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Overview

Our group has been involved in the development of a variety of novel affinity ligands for the purification of high value therapeutic recombinant proteins since 1970. The global market for biopharmaceuticals is projected to reach US$182.5 billion by 2015. These 

products comprise monoclonal antibodies (mAbs), vaccines, erythropoietin, recombinant human insulin, cytokines, hormones and other biomolecules. For example, mAbs constitute one of the most rapidly growing categories of biopharmaceutical, with more than 25 antibodies approved by the FDA and 240 currently in clinical trials. However, issues, such as healthcare reform and the increased demands upon healthcare budgets are forcing the pharmaceutical industry to reduce manufacturing costs. Moreover, due to the development of new approaches for upstream processing and the increase in the protein production yields from mammalian cells (~5g/L), the production bottleneck for some high volume products is shifting towards downstream processing,  which can constitute up to 80% of total manufacturing costs.

                  The concept of the "well-characterised biologic" requires that the biological molecule has to be characterised for its identity, purity, impurity profile and potency. Regulatory reform is likely to stimulate the development of new high-resolution separation processes.  Of particular concern is the resolution and purification of variants of the target itself, resulting from differences in glycosylation, folding, sequence, oxidation and a multitude of other post-translational modifications. The ability to resolve multiple glycoforms of therapeutic proteins, for example, is crucial since product registration is based on a particular isoform composition.

Among the different classes of biopharmaceuticals, our current research is focused on the development of synthetic affinity ligands for immunoglobulins, glycoproteins, erythropoietin and vaccines.

The aim is to implement cost-effective and highly efficient purification protocols, in order to reduce the purification and polishing steps during the downstream processing, and replace the biomolecules currently used in affinity chromatography. Biological affinity ligands, such as the IgG-binding proteins, protein A, G and L, and sugar-binding proteins, i.e. lectins, suffer from a wide range of limitations: They are expensive to produce and purify, may be contaminated with host DNA and viruses, show lot-to-lot variation, and low scale-up potential, and they may be degraded during conventional sterilization-in-place (SIP) and cleaning-in-place (CIP) procedures and lead to ligand leaching and subsequent contamination of the end product with potential toxins.

                  The synthetic affinity adsorbents are based on "biomimetic" ligands and the notion of de novo ligand design and intelligent combinatorial libraries. Ligand synthesis follows a defined five-part strategy which comprises: (i) Identification of a target site and design of a complementary ligand based on X-ray crystallographic studies of complexes between the natural target protein and the biological ligand; (ii) solid-phase synthesis and evaluation of  an intentionally  biased  combinatorial library of related ligands; (iii) screening of the ligand library for binding the target protein by affinity chromatography; (iv) selection and characterisation of the lead ligand, supported by in silico molecular modeling and docking of the ligand into the target protein, and (v) optimisation of the adsorbent and chromatographic parameters for the purification of the target protein.

              We are currently applying a new approach for the solid-phase synthesis of the ligands based on the multicomponent Ugi reaction, which is a four-component reaction in which an oxo-component (i.e. aldehyde-activated agarose beads), a primary or secondary amine, a carboxylic acid and an isonitrile group are condensed, in a one-pot reaction conducted at a constant temperature (50°C), to yield a di-amide scaffold product. The Ugi components can be varied to mimic key amino-acid residues of the biological ligand involved in its interaction with the target molecule. This approach also introduces the possibility of developing branched, cyclised and 3D affinity ligands.

              We have developed various Ugi ligands mimicking different IgG-binding proteins, namely, protein L and protein G. The protein L mimic was developed for the isolation of IgG from crude extracts, and more specifically for its ability to bind Fab fragments.

Moreover, we have recently synthesised and characterised a protein G mimic for the purification of mammalian immunoglobulins derived from different species and particularly the non-conventional camelid IgGs.

Camelid IgGs, present in the serum and milk of Camelus dromedaries, contain immunoglobulin subclasses that naturally lack light chains; they are termed “heavy-chain” antibodies or HCAb and display a molecular weight of ~95kDa instead of 160kDa for conventional mammalian antibodies. Their heavy chain also lacks the CH1 domain and the heavy chain only variable domain (VHH) is connected directly to the constant domain (CH2 and CH3) via the hinge region. The small size of these VHH domains (~15kDa) allows greater access to buried epitopes and recognition of antigenic sites in clefts that generally could not be reached by larger conventional antibodies and hence they are effective enzyme inhibitors.

Furthermore, we are currently focusing on the development of a variety of branched and complex Ugi ligands for different immunotherapeutic proteins and glycoproteins of interest. The relative ease of conducting the Ugi reaction suggests that this work may become the gold standard procedure in use worldwide for selective processing of high value biopharmaceuticals. 

List of Projects and Members:

Purification of Glycoproteins – Miss Chen Chen

Purification of EPO – Miss Basmah Khogeer

Purification Platform for Influenza Vaccine – Mr Shaleem Jacob

Solution-phase Synthesis of Ugi-based Biomemtic Ligands – Mr Kheng NG