Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant beta-secretase.
beta-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant beta-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant beta-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of beta-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 x 10(6) cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of beta-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.
Lüllau
E
E
AstraZeneca Biotech Laboratory, Enabling Science Technology Biology, R&D Södertälje, Sweden. elke.lullau@astrazeneca.com
Kanttinen
A
A
Hassel
J
J
Berg
M
M
Haag-Alvarsson
A
A
Cederbrant
K
K
Greenberg
B
B
Fenge
C
C
Schweikart
F
F
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000254:growth & development* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010865:Pilot Projects; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D011993:Recombinant Fusion Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human