Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept

Abstract

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columnsin theoryjust broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25–150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 μm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody–drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography

“…A regression line is added, and square root fits to illustrate the difference between measured and expected trends. Figure S2B shows two representative examples on the evolution of peak capacity vs column length at t G = 15 min and at F 1 = 1 mL/min and F 2 = 0.3 mL/min] These experimental results further support the discussion presented in the first part of this series, 15 namely, that column length has less impact on separation efficiency for large molecules compared to small molecules. It is also interesting to notice that with the very short, 2 mm long column, still a relatively high peak capacity can be achieved.…”

“…Half height peak widths ( w 1/2 ) were measured for all conditions and plotted against intrinsic gradient steepness ( b = S ·Δφ· t 0 / t G , where S is the slope of the linear solvent strength-curve and Δφ is the difference between the initial and final mobile phase composition during the gradient). To illustrate the fraction of column length which is occupied by the peak at elution, a measure of relative peak width ( w rel ) was used …”

“…All of these possible benefits motivated us to revisit the use of short columns for the analysis of biopharmaceutical products such as monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). In the first part of this series, the impacts of column length on solute retention, migration speed, and band broadening were studied for compounds possessing various molecular masses (0.2–150 kDa) . A few preliminary experiments were also performed on 5 × 2.1 mm reversed phase (RP) commercially available guard columns to demonstrate the promise of methods based on short columns.…”

“…In the first part of this series, the impacts of column length on solute retention, migration speed, and band broadening were studied for compounds possessing various molecular masses (0.2−150 kDa). 15 A few preliminary experiments were also performed on 5 × 2. demonstrate the promise of methods based on short columns. After systematic optimization, fast, 30 s separations were performed for the reduced ADC sample and mAb fragments.…”

“…To illustrate the fraction of column length which is occupied by the peak at elution, a measure of relative peak width (w rel ) was used. 15 To estimate the occupied column length fraction in practice, a relative peak width (w rel ) can be introduced. It can be written as where peak width is measured in time unit and assuming that the solute is virtually unretained once it starts migrating (travels with the mobile phase velocity, u 0 ).…”