High-throughput directed evolution to engineer thermostable therapeutic proteins

Project Information

HTS-STABTHERAPRO

Grant agreement ID: 795539

DOI

10.3030/795539

Project closed

EC signature date 12 April 2018

Start date 1 August 2018

End date 22 October 2020

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 195 454,80

EU contribution

€ 195 454,80

Coordinated by

UNIVERSITY COLLEGE LONDON
United Kingdom

Periodic Reporting for period 1 - HTS-STABTHERAPRO (High-throughput directed evolution to engineer thermostable therapeutic proteins)

Reporting period: 2018-08-01 to 2020-07-31

Summary of the context and overall objectives of the project

The aggregation of protein-therapeutics is a major hindrance to the development of successful drug candidates. The propensity to aggregate can significantly decrease purification yields, shorten shelf-life and increase the risk of anti-drug immune responses. This project will seek to establish and implement a very high-throughput platform for the directed evolution of therapeutic proteins (TPs) towards increased thermal stability and lowered aggregation propensity. TPs have become one of the fastest growing classes of approved pharmaceutical products, and yet pose significant challenges in their manufacture and formulation due to molecular instability and the formation of aggregates. This can lead to adverse immunogenicity and life threatening situations, or limited efficacy.

This is important for society because this action will introduce novel technologies and new ideas, and enable new modalities of biopharmaceuticals to reach the market, including engineered mAbs, fusion proteins, and bispecifics. Thermostable TPs will facilitate their efficient functioning inside body, improve their long-term storage stability, and decrease their aggregation tendency resulting in less immunogenicity. After a large number of variants screening to produce a stable TP, it will benefit the production of a potentially safer drug with a lower treatment costs.

The overall objective of this project:
The project will establish a novel high-throughput directed evolution platform for evaluating large libraries of TP variants for increased thermostability, slower global or local unfolding rates, and minimised aggregation propensity. DE can be carried out by encapsulating single plasmids, each encoding a unique protein variant, into droplets of an emulsion, then using cell free expression to generate the protein in each droplet. This project will extend this approach to incorporate non-natural amino-acid fluorophores into the protein to create a FRET-based signal that would give a direct report on protein expression levels. Such a platform would be used to directly evolve TPs with increased thermostability, and then slower local unfolding, which will combine to decrease aggregation propensity. An enzymatic system will be used initially to establish and validate the set-up, taking advantage of the simple fluorescent assay for enzyme activity. Once established, the set-up will then be applied to therapeutic antibody fragment proteins, using FRET to screen for retention of folded structure upon heating.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The research was carried out in 3 primary work packages:
WP1 – Screening of functionally active bovine enterokinase variant.
WP2 – Directed Evolution of Therapeutic protein: Fab fragment.
WP3 – Determination of aggregation propensity of thermostable Fab fragment.

WP1 work package was successfully finished, using TK instead of BEk as that system had also recently been shown to be amenable to NNAA incorporation. Work on the Fab protein was started as per WP2, and WP3, but were terminated early because the of the COVID-19 lockdown which blocked access to laboratories in March 2020. The successful implementation of WP1 ensured the establishment of the platform on which we planned to identify and express a thermostable variant of therapeutic protein in WP2 and WP3.
A career development plan and a data management plan were prepared and reviewed every 6 months along with an annual appraisal with supervisor and department.
All training objectives were reached during the fellowship. The fellow received training in protein engineering such as NNAA incorporation and DE; training in biophysical methods and biochemical assays such as FRET based assay; training in microfluidics chip preparation, single and double emulsion droplet preparation, droplet sorting technique on FACS Jazz, expertise in managing interdisciplinary research projects; and development of leadership and supervising skills which was required for implementation of the project.

Overview of the research results
There was a small deviation in the part of protein engineering platform development from the proposed plan. The original proposal was to use BEk for initial validation, but instead this was done using the TK enzyme system. The appropriate sites within TK were identified for NNAA incorporation after SDM to introduce amber stop-codons. The structural changes of TK were studied on different external effects such as denaturing agents, temperature etc. This was studied through the FRET signal generated by the dual fluorophores incorporated into the engineered TK.
A stable TK variant was identified followed by preparation of a TK genetic library using directed evolution to produce a larger repertoire of probable more stable TK variants.
CFPS of the TK enzyme was also optimized prior to validating the microfluidics based screening.

Exploitations & Disseminations
The fellow participated in 4 national and international conferences or workshops.
1. MiBIO 2018, University of Cambridge, UK
2. SynBio London at Birkbeck College, 2019.
3. UCL MBI module: Downstream processing- Chromatography, 2019
4. UCL MBI module: Quality by Design for Effective Bioprocess Characterisation and Validation, 2020.

Publications

As a result of the fellowship the fellow is the co-author of the following publications. These publications are in final stage of submission.
Mukhopadhyay, A; Dalby, PA.; Genetic mapping towards transketolase stability using FRET based evolutionary techniques. 2020.
Mukhopadhyay, A; Dalby, PA.; Next-generation laboratory platforms for molecular evolution and engineering of proteins. 2020.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Impact
The fellow has a clear objective to reach an independent research position and become a principal investigator in the academic sector. This fellowship provided the opportunity to raise the fellow’s research profile to represent an attractive and convincing prospect when applying for independent funding schemes. The fellowship helped the fellow to acquire diverse scientific skills and complementary training needed to address problems in the multidisciplinary field of synthetic biology. As part of the training received in interdisciplinary project the fellow handled the project with the Chemistry department and trained in MS-ESI. This fellowship provided experience in organizing project meetings and reporting schedule and risk management.
The fellow co-supervised one student, working on genetic modification of TK and expression followed by FRET measurement, which provided an opportunity to gain experience in supervising students, which included planning experiments, ensuring safe laboratory work and preparation of reports.
Regarding public engagement the fellow is the departmental in charge of organized weekly seminar organization committee and he successfully organize weekly seminar series where not UCL researchers, scientists, but also eminent scientists from outside are invited and present their talks. The fellow is a member of PDRA association where he takes part in organizing several public engagements, such as summer science show, pub quiz, scientific debates, monthly seminar series, etc. To communication with a broader audience through public engagement events, the fellow participated in few events organized by departmental outreach facilities. These events were held to the general public including high school students and involved a game depicting how therapeutic protein works.

Overview of the research plan

Click Chemistry

FRET-FLI

Plasmids

DLS study

3D structure of enzymes