Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules

Abstract

The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiolreactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions.

Molecular Separation by Using Active and Passive Microfluidic chip Designs: A Comprehensive Review

“…They claimed that their results were consistent with those obtained from a microscope or conventional chromatography system. [ 117 ] Afterward, in a 2019 publication, [ 118 ] they designed a microfluidic system composed of three individual chambers packed with different chromatography beads, such as protein A, CM Sepharose, and Q Sepharose, to determine recovery yield and purities quantitatively. Here, an artificial mixture, containing immunoglobulin G (IgG) and BSA, was used to evaluate the quantitative performance of their system.…”

“…Compatible with the use of complex matrices such as cell culture supernatants, this technique can also be integrated with valves and optical sensors. [ 118 ]…”

“…Compatible with the use of complex matrices such as cell culture supernatants, this technique can also be integrated with valves and optical sensors. [118] Landers et al [119] introduced a microfluidic device based on the pH-responsive charge of chitosan for low-volume DNA extraction from complex biological fluids, such as human blood. The whole blood DNA extraction efficiency of microchip was found to be 75 ± 4%.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…The study report suggested that this strategy can potentially be utilized as a predictive analytical tool in the context of purification of mAb (Pinto I. F. et al, 2019). The benefits of a HT chromatography system include its availability to predict design space for dynamic binding capacity (DBC), collect data for the prediction of elution behavior, and allow significant investigation using DoE (McDonald et al, 2016).…”

Simultaneous Recovery of Bovine Serum Albumin and Bovine Immunoglobulin G with Dual-Ligand Hydrophobic Charge-Induction Chromatography

“…Chromatography can be categorized based on the type of ligand employed, such as ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and mixed-mode chromatography. − The performance of chromatography is greatly affected by the type and operating mode of ligands. , Therefore, the discovery and optimization of ligands have always been a major focus of research in chromatography development. − However, the rate of discovery of new effective ligands has recently decreased. Fortunately, some studies have determined promising results by optimizing ligand operation modes. − For example, the grafting of ligands to create a three-dimensional distribution has been shown to increase chromatographic adsorption capacity. ,− Sun and colleagues , conducted a systematic study on an anion exchanger of polyethylenimine (PEI)-grafted Sepharose FF and demonstrated enhanced adsorption capacity, effective pore diffusion, and an upgraded adsorption rate of bovine serum albumin (BSA).…”