profile, a three-step chromatography purification process was developed.
The multi-step purification strategy provided multiple opportunities for impurity removal and enabled the downstream process to progressively improve product quality.
Rather than relying on a single purification step to simultaneously address all impurity species, the process was designed as a sequence of chromatography operations with complementary purification functions.
- Mixed-mode Chromatography for Impurity Removal
A mixed-mode chromatography resin was incorporated into the purification process to support the removal of challenging impurities.
Mixed-mode chromatography combines multiple interaction mechanisms and can provide an additional separation mechanism when conventional chromatographic approaches alone may not provide sufficient selectivity.
In this project, the mixed-mode step was used as part of the three-step purification strategy to improve the removal of protein-related impurities, particularly in the context of the elevated fragment and aggregate levels observed after expression.
- Balancing Purity and Recovery
The purification process was optimized not only for impurity removal but also for overall process recovery.
During process confirmation, the developed process was evaluated using both purity and recovery as key performance indicators. This allowed the team to establish a purification process capable of achieving high product quality while retaining a substantial proportion of the target molecule throughout downstream processing.
This balance was particularly important for the project because aggressive impurity removal could otherwise result in unnecessary product loss.
Results
High Purity with Approximately 73% Overall Downstream Recovery
The developed three-step chromatography process successfully addressed the challenging impurity profile of the bispecific antibody program.
During process confirmation, the downstream process achieved:
Overall Downstream Recovery
~73
%
Fig. 1. Schematic representation of the three-step chromatography strategy developed for impurity removal and product recovery.
These results demonstrated that the developed purification process could achieve high levels of product purity while maintaining an overall downstream recovery of approximately 73%.
Key Outcomes
High Product Purity
The optimized downstream process achieved SEC purity above 98% and nrCE purity above 96%.
Effective Impurity Removal
A three-step chromatography strategy incorporating a mixed-mode resin was developed to address the elevated fragment and aggregate levels.
Balanced Process Recovery
The process achieved approximately 73% overall downstream recovery, while effective target molecule recovery during filtration reached 80%.
Project Outcome
The project demonstrated an effective approach to downstream process development for a complex bispecific antibody with elevated protein fragment and aggregate levels.
Under a tight development timeline, Protheragen developed a three-step chromatography purification process incorporating mixed-mode chromatography to address the challenging impurity profile.
Process confirmation showed that the developed process achieved SEC purity above 98% and nrCE purity above 96%, while maintaining an overall downstream recovery of approximately 73%. The effective recovery of the target molecule during filtration reached 80%.
The results demonstrate that a carefully designed multi-step purification strategy can effectively balance impurity clearance, product purity, and process recovery in complex antibody downstream process development.
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