(46h) Strategy to Control Aggregate Levels in an Fc-Fusion Protein Purification Process
AIChE Annual Meeting
2014
2014 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Bioseparations and Downstream Processing
Monday, November 17, 2014 - 10:42am to 11:00am
Fc-fusion proteins often show a propensity for aggregation under conditions typically used for mAb purification, such as low pH for elution from Protein A and viral inactivation. Here we describe modifications made to a process for an Fc fusion protein that forms high levels of aggregate at low pH. Changes were made to reduce exposure to low pH conditions and to remove any aggregate formed during processing. A direct neutralization method was developed to minimize exposure of the Fc-fusion protein to low pH following Protein A elution. The neutralization method enabled the use of platform Protein A elution buffers and controlled aggregate levels below 1%. A DoE approach was used to optimize the Protein A elution and neutralization buffers and to ensure robust pH control and minimize aggregation. Optimization of the step also enabled direct loading of the neutralized Protein A product pool onto the subsequent anion exchange column without additional pH or conductivity adjustment. Low pH viral inactivation was replaced by detergent inactivation with Triton X-100. Hydrophobic interaction chromatography was used as a final polishing step to remove residual aggregates and achieve a product purity of >99% as measured by size exclusion chromatography. The combination of direct Protein A product neutralization, viral clearance by detergent inactivation and aggregate removal by hydrophobic interaction chromatography represents an effective aggregate control strategy for Fc-fusion proteins.