High‐throughput process development of chromatography steps: Advantages and limitations of different formats used

Abstract

In the past, development of a chromatographic separation method has been accomplished by performing a series of experiments using either manual or automated chromatography systems. The screening of a vast experimental space became very expensive because all experiments had to be performed in a serial manner, and the chromatography systems used were designed for relatively large columns and, therefore, the experiments required large sample volumes. To address these issues, high-throughput miniaturized methods employing different operating principles and/or formats have been introduced. Herein, a technical review of the most common high-throughput formats used for the development of chromatographic purification steps is presented. The formats considered include minicolumns, prefilled pipette tips, and microtiter filter plates prefilled with chromatography resins. Advantages and limitations of each format are discussed through the prism of chromatographic theory, engineering principles, and known mass-transfer mechanisms. A roadmap for applicability of the different formats for process development purposes and implementation of a Quality by Design initiative for designing/optimization of chromatography steps is also discussed.

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model

“…In recent years, significant advances have been made in the determination of protein binding as a function of protein concentration and mobile phase composition over broad ranges of conditions both by using analytical tools that allow measurements with very low sample volumes as well as by implementing automated, high throughput screening (HTS) approaches [18][19][20][21]. While these approaches can generate large amounts of data, the quantitative connection between the data and column performance is not always straightforward.…”

Adaptation of the pore diffusion model to describe multi-addition batch uptake high-throughput screening experiments

“…For linear adsorption isotherms, or cases where uptake is expected to be enhanced by homogeneous diffusion of bound protein, the assumption of bound protein remaining in place on the surface between steps may no longer be appropriate. For systems exhibiting very rapid diffusivity, two challenges may be encountered: achieving sufficiently-rapid external mass transfer from the fluid to the particle surface within the microplate environment [18,42] and the intrinsic time scales involved in performing protein addition, incubation, and separation steps on a high-throughput system. If equilibrium is reached before separation may be performed in a given step, only a lower limit on diffusion rates will be extractable from the collected data.…”

“…Further, the incubation time was significantly greater than the dead time between incubation and protein separation and re-addition. Analysis of the Biot number performed by Lacki [42] for mAb uptake in an agitated micro-plate on a macroporous ion exchange resin suggests that external mass transfer is sufficiently-capable of delivering protein to the particle surface (Bi = 10 2 ) based on an effective diffusivity to free solution diffusivity ratio (D e /D 0 ) of 0.1.…”

Automated small‐scale protein purification and analysis for accelerated development of protein therapeutics

“…Specifically for small proteins, it has been reported that velocity‐dependent mass transfer becomes the rate‐limiting step, limiting the ability for miniature columns to be predictive of higher capacity purification schemes. However, it is also noted for preparative applications, velocities typically fall well within the range to be predictive of higher capacity purification schemes . Additional concerns reported to impact the predictive capabilities of miniature columns include geometric factors, including bed volume heights leading to reduced separation capacities, residence times and flow rates, “wall effects” from a smaller inner‐diameter, and nonlinear elution gradients.…”