Robust screening cascade process for accelerating innovation in biocatalysis
Several emerging strategies leveraged by automated labs, aim to improve discovery, efficiency and reliability in designing chemical routes.
High throughput screening (HTS) and experimentation (HTE) are techniques which allow the execution of large numbers of experiments to be conducted in parallel while requiring less effort per experiment when compared to conventional means of experimentation. A reiterative process of design, execution, data analysis, and hit identification enables rapid exploration of conditions for reaction discovery and optimization.
The purpose of automated, parallelized experiments is not to reduce the number of experiments conducted, but rather to significantly increase their volume and speed in order to gain full awareness on the targeted (bio)transformation. These high-throughput techniques originated in biology in the 1950s1 and have since become standard practice in that field. However, high-throughput experimentation (HTE) in chemistry is still less advanced. Despite some hurdles, such as the use of volatile organic solvents, which can cause issues like material incompatibility and solvent evaporation, the past decade has seen considerable progress in adapting equipment and methods to better suit the needs of synthetic chemistry2.
Biocatalytic Process Implementation
In the objective of implementing an innovative biocatalytic process for manufacturing chemical products, below, we present our strategic workflow customized to deliver biosolutions. This includes delivering high-quality results with increased efficiency, generating robust data across a broad chemical space, and ensuring process development through our proven screening cascade methodology. At the cornerstone of this workflow stands the critical role of fit-for-purpose analytical methods to ensure reliability and speed through the different stages.
Simplified and standardized workflow from discovery to enzymatic process development
Typically, for enzyme discovery or enzyme improvement, large set of experiments may be required. At SEQENS, our approach is first to navigate through our genomic data collection by conducting bioprospection with our computational tools and proceed rational analysis. After selecting the panel of potential candidates, experimental evaluation is required. The key to success is to identify where automation brings value.
Primary Screening – enzyme hit(s) identification
High-throughput screening (HTS) technique enables the rapid testing of numerous enzymes to identify a pool of best candidates for further development. Large set of data generation must be obtained rapidly and cost effectively. By automating and miniaturizing assays, HTS is a hit-identification strategy which involves the use of sensitive and miniaturized methods for screening smart to large libraries of enzymes in a practical and efficient manner.
Depending on the purpose of the screening campaign, different attributes can be considered, including enzymatic activity, functional activity, selectivity, and specificity. Beyond the physical & chemical nature of substrate/product, each attribute will require the development of a compatible HTS assay and hit detection technology to reach the expectations.
Modern HTS is a highly multidisciplinary approach, and the success of screening programs relies heavily on experiences in automation, analytics, biology, chemistry, data science, chemo- and bio-informatics and operational efficiencies to ensure identification of the most promising hit series.
Secondary Screening – confirmation & validation
The purpose of this second program is to select the most robust enzyme candidates by challenging some key parameters, such as pH, substrate concentration, temperature.
Once potential active enzymes are identified in the primary screening, they undergo hit confirmation through methods such as confirmatory testing and secondary assays. The success of identifying true hits depends heavily on the quality of the assay and the reliability of the automated systems used. Since hits in HTS are often based on single-concentration assays, further evaluation may be required to confirm their true activity. This begins with confirmatory screening under the same conditions to ensure reproducibility, followed by additional tests to assess the best candidates. These steps are vital to reduce false results and ensure that only the most promising enzymes move forward in the development process.
Once the best hits are identified through HTS, the next challenge is to optimize their activity under process-relevant conditions. This is where HTE becomes essential.
Tertiary Screening – optimizing experimental conditions viable/suitable for process development
HTE is a rational, hypothesis-driven extension of traditional experimentation, enabling rigorous exploration of every combination of experimental parameters. This technique uses microscale experiments and fast analytical techniques to quickly generate results. The true power of HTE lies in its ability to challenge complexity and to test in parallel multiple parameters, such as running Design of Experiments (DoE) programs. It allows for a more detailed understanding and control of chemistry by testing a large array of experimental conditions and examining how reaction components affect the outcome. Technologies such as automated liquid and solid dispensing allow chemists to focus on more challenging tasks than repetitive and labor-intensive experimentation, while providing high reproducibility and evading the risk for human error in setting up large arrays of experiments.
Much effort has already paved the way to show the great potential in tackling complex synthetic chemistry problems3 by enabling the rapid execution of large, rationally designed experiments for multidimensional hypothesis testing and extensive data collection.
Within HTS or HTE use, large datasets need to be analyzed. Implementing standardized protocols and data formats, helping to simplify the operations and process at all stages of this cascade methodology. Overall, this lead to experimentation acceleration and reduce costs.
Fit-for-purpose analytical methods
For chemo-enzymatic process implementation and development, a significant number of datasets is required as early as possible to de-risk the outcomes through the different stages. Consequently, developing and optimizing assays for high throughput series is essential to ensure accuracy, reproducibility, and minimal interference, which helps reduce false positives and negatives. Assays must be both robust and sensitive. The focus is generally on maximizing signal clarity, minimizing variability, estimating statistical noise, and setting thresholds to distinguish active enzymes from inactive ones.
The preferred solution is to have access to analytical methods that include speed, universality, sensitivity, and accuracy. Still, one must strike a balance between the required analysis speed for a given application and the quality or level of information of the generated data, respectively.
For detection many approaches rely on spectroscopic methods like absorbance, luminescence, or fluorescence. While UV absorbance can detect many compounds, fluorescence offers higher sensitivity. Custom-designed colorimetric or fluorogenic probes, including “turn-on” probes that increase signal proportionally with product formation, are alternative strategies for improved detection.
Fast analytical techniques are also generally associated with minimal workup for generating results quickly, including chromatographic methods, such as HPLC and UPLC for the determination of selectivity, conversion or yield. UV detection is generally applicable, and MS analysis is a powerful tool for giving insights on structural properties. For compounds without chromophores, fast GC analysis or HPLC CAD can be used.
At SEQENS, we develop high throughput HPLC/MS methods for HTS and HTE programs.
In the following example, we have identified and optimized an enzyme ERED within one month in order to implement a bio-reduction process for an enone derivative.
Concluding Remarks
High-throughput robotic tools associated with analytical instrumentation and techniques, along with statistical modeling (including DoE programs), and computational tools are significantly boosting innovation, research and development productivity. These tools can assist scientists to embrace challenges.
At SEQENS, we have developed a robust screening cascade process to accelerate the identification, confirmation, validation and cost-effective conditions for optimal enzymes performance along with the design of biocatalytic processes.
HTS is used to rapidly identify promising enzyme candidates. These hits are then passed into HTE workflows, where reaction conditions such as solvent, temperature, concentration and pH are systematically optimized. Together, HTS and HTE form a feedback loop that accelerates both discovery and process development.
This approach is also adapted for developing multi-step catalysis synthesis. Within our problem-solving mindset, we have developed customized high throughput analytical methods to fit a broad range of applicability on compounds.
Based on our +25 years’ experience and our multidisciplinary expertise, we design and develop chemo-enzymatic processes with the ability to significantly reduce time to market and manufacture at different scales, including in GMP environment.
At SEQENS, we aim at accelerating the development of competitive and sustainable processes. We have cutting-edge equipment, including:
- an automated screening platform, allowing to run up to 5000 tests in parallel per day. Mainly dedicated to enzyme discovery & optimization
- an HTE platform with the BIG KAHUNA from Unchained Labs coupled to an ORBITRAP MX from ThermoFisher for accelerating innovative proof of concepts in synthetic chemistry and optimization
Our Analytical Park includes:
- 4 Acquity Arc UHPLC devices (microplate samplers) Detection: UV, RI, ELSD, MS – Throughput: up to 1000-1400 analysis per day per equipment
- 3 Agilent FID devices (microplate samplers) Throughput: up to 400-500 analysis per day per equipment
Explore our Biotechnologies offer, we have biosolutions for every application !
References
1. SM Mennen et al. The evolution of high-throughput experimentation in pharmaceutical development and perspectives on the future, Org. Process Res. Dev. 2019, 23, 6, 1213–1242
2. Farkas, E. Microtitrations in Serology and Virology. Curr. Contents/Life Sci. 1992, 30, 10 and references cited therein.
3. Michael Shevlin, Practical High-Throughput Experimentation for Chemists, ACS Medicinal Chemistry Letters 2017 8 (6), 601-607 DOI: 10.1021/acsmedchemlett.7b00165
