Hydrophilic interaction chromatography (HILIC) analysis of monoclonal antibodies (mAbs) using innovative combination methodology

Berlin: – Molnár-Institute for Applied Chromatography has been able to apply its co-pioneered innovative methodology to speed up the chromatographic characterization of anti-COVID protein therapeutics using a combination of automated, systematic modeling and ultra-short liquid chromatography (LC) columns to hydrophilic interaction chromatographic (HILIC) separations.

While the study has yielded highly encouraging results across a range of relevant chromatographic techniques in protein analysis, HILIC is considered particularly significant in the context of characterizing sugar moieties of monoclonal antibodies (mAbs).

COVID-19 Imperative

The study, ‘Expediting the chromatographic analysis of COVID-19 antibody therapeutics with ultra-short columns, retention modeling and automated method development’ is published in the Journal of Pharmaceutical and Biomedical Analysis [1].

The research, led by Bastiaan Duivelshof from the University of Geneva, also involved Molnár-Institute application specialist Arnold Zöldhegyi and Waters Corporation scientists Szabolcs Fekete and Matthew Lauber. It was launched as a joint response to the need exposed by the COVID-19 pandemic to accelerate emergency use authorization (EUA) of new therapeutics and vaccines that included several monoclonal antibodies (mAbs).

To overcome the problems, including lengthy traditional turnaround times for LC characterizations and urgent need for scientific data on drug candidates to be collated in parallel with drug development, that typically slow EUA submissions, the team set out to establish a faster method for chromatographic characterization of protein therapeutics.

Software-assisted workflow

The presented study combined an automated, analytical quality-by-design (AQbD) workflow along with the use of innovative ultrashort LC column formats to accelerate optimized high-throughput separations, using two COVID-19-related monoclonal antibodies (mAbs), casirivimab and imdevimab as their focus for study.

The study discovered, that using this new, synergistic approach, both mAb-substances could be characterized by each analytical methods in as little as a few minutes run time, making it feasible to run a full suite of four LC profiling techniques —reversed phase (RP), ion-exchange (IEX), HILIC and size-exclusion chromatography (SEC)— within a one-hour turnaround time.

Computerized modeling using Molnár-Institute’s DryLab® software platform played a key role in speeding up the entire analytical process by reducing the number of wet-lab experiments and quickly contextualizing complex chromatographic data, using retention modeling in its Design Space facility for quick visual comparison of underlying separation systems [2].

The other main innovation used in the study was the generic modeling strategy taking account of the propensity of large proteins to be extremely sensitive to the mobile phase composition, becoming eluted in many types of separations with only a very minor change in eluent strength, referred to as the “on-off” adsorption phenomenon or “bind and elute” retention mechanism. This effect is getting even more pronounced in HILIC: only a few %water determines whether the solute molecules are fully retained or released from the chromatographic column.

Another consequence of “on-off” elution behavior is, that only the first segments of a column will actively take part in peak retention and separation, allowing the use of short and ultrashort columns (from few mm to 2 cm in length) to speed up separations without compromising separation efficiency.

HILIC analysis of casirivimab and imdevimab subunits

While a range of ultrahigh-pressure liquid chromatography (UHPLC) separation techniques are well-suited to biopharmaceutical analysis of mAb purity and identity control measurements, reversed phase (RP) and hydrophilic interaction chromatography (HILIC) applications are paramount, as these techniques can be readily coupled with mass-spectrometry and thereby provide in-depth characterization data [3].

The study notes, that HILIC is a very efficient tool for the analysis of mAbs glycan heterogeneity, especially when put to practice at the subunit level.

“Compared to RPLC, HILIC can often separate additional peaks, and the sFc fragments of mAbs normally elute in multiple peaks, due to the separation of their glycovariants. In many cases, the elution order of the fragment species is reversed, compared to RPLC. This is not strictly predictable, though, given the complex retention mechanisms in HILIC, MS based identification of peaks from HILIC subunit separations thus plays a key role in confirming peak identifications” the authors observe.

HILIC-analysis was performed using the Waters ACQUITY™ UPLC™ system and a glycoprotein BEH™ Amide (2.1 mm × 50 mm, 1.7 μm, 300 Å) column. Direct communication and full control between the modeling software and the Empower instrument controlling chromatographic data system (CDS) was established with the all-new Automation Module of DryLab® 4.4 Software.

For the more sensitive HILIC-separations, the team used both a stronger additive in the mobile phase 0.1 % trifluoroacetic acid (TFA) in acetonitrile (ACN) for mobile phase A and 0.1 % TFA in water for mobile phase B as well as a further dilution (4 parts digest to 1 part 0.1 % TFA acidified ACN) applied to mobile phase B to avoid peak distortion or breakthrough effects upon injection. Intact mAb analysis was performed by dilution with water to 1 mg/mL prior to injection.

Accurate modeling of the underlying separation system was performed by performing systematic Design-of-Experiments (DoE), including changes of relevant method parameters: Gradient time was set to tG1 = 4 and tG2 = 12 min (15–35 %B gradient), and column temperature to T1 = 65° and T2 = 90°C.

HILIC modeling results

After performing the model input runs, HILIC separations were also optimized using the 2-dimensional critical resolution map. The model showed consistency in peak areas, suggesting appropriate recovery can already be achieved at 65^◦C, selected for optimal method, since selectivity and resolution were maintained across broad ranges of the modeling map. When further optimizing gradient steepness and initial- and final mobile phase compositions, the team found, that a linear gradient of 26 – 34 %B in five minutes at flow rate (F) of 0.7 mL/min resulted in appropriate separation of both samples. Finally, an initial short steep gradient ramp was added—prior to the analytical gradient—to avoid potential breakthrough effects and peak distortion, that can occur with larger injection volumes and more concentrated samples.

This separation was also used to test the application of a prototype, ultra-short, HILIC column hardware designs.

Achieving a HILIC analysis of antibody-subunits on a 2.1 × 50 mm column within 6 min represented a sizeable improvement in method throughput, comparing with 10–20 min run times for mAb subunits analyzed using previous methods on 2.1 x 150 mm HILIC columns.

Speeding-up the comprehensive characterization of mAbs

The team identified practical benefits of combining retention modeling and automated experiments with use of short protein columns for mAb analysis.

“Retention modeling enables the in-silico optimization of mAb (or any protein) separations within a few minutes. The automation module of DryLab® software significantly simplifies manual work and thus speeds up the method development procedure” the study concludes.

It suggests, that the whole process to develop and optimize 4 chromatographic methods (also including RPLC, IEX, and SEC) for two mAb samples might require only two days of experimental work, compared with at least a week using current methodology. By using both short and ultrashort protein columns (15–20 mm long for RPLC and IEX), analysis times in the range of 1–2 min were obtained, marking a significant time savings in comparison to 10–30 min long traditional methods.

“Overall, we have shown that conventional chromatographic techniques can quickly be transferred in silico to short column, higher throughput parameters and to low dispersion chromatographic systems without the need for further experiments. These approaches might be valuable for turning around scientific data on drug candidates during critical times when the preparation of filing information must be accelerated” the study concludes.

References

1. ‌B. Duivelshof, A. Zöldhegyi, D. Guillarme, M. Lauber, Sz. Fekete (2022). Expediting the chromatographic analysis of COVID-19 antibody therapeutics with ultra-short columns, retention modeling and automated method development. Journal of Pharmaceutical and Biomedical Analysis, 221, 15039 doi: 10.1016/j.jpba.2022.115039.
2. J. den Uijl, P. J. Schoenmakers, B. W. Pirok, M. R. Bommel (2020). Recent applications of retention modelling in liquid chromatography. Journal of Separation Science, 44(1), 88–114, doi:10.1002/jssc.202000905.
3. D’Atri, A. Goyon, B. Bobály, A. Beck, Sz. Fekete, D. Guillarme, Protocols for the analytical characterization of therapeutic monoclonal antibodies. III – denaturing chromatographic techniques hyphenated to mass spectrometry, J. Chromatogr. B, 1096 (2018) 95–106, https://doi.org/10.1016/j.jchromb.2018.08.013.

Founded in 1981, the Molnár-Institute develops DryLab®4, a software for UHPLC modeling for a world-wide market. Its powerful modules gradient editor, peak tracking, automation, robustness and Design Space Comparison allow the most sophisticated method development as required across modern pharma industries. Analytical scientists use DryLab®4 to understand chromatographic interactions, to reduce analysis time, to increase method robustness, and to conform to AQbD-principles, according to the recently published ICH Q14-regulatory framework.

The Molnár-Institute is a registered partner to the US-FDA, CDC and many other regulatory bodies. DryLab®4 has pioneered AQbD long before regulatory agencies across the world encouraged such submissions. Widely implemented by thought leaders, the software contributes substantially to the paradigm shift towards a science and risk driven perspective on HPLC Quality Control and Quality Assurance in pharmaceutical analysis.

Further information at: http://www.molnár-institute.com/

Resources

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