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

Abstract

In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths. It was found that on a low dispersion ultrahigh-pressure liquid chromatography instrument, columns between 10 and 20 mm were most effective when made with 2.1 mm i.d. tubing. However, with the same LC instrument, 3 mm i.d. columns as short as ∼5 to 10 mm could be effectively used. In both cases, it has been found to be best to keep injection volumes below 0.6 μL, which presents a potential limit to further decreasing column length, given the current capabilities of autosampler instrumentation. The additional volume of the column hardware outside of the packed bed (extra-bed volume) of very small columns is also a limiting factor to decrease the column length. For columns shorter than 10 mm, columns’ extra-bed volume was seen to make considerable contributions to band broadening. However, the use of ultra-short columns seemed to be a very useful approach for RPLC of large proteins (>25 kDa) and could also work well for ∼12 kDa as the lowest limit of molecular mass. In summary, a renewed interest in the use of ultra-short columns is warranted, and additional method development will be to the benefit of the biopharmaceutical industry as there is an ever-increasing demand for faster, yet accurate assays (e.g., high-throughput screening) of proteins.

Denaturing and Native Mass Spectrometric Analytics for Biotherapeutic Drug Discovery Research: Historical, Current, and Future Personal Perspectives

“…280 Applying this acoustic sample introduction technology to Mw screening of early protein engineering panels 29 could clearly further throughput gains over those already realized using SPE-MS-based systems 170,253 and high-throughput rpLC−MS methods. 281,282 Nucleic Acid Based Therapeutics. There are therapeutics that are not antibody related.…”

“…Acoustic MS has developed rapidly since 2016 when originally described for substrate-to-product label free detection; identifying inhibitors of a human histone deacetylase from a two-million compound library to current applications demonstrating a sampling rate of 6 Hz . Applying this acoustic sample introduction technology to Mw screening of early protein engineering panels could clearly further throughput gains over those already realized using SPE-MS-based systems , and high-throughput rpLC–MS methods. , …”

Expediting the chromatographic analysis of COVID-19 antibody therapeutics with ultra-short columns, retention modeling and automated method development

“…On ultra-short columns, very small column peak variances and short analysis times (e.g. ≤ 2 min) are expected, therefore extra-column band broadening and gradient delay of the system should be minimized to obtain the full benefit of ultra-short columns [24] . First, the optimal conditions found on 2.1 ×50 mm columns were geometrically transferred to 2.1 ×20 mm (RPLC) and 2.1 ×15 mm (IEX) columns.…”

“…In this regard, the linear solvent strength (LSS) model is frequently applied to describe protein retention. A consequence of “on-off” elution behavior is that only the first segments of a column will actively take part in peak retention and separation [24] . Therefore, very short columns can be used without comprising the separation efficiency.…”

Monitoring mAb proteoforms in mouse plasma using an automated immunocapture combined with top‐down and middle‐down mass spectrometry

“…Nonetheless, it should be noted that almost all the work published so far is based on the use of a large amount of starting material (a few micrograms), which is not sustainable for the analysis of in vivo biotransformation products. Improvements in stationary phases [49][50][51][52][53][54][55], low-flow chromatography, and alternative front-end separations [56][57][58] have recently opened the way to higher sensitivity studies. For instance, both the qualitative and quantitative top-down and middle-down analysis of biotherapeutics extracted from plasma has been described in the last years [59][60][61][62][63].…”