Karol Lacki

High‐Throughput Process Development: Determination of Dynamic Binding Capacity Using Microtiter Filter Plates Filled with Chromatography Resin

Steadily increasing demand for more efficient and more affordable biomolecule‐based therapies put a significant burden on biopharma companies to reduce the cost of R&D activities associated with introduction of a new drug to the market. Reducing the time required to develop a purification process would be one option to address the high cost issue. The reduction in time can be accomplished if more efficient methods/tools are available for process development work, including high‐throughput techniques. This paper addresses the transitions from traditional column‐based process development to a modern high‐throughput approach utilizing microtiter filter plates filled with a well‐defined volume of chromatography resin. The approach is based on implementing the well‐known batch uptake principle into microtiter plate geometry. Two variants of the proposed approach, allowing for either qualitative or quantitative estimation of dynamic binding capacity as a function of residence time, are described. Examples of quantitative estimation of dynamic binding capacities of human polyclonal IgG on MabSelect SuRe and of qualitative estimation of dynamic binding capacity of amyloglucosidase on a prototype of Capto DEAE weak ion exchanger are given. The proposed high‐throughput method for determination of dynamic binding capacity significantly reduces time and sample consumption as compared to a traditional method utilizing packed chromatography columns without sacrificing the accuracy of data obtained.

High-throughput techniques to evaluate the effect of ligand density for impurity separations with multimodal cation exchange resins

Scale‐down, high‐throughput screening techniques are well on their way to becoming a commodity in downstream bioprocess development, especially for the rapid development of chromatography process steps. This work used both resin slurry plate and miniature column high‐throughput screening methodologies to identify the best resin properties for mAb separations utilizing a multimodal chromatography ligand interaction. A ligand with both cation exchange and hydrophobic interaction properties was studied at several ligand densities and compared to a commercially available multimodal resin with a larger particle size at high ligand density. The resins were screened with mAbs containing distinct process impurities (aggregates and a hydrophobic variant), and optimized conditions provided more than a log of clearance of both types of impurities for the different resins screened. These studies reveal that while a smaller particle size is generally preferable, optimal ligand densities can be different depending on the properties of both the mAb and impurity studied.

A Methodology for the Comparative Evaluation of Alternative Bioseparation Technologies

Advances in upstream technologies and growing commercial demand have led to cell culture processes of ever larger volumes and expressing at higher product titers. This has increased the burden on downstream processing. Concerns regarding the capacity limitations of packed‐bed chromatography have led process engineers to begin investigating new bioseparation techniques that may be considered as “alternatives” to chromatography, and which could potentially offer higher processing capacities but at a lower cost. With the wide range of alternatives, which are currently available, each with their own strengths and inherent limitations, coupled with the time pressures associated with process development, the challenge for process engineers is to determine which technologies are most worth investigating. This article presents a methodology based on a multiattribute decision making (MADM) analysis approach, utilizing both quantitative and qualitative data, which can be used to determine the “industrial attractiveness” of bioseparation technologies, accounting for trade‐offs between their strengths and weaknesses. By including packed‐bed chromatography in the analysis as a reference point, it was possible to determine the alternatives, which show the most promise for use in large‐scale manufacturing processes. The results of this analysis show that although the majority of alternative techniques offer certain advantages over conventional packed‐bed chromatography, their attractiveness overall means that currently none of these technologies may be considered as viable alternatives to chromatography. The methodology introduced in this study may be used to gain significant quantitative insight as to the key areas in which improvements are required for each technique, and thus may be used as a tool to aid in further technological development.

Definition and dynamic control of a continuous chromatography process independent of cell culture titer and impurities

Advances in cell culture technology have enabled the production of antibody titers upwards of 30g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31g/L. Initial experimentation showed a challenge with determining a difference in change in UV280nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beers Law given a known absorbance of the media background and impurities or experimentally determined using various UV280nm cell pathlengths. Based on these results, a 0.35mm pathlength at UV280nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations.

High-throughput process development: Chromatography media volume definition

Prediction of chromatographic separations in liter scale with the help of data gathered using a microliter format is challenging, as both scale effects and differences in the formats used (plates vs. packed minicolumns) need to be addressed. The use of microplates in high‐throughput process development (HTPD) studies has become a routine. While this method is typically used for investigating chromatography conditions such as salt and pH, it can also be used for screening of different chromatography media (resins). In this study, the influence of the physicochemical properties of various media on preparation of medium‐containing microplates is discussed, and examples are presented that show how these properties can influence the conclusions drawn in benchmarking studies done using this HTPD format. The results indicate that differences in chromatographic media volume between microliter sampling techniques and packed chromatography columns do exist. Regardless of the chosen chromatography media, sampling technique or volume definition method, the simplest way to address this issue is by calibrating the microliter scale with packed‐column volumes in appropriate dry weight experiments.