Membrane-based systems have utility within a number of unit bioprocesses, and in purification offer some advantages over chromatography including overcoming solute diffusion limitations. Membrane chromatography can be used for purification of mAbs and other biomolecules. In membrane chromatography, the ligand, for example Protein A, is covalently attached to a membrane of regenerated cellulose. The pore size of the membranes is larger than those of chromatography beads enabling capture and polishing at much higher flow rates. This project will investigate integration of these technologies into biopharmaceutical manufacturing processes utilising model biopharmaceuticals.

Lead investigator	Associate Professor Linda Lua
Postdoc	Dr Balaji Somasundaram
Industry Partner	GE and Patheon joint project; PIs, O’Meara (GE), Shave (Patheon).

The metabolic demands of a bioproduction cell line are inherited genetically, however gaining an understanding of these demands can have a significant impact on the quantity and quality of the product. As new technologies evolve around complete profiling of nutrients and metabolites in spent media analysis, the mantra is towards automation of current off-line process analytical technology (PAT) as an on-line feedback mechanism. This project will investigate real time control of critical bioprocess parameters, as a value proposition in the attempt to develop an automated bioprocess system.

Lead investigator	Dr Esteban Marcellin Saldana
Postdoc	Dr Veronica Martinez Salazar
Industry Partner	Patheon; PI, Crowley (Patheon)

This project will develop and evaluate a periodic counter current (PCC) chromatography and straight-through processing (STP) continuous three-step mAb purification process. Process intensification by implementing continuous or semi-continuous downstream processes in mAb production, for example, can contribute to significant cost-savings and improved throughput. Continuous processing also offers the possibility of increased automation of the process. The project will develop these bioprocesses using model proteins, thereby developing a new, innovative bioprocess platform for the large scale production of mAbs.  

Lead investigator	Associate Professor Linda Lua
Postdoc	Dr Balaji Somasundaram
Industry Partner	Collaborative project with GE and Patheon.

This project is focussed on upstream bioprocessing and investigates the similarity (identity) of recombinant protein biopharmaceuticals produced in batch culture compared to the same proteins produced in continuous culture. It will involve extensive physico-chemical characterisation using a variety of highly sophisticated analytical techniques. Of specific interest is the extent to which product characteristics can be manipulated using continuous process parameters (i.e. perfusion rate, bleed rate, temperature, pH, and critical media components for quality issues e.g. sugars).12, 13 The project could explore the design envelope for continuous bioprocesses and the potential for these to better meet biosimilar specifications. A successful outcome to this project will be knowledge into the effect of manufacturing process on the molecular identity of the therapeutic protein product, allowing the manufacturer to understand the potential impact of changes to the manufacturing process.  

Lead investigator	Dr Christopher Howard
Postdoc	Dr Lucia Zacchi
Industry Partner	CSL; PIs, Glover, Sandford and Lee (CSL).

This project will investigate and compare the differential expression of different classes of proteins, namely antibodies and coagulation factors. Expression of antibodies is well characterised, with consistent yields of 3 g/L or better in optimised conditions. Commercial production of antibodies is therefore a fairly generic process. Conversely, coagulation factors are extremely difficult to express at high levels and each factor represents a unique manufacturing challenge. The project seeks to understand what cell machinery / processing mechanisms create the bottlenecks in production of recombinant coagulation factors. 9, 10 The project will involve metabolomic analysis, as well as protein and cell engineering. A successful outcome to this project will lead to yield improvements for the commercial production of recombinant coagulation factors (and potentially other therapeutic proteins), resulting in improved productivity and reduced manufacturing costs.

Project Leader	Dr Benjamin Schulz
Postdoc	Dr Lucia Zacchi
Industry Partner	CSL

Typing reagents for hybrid glycophorin blood groups that are rare in Caucasians, but with prevalence of up to 8% in East Asian ethnic groups, are unavailable despite repeated attempts by various groups to use conventional approaches to produce useful mAbs. We will utilise naive human phage libraries to develop reagents that react with RBC with a phenotype defined by the most common hybrid glycophorin but not with pooled human RBC that do not display these antigens. The usefulness of the mAb produced as a typing reagent will be assessed by binding to the target hybrid glycophorin detected by flow cytometry. Subsequently reactivity using the standard immunohaematological agglutination based techniques will be assessed as well as characterising other features required for typing reagents, such as long term stability at room temperature.  

Lead investigator	Dr Martina Jones
Postdoc	Dr Xuan Bui
Industry Partner	ARCBS; PIs, Flower and Irving (ARCBS)

Integral membrane proteins are attractive targets for research, diagnostic and therapeutic applications, as they act as biomarkers to define a particular cell type, developmental stage or disease type. As such, mAbs against cell-surface biomarkers are highly sought after as biological therapeutics, laboratory reagents or diagnostic reagents. Based on antibody phage display methodologies.6 AIBN has developed novel whole-cell biopanning techniques to improve the efficiency of screening antibody libraries on whole cells displaying biomarkers. The proposed collaborative project aims to pool the skills of AIBN and CSL researchers to further optimise the whole cell biopanning technique to isolate new mAbs against specific cell surface biomarkers that are of interest to CSL. The outcomes of this project would be further innovations in whole cell panning methodologies, as well as isolation of new antibodies of therapeutic significance.

Lead investigator	Dr Martina Jones
Postdoc	Dr Sumukh Kumble
Industry Partner	CSL; PI, Panousis (CSL)

Using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, ARCBS has identified a unique murine pedigree with a splice-site mutation in a functionally important domain of a gene encoding an acetyltransferase. Red Blood Cells (RBC) from homozygous mutants from this pedigree demonstrate significant modifications of carbohydrates and lack the erythroid lineage marker TER-119. We plan to investigate the extent to which these changes modify the susceptibility to infection for pathogens that target developing and mature cells of the erythroid lineage. We will compare RBC from homozygous and wild type (WT) mice to characterise the RBC surface and identify novel RBC targets to be used as a basis for the development of biopharmaceuticals for treatment of diseases where infectivity is mediated through RBC receptors (e.g. Malaria, Parvovirus (B19)).

Lead investigator	Prof Stephen Mahler
Postdoc	Dr Xuan Bui
Industry Partner	ARCBS; PIs, Dean, Flower and Irving (ARCBS)

The Patheon process for high density cell culture (Patheon XD® Upstream Processing (USP) technology) can result in up to a 25 fold increase in bioreactor output (cell density). CHO cell lines can be engineered to deliver maximal performance and productivity. The proprietary Patheon XD® USP bioprocess may be improved for suitability to high density culture, by engineering CHO cell lines with acquired properties that favour high density. In this project, development of a cell line(s) optimised for high density cell culture, utilising CRISPR and other cell line engineering techniques, will be investigated.  

Lead investigator	Dr Esteban Marcellin
Postdoc	Dr Veronica Martinez Salazar
Industry Partner	Patheon; PI, Shave (Patheon)

The majority of proteins generated by the Recombinant Protein Group within CSL to date for in vivo use are mAbs that are not dependent on post-translational modifications for activity. However, CSL now has several projects that are not antibody-focussed and require the generation of complex proteins, including FVII-HSA, FVIII, AAT and C1Inhibitor. The correct glycosylation and, in particular, sialylation of these recombinant therapeutic proteins are important for in vivo animal studies, as glycostructures can influence the pharmacokinetics and immunogenicity of the protein.11 The project aims to understand the effect of host cell line, manipulation of enzymes in the protein glycosylation pathway and culture conditions on glycostructures in recombinant therapeutic proteins. The project will also compare proteins expressed in the Wave Bioreactor to those in a stirred tank bioreactor, with respect to posttranslational modifications and quality.

Project Leader	Dr Benjamin Schulz
Postdoc	Dr Lucia Zacchi
Industry Partner	CSL

Human neutrophil antigens (HNAs) are a group of 5 glycoproteins that are expressed on human neutrophils, and in some cases, also on other cells and tissues. Endogenous antibodies against HNAs have been implicated in cases of alloimmune neonatal neutropenias, autoimmune neutropenias, febrile transfusion reactions and transfusion-related acute lung injury (TRALI). The availability of mAbs to HNAs permits the use of solid phase assays to detect endogenous antibodies specific to these antigens. While mAb reagents against HNA-1, HNA-2, HNA-4 and HNA-5 antigens are available, there are not yet any mAbs available for HNA-3. This project aims to develop mAbs against the two alleles of HNA-3 that can then be used to develop cell-based assays to detect anti-HNA-3a and anti-HNA-3b antibodies in blood donors. Furthermore, this project aims to use the anti-HNA-3 mAbs in existing in vitro transfusion models to help understand the mechanisms by which TRALI develops.

Lead investigator	Dr Martina Jones
Postdoc	Dr Xuan Bui
Industry Partner	ARCBS; PIs, Tung, Flower and Irving (ARCBS)

Intracellular processes including protein transport, transcription and signalling present a range of new potential targets that could have application in the treatment of a variety of disease indications. The screening of chemical libraries for binders to intracellular targets has had limited success due to (i) the relative lack of binding clefts and hydrophobic pockets compared to those located on cell surface proteins and (ii) the small “footprint” to which a small molecule can bind on an extensive protein surface interface. As an alternative, antibody fragments are being explored as molecular entities that are capable of disrupting protein-protein interactions within the cell, traditionally thought to be undruggable.5 This project will investigate strategies for intracellular delivery of antibodies and antibody fragments, along with assessing the resulting impact on cellular processes. An antibody against the transcription factor SOX18 (created at AIBN and IMB) will be used as the initial model system for intracellular antibody delivery. A successful outcome of this project will be the establishment of platform technology for the delivery of antibody fragments to intracellular targets.  

Project contacts

Project Leader	Dr Mathias Francois
Postdoc	Dr Sumukh Kumble
Industry Partner	CSL