Small Scale Production of European Pharmacopeia Compliant Erythropoietin & Purification from Recombinant CHO Cell Line
B I O T E C H
Md. Shawkat Hossain1, Rubayat Jamal1, Md. Tariq Hasan1, Abdullah Al Maruf1, Kawsar Hossain1, Dr. Mohammad Sorowar Hossain1, Dr. Md. Iqbal Hassan Khan1 1
Research & Development, Biotechnology Derived Product Facility, Incepta Pharmaceuticals Ltd, Dhaka, Bangladesh * Correspondence to:
[email protected]
ERYTHROPOIETIN: PHYSIOLOGY AND BACKGROUND INFORMATION Erythropoietin (EPO) stimulates the proliferation and differentiation of erythroid cells, leading to an increase in the number of reticulocytes in the blood, which consequently increases the blood’s haemoglobin concentration. This hormone is mainly produced in the kidney in human adults, and anaemia associated with renal failure often results from decreased levels of Erythropoietin. In this poster we have demonstrated the small scale production and purification process of EPO
Courtesy: Investors_2004 Annual Report
EPO exists as a mixture of isoforms. This micro-heterogeneity is related to the charged carbohydrate moiety of the protein and is associated with the presence or absence of terminal N-acetyl neuraminic acid residues with varying amounts of acetylation as well as the presence or absence of N-acetyl lactosamine extensions. Therefore the degree of sialylation of the polysaccharide chains of the protein strongly influences the bioactivity of this glycoprotein.
Dr. Goldwasser, working at the University of Chicago, isolated and purified erythropoietin in 1970s. He then shared his findings with a young biotechnology company, which figured out how to produce larger amounts of the protein using genetic engineering. That company, AMGEN, became the world’s biggest biotechnology company on the basis of EPO. Sales of Erythropoietin under names like Epogen, Procrit and Aranesp amounted to billions of dollars a year for Amgen, as well as Johnson & Johnson and Roche. (Picture to the left and right Amgen leaders past and present) From left to right, former CEO Kevin Sharer, founder Franklin “Pitch” Johnson, former CEO Gordon Binder, former CEO George Rathmann (seated) and founder Bill Bowes.
Courtesy: www.pnas.org/F1
Courtesy: McGraw Hill Companies, Inc.
PRODUCTION & PURIFICATION In Roller Bottle based technology cells are attached to surface of the Roller Bottle and the shaft of the incubator turns them in a particular speed. Initially the EPO producing CHO cells were selected by using the selection pressure of Methotrexate and after that only the surviving cells were transferred to growth media to increase in number. At confluence cells were transferred to Roller Bottle. In 4-5 days cells reached confluence and the media is transferred to production media which is free from serum and antibiotic to make purification process easier. From the following day EPO harvest collection starts and replace with fresh media. Final purification starts after combining all 7 days harvested and semi purified crude proteins.
0 hour T-175, Growth Media
“EP” COMPLIANT ERYTHROPOIETIN European Pharmacopeia guideline has details regarding the approval of rhErythropoietin
24 hour T-175, Growth Media
TEST NAME
96 hour T-175, Growth Media
72 hour T-175, Growth Media
PARAMETERS
RP-HPLC
Peak of standard (EPO BRP) and the sample should have the same retention time
SDS-PAGE, WESTERN BLOT
Electropherogram of the test solution should be a single diffuse band corresponding in position and intensity of the reference solution
IEF, CZE
Isoelectric pattern should be same as the reference solution in terms of permissible range of band number
PEPTIDE MAPPING
Peptide map of the sample should be qualitatively identical to the reference solution
N-TERMINAL SEQUENCE
First fifteen amino acid should be tested for identical results of the published data
TEST FOR DIMERS
Product should be free form dimer formation
SIALIC ACID CONTENT
Each mole of Erythropoietin should have more than 10 mole of Sialic Acid
BIO ASSAY
Estimated potency will be between 80% to 125%
LAL TEST
Maximum Permeable range of bacterial residual endotoxin is 20IU/10000IU of Erythropoietin
Purification Approaches
Final QC Report
Affinity
Desaltation
Anion Exchange
Desaltation
Cation Exchange
Chromatography
Chromatography
Chromatography
Chromatography
Chromatography
MW 30 kDa
Courtesy: geproteinskills.blogspot.com
CURRENT AND FUTURE APPROACHES Batch 1 14 SP:11_UV1_280nm Batch 1 14 SP:11_EditedBaseline
Batch 1 14 SP:11_Cond
Batch 1 14 SP:11_Conc
Batch 1 14 SP:11_pH
Formulation Strategy: Formulation is another challenge after successful development of a therapeutics. As we are developing a biosimilar version of erythropoietin-alfa therefore we have chosen the two formulations from the innovator to follow- (1)Epogen(Amgen) (2) Eprex (J&J). We have already start to check the necessary excipients effect and full formulation accelerated outcome by using standard quality test methods. After completing this step we will have both the formulations in hand and will go for pre-clinical & clinical study.
Batch 1 14 SP:11_Logbook
mAU
2500
2000
1500
1000
245.70
100%B
500
0 0
50
100
150
200
250
300
ml
Yield Calculation: In pilot scale level (10 roller batch size) we have completed three successful batches. Recently completed batch had extended harvest time (12 days) which is 5 days more from the usual 7days harvest. We did that extension in trial basis considering the scale up plan in near future. In each day of harvest, used media from rollers containing crude protein collected, filtered and semi purified through affinity chromatography. Only the glycosylated proteins were separated in affinity process and passed through the desaltation process to lower down the conductivity. Until the completion of overall harvests, desalted products were stored at -20°C freezer. Finally desalted products were pooled and passed through anion exchange, desaltation and cation exchange process sequentially to complete the final purification. A representative yield calculation is given below from the latest completed batch:
Courtesy: rxlist.com
Future Goal: Start from the bench top research and development to Concluding Remarks: Currently five EP compliant biosimilars of erythropoietin (EPO) deliver in market shelf Incepta has fixed some milestones: are being sold in European market under five different brand name and all of them were got approval in 2007. Therefore Incepta Biotech look forward to launch the developed EP compliant EPO in the local market first then subsequently in the European market and other continental markets as well. Meanwhile EPO beta and PEG beta development in bioreactor platform still in pipeline.
