Industry
Pharma
Company Size
~72 000 employees
Sites
New Jersey (USA) + Global
The number of biopharmaceuticals sold on the market has quadrupled since 2006 and this trend is expected to continue. Biopharmaceuticals are structurally complex, which poses several challenges in drug development as they require complicated analytical assays and multistep purifications. To overcome these challenges, companies are developing strategies to simplify method development for large molecules.
A Strategy to Simplify Method Development
Luca Losacco and his teammates on the method screening purification group at Merck use the LC Simulator module from ACD/Labs (part of the Method Selection Suite) to manage their screening process. They have introduced a dual strategy integrating computer-assisted analysis into screening platforms with in silico modeling to simply method development. Using the software, they analyze a comprehensive set of conditions to determine the best combinations of columns, stationary phases, and chromatographic techniques most suited for the sample.
Losacco and his team combine automated method development strategies for various separation techniques including ion exchange chromatography (IEX) and chiral ultra-high performance liquid chromatography (UHPLC) with in silico modeling to accelerate their method development process for small and large molecules. The strategy implemented by the team has proved to be simple, practical, and versatile as they have been able to successfully apply it to these different techniques.
“We’re starting to rely more on the utilization of modeling tools to dramatically reduce the number of experiments, to quickly identify the best set of combinations that give us the final separation, and the baseline resolution of all our peaks and components.” – Luca Losacco, Postdoctoral Fellow, Associate Scientist, Merck & Co.
Goals
Combined Automated Screening and In Silico Modeling Workflow
Save Time
Minimal Manual Intervention
Minimize the Number of Experiments
Streamlining Purification of Biopharmaceuticals with Minimal Manual Intervention
As part of Merck’s workflow, and to reduce laborious IEX method development, the team conducts multicolumn IEX screening in conjunction with computer-assisted simulation. With the aim to develop methods and purify biopharmaceuticals efficiently. This practical workflow successfully separates, analyzes, and purifies nucleotides, peptides, and proteins.
- 1 12 different columns & 24 mobile phases were sequentially operated for cation and anion exchange modes (CEX and AEX)
- 2 3 different classes of multicomponent mixtures (nucleotides, peptides, and proteins) commonly encountered in biopharmaceuticals were screened and tested under CEX and AEX conditions, through all columns and different mobile phase pHs
- 3 The appropriate factors for elution profiles (gradient, pH, and different types of concentrations of salts) were determined
- 4 Automation allowed the team to perform 96 injections (for each mixture) overnight, with minimal manual intervention, giving optimal and robust conditions with the best separation
“The IEX screening workflow has facilitated rapid identification of the best mobile phases pH and column combination for baseline separation overnight, without manual intervention”.1
To save time and effort in method optimization, the team uses the LC Simulator module to perform in silico modeling. Using the software, they conduct multifactorial optimization of the IEX method, minimizing the number of experiments, and analyzing the impact of multiple chromatographic parameters. The LC Simulator module allows visualization of a resolution map cube modeling pH, time, and temperature, showing the condition(s) of maximal resolution and minimal analysis time. Experimental verification shows an excellent correlation between the simulated and experimental chromatographs, with a difference in retention time of less than 0.5%. The workflow has successfully enabled more efficient analysis and purification of different nucleotides, proteins, and peptides.
Automating Screening to Reduce the Time Spent Developing Ultrafast Chiral Separation Methods
Chiral separations are some of the most challenging for chromatographers, requiring extensive screening and a comprehensive evaluation of all possible column and mobile phase combinations. There is also a critical need for these separation assays to be delivered quickly. The versatility of the workflow means the team can also apply it to develop ultrafast chiral separation methods.
“This chiral UHPLC screening platform not only provides the possibility to quickly identify the best analytical conditions needed to efficiently separate enantiomers, but it also lays the foundation for streamlined development of fast-paced enantiopurity assays”.2
- 1 A multicolumn UHPLC screening workflow is automated to run overnight (taking approx. 6 hours per compound), operating under various conditions with minimal manual intervention
- 2 A comprehensive set of conditions was obtained
- 3 To verify the workflow, automated screening was then set up for 18 chiral compounds (mostly classic synthetic APIs and amino acids)
- 4 Each racemate was tested on all 14 columns using different combinations of chromatographic conditions. The team was able to screen the experimental conditions in a reduced timeframe, identifying at least one stationary and mobile phase combination for baseline resolution.
Next, the team performs in silico modeling for method optimization. A crucial step in the optimization process for large biomolecules is to ensure that the correct regression retention model is being deployed. The team uses the LC Simulator module to generate a second-degree regression model and creates a resolution map using 9 combinations of the gradient slope and column temperatures for the enantiomeric mixtures. From this, optimal experimental conditions are obtained. The results show there was an excellent correlation of retention time between experimental and simulated experiments, with a difference of less than 0.5%. The workflow has helped to reduce the amount of time spent developing enantiopurity assays.
“We have introduced an automated enantioselective UHPLC screening workflow that dramatically reduces the time spent developing enantiopurity essays.”2
Impact
Benefits Being Realized from the Workflow
Efficiency
The simple and practical workflow uses in silico modeling to optimize method parameters, minimizing the need for manual intervention and accelerating method development
Time Savings
Automation of the screening workflow and modeling has reduced the time taken to screen the experimental conditions
Sustainability
In silico multifactorial optimization minimizes the number of experiments conducted, saving time and resources
Robust Methods
Excellent correlation of retention time between experimental and simulated experiments (differences of less than 0.5%)
Successful Development of Robust Methods
Losacco and his team have shown how method development can be simplified and streamlined by efficiently integrating automated screening platforms with computer-assisted in silico modeling in different applications.
The workflow has helped to reduce the time spent developing enantiopurity assays and successfully enabled more efficient analysis and purification of different nucleotides, proteins, and peptides. In both applications, the results show an excellent correlation of retention time between experimental and simulated experiments.
Automation of screening platforms has significantly reduced the amount of manual intervention required while also saving time. In silico modeling with the LC Simulator module has proven to be an effective tool to conduct fewer experiments and identify optimal conditions, improving the overall screening outcome, and generating robust LC methods faster.
“Using in silico modeling, we were able to optimize three parameters at the same time, in a single morning. Normally, with other approaches, this takes days. We were able to reduce the entire method development from days to just half a day.” – Luca Losacco
Download the Case Study
Learn more about In Silico Modeling and Method Optimization
References
- G.L. Losacco, M.B. Hicks, J.O. DaSilva, H. Wang, M. Potapenko, F.-R. Tsay, I.A.H. Ahmad, I. Mangion, D. Guillarme, and E.L. Regalado. (2022). Automated ion exchange chromatography screening combined with in silico multifactorial simulation for efficient method development and purification of biopharmaceutical targets – analytical and bioanalytical chemistry. Analytical and Bioanalytical Chemistry, 414, 3581-3591. https://link.springer.com/article/10.1007/s00216-022-03982-z
- G.L. Losacco, H. Wang, I.A. Haidar Ahmad, J. DaSilva, A.A. Makarov, I. Mangion, F. Gasparrini, M. Lämmerhofer, D.W. Armstrong, E.L. Regalado. (2023). Enantioselective UHPLC screening combined with – ACS publications. Analytical Chemistry, 94(3), 1804-1812. https://pubs.acs.org/doi/10.1021/acs.analchem.1c04585