The market for biopharmaceutical based medicines in 1996 was USD10.7 billion in the USA alone. And 85% of new pharmaceuticals are as a result from biotechnological research and development. In South East Aisa, the annual growth of pharmaceutical markets is projected to be around 11% annually over the next 5 years of which will represent 8% of world market to be estimated around USD1 billion each year.

Human growth hormone (hGH) and erythropoietin (hEPO) are two biopharmaceuticals which currently and in future will have a very huge international market. In addition, the patents for hGH has already expired and the patent for hEPO will be expired in 2003. Therefore, it will undoubtedly open up a huge market for these two biopharmaceuticals.

Human growth hormone is produced in pituitary gland and secreted throughout a person's lifetime however, the level of its expression declines as the age progresses. The main function of hGH is to promote growth in children. Signs or symptoms when a person is lack of hGH are short stature or dwarfism syndrom, memory loss, increase anxiety and psychological imbalance. In addition, there is an indication that hGH may play a role in slowing down and might even reverse the aging process.

Human eryhtopoeitin is produced in kidney. The main function of hEPO is to stimulate and increase the production of red blood cells and also to promote and enhance maturation of eryhthrocytes into functional red blood cells. Several pathophysiological conditions can reduce the level of hEPO circulating in our blood. Such conditions include kidney damage, anemia and certain medications. As a result of the low level of hEPO, there will be a vast reduction in the amount of circulating mature red blood ceels, therefore, human body will not be able to utilise oxygen for breathing and eventually death will occur.

Taking together all these factors, in addition to the available experties in Malaysia, the main objectives of the projects are (i) to isolate hGH and hEPO cDNAs and clone into vectors of expression systems, (ii) to cultivate the recombinant clones producing hGH and hEPO in small scale flasks and bench scale bioreactor, (iii) to purify recombinant hGH and hEPO and evaluate the system efficiency, (iv) to scale-up cultivation of recombinant system producing hGH and hEPO in pilot scale bioreactor, and (v) to develop large scale purification procedure and optimized chromatography process.

The approach or methodology taken to achieve the objectives listed above can be outlined in five major steps as follows:

1. Isolating and constructing hGH and hEPO cDNAs
Full length hGH and hEPO genes will be isolated from human pituitary gland and kidney cDNA library respectively, utilising PCR based method using primers based on the published sequence. The PCR products will be cloned into pGEM-T vector for the purpose of propagation and subsequently precisely engineered into appropriate expression vectors.

2. Constructing expression cassette with hGH and hEPO cDNAs inserts
hGH and hEPO cDNAs will be cloned into appropriate expression vectors. Pichia pastoris and prokarotic expression systems, and Pichia pastoris and eukaryotic expression systems will be utilised for the expression of hGH and hEPO, respectively.

3. Cultivating the recombinant clones in smale scale flask/bioreactor
Upon completion of the cloning work, the recombinant clones will be cultivated in small scale flask. The expression of gene of interest will be induced and optimised. Purification of the biopharmaceutical at bench scale level will also be conducted to evaluate the expression system efficiency.

 

 

EXECUTIVE SUMMARY

Project Title:
Production of recombinant biopharmaceuticals in pilot scale bioreactor and purification of
product using downstream processes

 

 

4. Producing the hGH and hEPO in pilot scale bioreactors Once the optimised parameters have been achieved at laboratory scale, the recombinant clones will be cultivated using the most efficient system in pilot scale bioreactor. All bioprocess parameters which are relevant to the growth of the recombinant clones will be investigated.

5. Developing large scale purification procedure and process chromatography optimization
Large scale recombinant biopharmaceuticals purification will be carried out using pilot scale using tangential flow filtration system and low pressure liquid chromatography available at the Fermentation Technology Centre (UPM). The steps involved are; capture step using ion exchange or affinity chromatography, intermediate purification using hydrophobic or competitive interaction and polishing step using gel filtration.

Four main expected outputs have been identified which are (i) established clones of recombinant hGH and hEPO, (ii) pilot scale process for the production of recombinant hGH and hEPO, (iii) purified recombinant hGH and hEPO, and (iv) technology on large scale production of recombinant biopharmaceuticals. Eventually, it is hoped that the clones for hGH and hEPO will be patented and the purified hGH and hEPO will be commercialised once the necessary facilities are available in Malaysia (cGMP plant).

Period of the project : 3 Years (2001 - 2004)

Achievements

Progress/Achievements for year one

New Products :

1- 3 putative hEPO cDNA were successfully amplified from EST clone purchased from
ATCC with the size of 580bp.

2- A CHO cell line is in the mamalian cell cultivation process of being acquired

3- The human growth hormone will be expressed in recombinant E.coli and also in yeast, hence fermentation condition for E.coli cultivation is being developed.

New Process :

1- The EST clone was used in exchange for amplication of the cDNA clone from foetal kidney library.

2- The sequencing data revealed mutations occured in all 50 clones of hGH ranging from 5 to 20 mutations and the least number of mutations occured in a single clone was 5 and this clone was selected for repair.

3- Mamalian cell cultivation study is being carried out in 2L bioreactor system at the Fermentation Technology Unit, Institute of Bioscience, UPM

4- Optimization of fermentation condition of recombinant E.coli carrying a human protein using 2L bioreactor. Work is being carried out to optimize the media formulation for E.coli cultivation and mode of fermenter operation is also being investigated. Initial improvement of dry cell weight from 0.04g/L to 4g/L was achieved by using fed_batch fermentation mode in a 2L bioreactor system.

Discovery :

1- By using PCR assembly approach, a total of 50 hGH cDNA clones were successfully assembled using 20 oligonucleotides with the size of 610bp

2- Sequencing data revealed two of hEPO clones were free of mutation. The hEPO cDNA was then successfully cloned into the expression vector.

3- Four of the mutations were successfully repaired using site-directed mutagenesis approach.