Design and Fabrication of an Artificial Golgi Reactor
Lead Research Organisation:
Imperial College London
Department Name: Chemical Engineering
Abstract
Modern pharmaceuticals are largely based on the production of biorelevant and therapeutically useful proteins to improve drug effectiveness and patient response. Monoclonal antibodies are part of this group of molecules. It is expected that 30-40% of the new recombinant drugs will be glycosylated monoclonal antibodies. Glycosylation, the process of adding sugars to molecules such as a protein has an impact on the functionality, folding and specificity of these antibodies as it contributes to their interaction with a pathogen or antigen as well as with the immune system of the patient.
A great challenge for the pharmaceutical industry is the production of bespoke, effective, glycosylated therapeutic proteins in an economical manner. Working in a cell free environment provides the ability to access targeted glycosylation pathways enabling tailored antibodies to be synthesized. Furthermore, by targeting individual pathways and controlling the environment only the desired product is produced. This is in contrast to the production of these drugs in an in vivo environment where the natural heterogeneity produces a range of molecules lowering the overall efficacy of the drug. Reactions in cell free microenvironments are manipulated using minimal resources to determine the conditions required to achieve optimal yields. Such efforts contribute to scaling the process leading to economies of scale.
In line with the current needs, this project will consist of designing and optimizing the fabrication of an artificial Golgi reactor in a cell free microfluidic environment to control N-linked glycosylation. This is the process of producing complex glycans by adding carbohydrates to Asparagine residues which naturally occurs in the Golgi apparatus. Our goal is to create an in vitro process to produce tailored glycosylated proteins. To do that we aim to express and purify active, recombinant versions of the enzymes enabling the selected N-linked glycosylation pathway. Following this we aim to immobilize these enzymes in microfluidic channels. This will allow their retention in specific compartments, to have sequential enzymatic reactions such as those that occur in the Golgi Apparatus.
A great challenge for the pharmaceutical industry is the production of bespoke, effective, glycosylated therapeutic proteins in an economical manner. Working in a cell free environment provides the ability to access targeted glycosylation pathways enabling tailored antibodies to be synthesized. Furthermore, by targeting individual pathways and controlling the environment only the desired product is produced. This is in contrast to the production of these drugs in an in vivo environment where the natural heterogeneity produces a range of molecules lowering the overall efficacy of the drug. Reactions in cell free microenvironments are manipulated using minimal resources to determine the conditions required to achieve optimal yields. Such efforts contribute to scaling the process leading to economies of scale.
In line with the current needs, this project will consist of designing and optimizing the fabrication of an artificial Golgi reactor in a cell free microfluidic environment to control N-linked glycosylation. This is the process of producing complex glycans by adding carbohydrates to Asparagine residues which naturally occurs in the Golgi apparatus. Our goal is to create an in vitro process to produce tailored glycosylated proteins. To do that we aim to express and purify active, recombinant versions of the enzymes enabling the selected N-linked glycosylation pathway. Following this we aim to immobilize these enzymes in microfluidic channels. This will allow their retention in specific compartments, to have sequential enzymatic reactions such as those that occur in the Golgi Apparatus.
Organisations
People |
ORCID iD |
| Cleo Kontoravdi (Primary Supervisor) | |
| Elli Makrydaki (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/N509486/1 | 01/10/2016 | 31/03/2022 | |||
| 1855070 | Studentship | EP/N509486/1 | 01/10/2016 | 30/09/2020 | Elli Makrydaki |
| Description | Most of the enzymes participating in the glycosylation of proteins, often compete against the same substrate. As a result, we get a mixture of glycosylated products instead of a homogeneous result. In this research and in order to address the issue of enzyme promiscuity we developed an enzyme immobilisation strategy where enzymes were immobilised on solid supports. Contrary to traditional techniques, using immobilized enzymes allows the removal of an enzyme from the reaction before adding the next one. This way we can have the desired outcome instead of a mixture of unwanted products Based on the above, we successfully immobilised 3 enzymes participating in the glycosylation of proteins. The activity of the enzymes was confirmed using artificial sugars chains. Briefly, the enzymes recognized the sugar chains as a substrate and extended it by adding or removing molecules. Analysis proved that enzyme immobilisation and sequential reactions allowed us to avoid the enzyme competition and have a final product of higher homogeneity. Finally, we were able to re-use the immobilized enzymes thus lowering the cost of these reactions. |
| Exploitation Route | So far, we demonstrated that immobilized enzymes can be used for targeted glycosylation of artificial glycans, protein fragments as well as whole antibodies. Finally we demonstrated that we can succesfully reuse our enzymes. Our immobilization system can also be applied to more enzymes regulating glycosylation, thus allowing more and complex reactions to take place. These findings can also be used for the glycosylation of therapeutic proteins coming from producing platforms such as mammalian cells as well as chaper protein production platforms such as bactreia or yeast. Finally, a key bottleneck in the production of therapeutic proteins in cells is the incomplete glycosylation the i.e. galactosylation or sialylation. Our system of immobilised enzymes can be used to push the glycosylation of therapeutic proteins towards completion . This is feasible due to the key advantage of the immobilised enzymes that they can be easily separated from the desired protein without harming or damaging it. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |