Award details

Manipulation of signal peptide sequences for enhanced synthesis and secretion of biotherapeutic monoclonal antibodies

Reference	BB/G01762X/1
Principal Investigator / Supervisor	Professor Christopher Smales
Co-Investigators / Co-Supervisors	Dr Diane Hatton
Institution	University of Kent
Department	Sch of Biosciences
Funding type	Skills
Value (£)	74,410
Status	Completed
Type	Training Grants
Start date	01/10/2009
End date	30/09/2013
Duration	48 months

Abstract

unavailable

Summary

There has been a large amount of work investigating the effect of different secretion signals on the secretory expression levels of recombinant proteins in yeast systems (e.g. see Ghosalkar et al, 2008). Whilst there has been some work undertaken in this area in mammalian cells, surprisingly little published work is publically available with regard to how much effect the choice of signal pepide has on the level of synthesis and secretion of recombinant biotherapeutic proteins expressed in mammalian cell systems. Currently a small number of classical signal sequences are utilised that originate from secretory proteins, it being accepted that these are likely to be sufficient to target polypeptides to the ER and secretory pathway. Further, the coding regions for signal peptides at the mRNA level are known to affect mRNA stability (Knappskog et al, 2007), and protein folding as the presence of non-optimal codons in this region can slow the rate of translation across N-terminal signal sequences to aid correct folding of the secreted protein (Zalucki and Jennings, 2007). Indeed, using a model luciferase reporter gene, Knappskog et al (2007) have shown that the choice of signal peptide dramatically changes the levels of secreted recombinant luciferase observed in the culture medium of CHO cells in both transient and stably transfected cells. A further study has shown that nonclassical secretion signals may be more efficient at secretion than classical secretion signals. As such there is the potential to enhance synthesis and secretory levels of biotherapeutic proteins produced from in vitro cultured mammalian cells by a systematic investigation and manipulation of the signal peptides utilised. This project sets out to test the hypothesis that the synthesis and secretory levels of recombinant biopharmaceuticals, specifically monoclonal antibodies, can be enhanced by manipulation of the N-terminal signal peptide. To achieve this we will use a model monoclonal antibody available in house at Medimmune and a series of classical and non-classical signal sequences which will be cloned onto the N-terminal end of the open reading frame of the light chain sequence of the model monoclonal antibody. We will also investigate the effect of placing the signal sequences at the N-terminal of the open reading frame of the heavy chain sequence. Initially we will undertake transient expression studies in a host CHO cell line and determine the levels of fully assembled monoclonal antibody in the culture medium and cell lysates. We will also determine the levels of half-antibodies and any free heavy or light chain (or dimers of each) in the medium and cell lysates. Once we have determined the best antibody yielding signal sequence we will test to see that this effect is generic for a number of different antibodies in our transient system. Following this, we will investigate the effect of introducing rarer codons into the signal sequence and determine the effect on translation rates by 35S-Met labelling and by monitoring the levels of fully assembled monoclonal antibody in the culture medium. Ultimately we will use these results to develop a signal peptide system that can be generically utilised to enhance monoclonal antibody synthesis and secretion in CHO cells in both transient and stable expression systems above the levels that are currently routinely achieved. REFERENCES Ghosalkar, A., Sahai, V., Srivastava, A. (2008) Protein Expression and Purification 60, 103-109. He, Z., Sun, X., Mei, G., Yu, S., Li, N. (2008) Cell Biology International 32, 367-373. Knappskog, S., Ravneberg, H., Gjerdrum, C., TroBe, C., Stern, B., Pryme, I.F. (2007) Journal of Biotechnology 128, 705-715. Zalucki, Y. M., Jennings, M. P. (2007) Biochemical and Biophysical Research Communications 355, 143-148.

Committee	Not funded via Committee
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Training Grant - Industrial Case

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