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A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase
Eduardo J S Bras1, Ruben R G Soares1, Ana M Azevedo2
1Instituto de Engenharia de Sistemas E Computadores-Microsistemas e Nanotecnologias (INESC MN) and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
This study introduces a microfluidic system for rapid screening of antibody purification using aqueous two-phase extraction (ATPE). The method significantly enhances antibody partitioning with LYTAG fusion proteins, improving efficiency for biopharmaceutical production.
Area of Science:
- Biotechnology
- Bioprocess Engineering
- Separation Science
Background:
- Biopharmaceuticals like antibodies require efficient and cost-effective purification.
- Aqueous two-phase extraction (ATPE) is a promising purification method but is challenging to model and optimize.
- Current optimization methods for ATPE are time-consuming and expensive.
Purpose of the Study:
- To develop a rapid screening methodology for optimizing antibody extraction conditions in ATPE.
- To demonstrate the utility of a microfluidic toolbox for high-throughput screening and integrated purification protocols.
- To evaluate the effectiveness of LYTAG fusion proteins as affinity tags for enhancing antibody partitioning in ATPE.
Main Methods:
- A microfluidic channel-based toolbox was designed for rapid screening of up to 8 ATPE conditions simultaneously.
- The system utilizes negative-pressure driven flow and minimal sample volumes (<20μL per phase).
- A second microfluidic structure was developed for integrating multi-step extraction protocols.
Main Results:
- The microfluidic toolbox enabled rapid optimization of antibody partitioning in ATPE.
- LYTAG fusion proteins significantly enhanced antibody partitioning, achieving a partition coefficient (K) of 9.2 with LYTAG-Z.
- This represents a 3.7-fold increase in partitioning compared to conditions without the affinity tag (K=2.5).
Conclusions:
- The microfluidic toolbox offers a miniaturized and versatile approach for rapid optimization of ATPE processes.
- The integration of affinity tags like LYTAG fusion proteins can substantially improve antibody partitioning in ATPE.
- This methodology facilitates efficient purification of high-purity biopharmaceuticals using ATPE.
