The Science

Summary
The resources expended in drug research grow exponentially, while the number of new drug molecules approved each year remains constant: the inevitable endpoint of research constrained to a fixed paradigm. A new class of drugs, known as biologicals, should have been precisely the paradigm shift needed to transcend this, as protein based structures modified through synthetic functionalization and glycosylation have a more complex mode of action and increased customizability in comparison to small drug molecules. They have, however, not lived up to this promise: their development and testing currently involves the complexity of the in vivo environment and is thus a slow and labor-intensive process.

Our project is to develop a streamlined in-line in vitro technology for the development and testing of biological drugs. We will build at first a module as a stand-alone device for pharmaceutical protein preparation and subsequent screening. We will include in-line process control for stringent quality control of the final product. While our project is limited to the development of biological drugs, and only the first module for a larger protocol and device, the results of this project will ultimately be applicable to all of biotechnology.


Objectives
Our long term goal is to develop a technology that will perform a task that currently can only be performed through the use of biotechnology through a far more efficient in-vitro protocol: development of functionalized proteins for a wide range of applications. We will refer to this technology as ‘integrated continuous – protein conjugate synthesis’ (IC-PCS).

Background
We need well-defined, fast methods to prepare protein-based drugs, easily add more functionality if needed, and the facility to evaluate them. The whole process needs stringent control from A-Z to eliminate batch-to-batch variations and to ensure the test results of the final product are reliable.

Biologics are medical biological products typically manufactured in living systems such as a microorganism, plant or animal cells (e.g. DNA, vaccine, blood-products, and proteins)[1,2]. Pharmaceutical protein development is hindered by the slow turnover of protein synthesis due to the use of living cells. Most pharmaceutical companies abandoned the use of transgenic animals[3,4] (turnover rates of 8-34 months) and focus on the industry standard of today: recombinant protein production in cells[5, 6]. The process, including clone selection, recombinant protein production, and the down-streaming processes, can take anywhere from 2 weeks up to 2 months (see figure below). Proteins reside in different environments, which may result in variations leading to missed lead-molecules or developing incorrect molecules for clinical trails. At the same time, the project life cycle of biologics is getting shorter, while the developmental phase remains the same[7,8]. The resources expended in drug research grow exponentially, while the annual number of new drug molecules approved remains constant[9,10]. This fixed paradigm needs new technologies to be disrupted.

In order to prepare, modify, purify, and evaluate new and better protein drugs variants new technologies are needed. A new technology must simplify, streamline, and transcend the process being conducted by the current state-of-the-art methods. Reducing the time necessary to produce synthetic functionalized proteins and validate their function within hours in an in-vitro environment of at least 100 biologics would achieves one important aspect. Gaining improved accuracy by the use of parallel streams on chip-like devices, process analytical technology (PAT), and clinical feedback through computational modeling and collaboration with the IDAAPM-database[11 would achieve the rest. 

Our Solution is two-fold
(1) Fail-faster to succeed faster, and (2) eliminate harmful variations at the same time. Our overall objective is to shorten the preparation and evaluation of protein based drugs several months to days. At the same time we will build-in PATs for process- and quality control in at every step, and build translational computational models. Our future goal is to prepare 100+ drug candidates in one day, evaluate them, re-engineer and start again.

Therefore we will apply a streamlined in-line in-vitro technological platform for the synthesis and testing of biological drugs to prepare, purify, formulate, and evaluate them on (i) toxicology, (ii) the interaction with the innate immune system, (iii) aggregation, (iv) adverse effects, and (v) binding to their respective targets (drug delivery). More importantly we will eliminate protein heterogeneity by providing feedback loops at every step in the process. In such a way we are not just faster; we will build a wide and deep understanding of our protein drug of interest from pre-production to the final formulations in systematic way never tried before.

In order to achieve this ambitious goal, we obtained crucial funding from the Academy of Finland to build a stand-alone module for continuous in-vitro protein synthesis and purification, and preparation of our first mathematical chemometric models. In this project we will guide experimental design via deep-learning models (DLMs) based on clinical data, then design and manufacture additional microfluidic screening modules, and link them together.


Novelty
The current state-of-the art in protein engineering and biotechnology lies in advanced molecular biology techniques and cell-based methods. Currently synthetic biology infringes into both fields, however new approaches for the needs of protein engineering and post-translational modifications are merely incremental improvements[2]. The novelty of our approach lies beyond the mere application of existing technologies: we will integrate interfacing capacities for sequential in-line preparation, modification, purification and characterization of pharmaceutical protein drugs. IC-PCS is comprised of (1) Cell Free Protein Synthesis (CFPS12), (2) Capture and Release, (3) Time Gated Raman spectroscopy (TG-Raman), (4) Surface Plasmon Resonance spectroscopy (SPR), (5) Micro fluidics, (6) in-line complement activation screening (IN-CAS), (7) in-line drug delivery to in-vitro screening modules, and (8) in-line toxicology in-vitro screening modules.

Why is being fast better?
Biologics, especially pharmaceutical proteins, are highly prone to degradation. Their success is due to their high specificity, but it is also a huge weakness: every protein behaves different. Proteins ‘age’ and behave differently in their production cycle and further lifetime. Proteins degrade in a process called aggregation, and proteins that show aggregation during pre-clinical development are excluded from further evaluation. This creates a paradox: some proteins that aggregate due to the many steps needed to prepare and evaluate them could be the most promising lead-molecules. On the other hand, some partly unfolding of proteins may be evaluated wrong and become a major problem in further clinical trials.

Proteins can aggregate due to any trigger: protein concentration, salt concentration, pH, bioconjugation, temperature, and protein-protein interactions to mention some of the most important ones. It is therefore imperative that proteins are screened for their activity as soon as they are produced and formulated in a fail-fast system which immediately reports at its interface any condition that is likely to indicate a failure: fail fast to succeed faster.

The current state of the art is biotechnology, or the process of employing cells to develop or make ‘products’[3 -5]. Many factors can influence the organisms and their productivity; therefore much effort is put into engineering more robust solutions; however, turnover rates are long and induces protein heterogeneity. We will bypass the use of cells by using in-vitro technology (CFPS[12]) and capture the protein during expression in an in-line module. We are currently evaluating several cell free systems (E. coli, tobacco plant, insects cells, wheat germ and mammalian) regarding compatibility with our microfluidic bioreactor.

Immediate feedback of protein preparation, purification via capture and release (i.e. protein amounts), secondary protein structures, and evaluation of protein drug delivery to cells, toxicology, activation of the innate immune system, and adverse effects are planned to be an integrated aspect in IC-PCS in the form of PA
T and screening assays. We will apply TG-Raman spectroscopy for chemical, compositional and structural information of biological materials [12-13], for toxicology assays[14], and as a supporting method to follow the binding to target cell lines in the functionality screening by use of Cell-Based Reporter Assays. SPR has been widely been used for monitoring bioaffinity reactions[13,15]. A method developed in our faculty, the interaction with the innate immune system will be adapted for use in microfluidic systems[16]. We will build computational models based on experimental data and bridge existing clinical data via Deep-Learning- and Machine Leaning-models. The proposed techniques are rapid, real-time, label-free and non-invasive and are a necessity for in-line monitoring tools. FTIR and LC/MS will be used for final protein amount analysis off-line and benchmarking experiments.

Impact and future applications
We hope to build a community via the IC-PCS website and other researchers, organize on-line workshops, and we will invite industry as well for participation. We intend to publish scientific papers in open access journals (green and gold standard) and train young researchers in this field. The impact of our project will be ensured by the fact that the project is not limited to the research that merely establishes the protocol: we will generate original science for microfluidic catalysis for protein drug formulation, and on-a-chip engineered purification strategies for the making of protein drugs. Furthermore, a first prototype will see the light at the end of the project, while fundraising to nurture offshoot projects and planned commercial involvement to oversee the commercial exploitation of the stand-alone modules, the IC-PCS platform as a whole, and its products. The impact will be seen first in assisting the development of a selections of protein drugs, by building expertise and knowledge through true interdisciplinary collaboration, inducing leadership, publicly accessible translational models via the IDAAPM-database[11] and our IC-PCS website, and increasing the innovation potential around these bright new fields within Europe.

In the longer term our ambition is for various IC-PCS platforms to streamline and cut the development time by orders of magnitude applicable for protein synthesis compared to current biotechnology. The impact in the field of biotechnology is to be broad and revolutionary. Thus, our strategy to ensure impact focuses around a broad dissemination and exploitation plan, engaging industry from the beginning, training future leaders in this novel field and creating a research community with a mission to further advance the in-vitro technology for the development of biological drugs.


References
[1] http://goo.gl/gvLwf7 (FDA definition of Biologics); [2] http://goo.gl/WMBVGf (EMA regulations of Biologics); [3] Walsh G. Nature Biotechnology, 2000, 18, 831-8333; [4] Walsh G. Biopharm International, 2013, 26, 54-56; [5] Casteleijn MG et al. Eur Pharm Rev 19: 12-9, 2014; [6] Casteleijn MG et al. , Int J Pharm 440: 39-47, 2013; [7] Bernard S. PharmExec, 25-28, 2013; [8] Stanley M. The US Healthcare Formula Cost Control and True innovation, June 16, 2011; [9] http://goo.gl/J51kVS (McKiney report 2014); [10] http://goo.gl/yMcvD0 (Markets and Markets, report 2015); [11] Ghemtio L et al. 2016. Journal of Cheminformatics, June 14;8:33; [12] Butler et al. Nat Protocols 4: 664-687, 2016; [13] Jönsson, U. et al. BioTechniques 11: 620-627, 1991; [14] Owen CA et al. J Cell Biochem. 2006 Sep 1;99(1):178-86; [15] R.B.M. Schasfoort, A.J. Tudos, Handbook of Surface Plasmon Resonance, The Royal Society of Chemistry, Cambridge, 2008; [16] Kari O et al. 2016. Drug Deliv Transl Res. Aug 4


 

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