Biomedical Engineering Reference
In-Depth Information
TABL E 3.2. Potential Critical Process Parameters and Quality Attributes Evaluated During
Development
Potential critical process parameters for product development
Gene design/codon usage
Osmolarity
Gene boundaries
Quantity
Secretion of product into fermentation media
Stability
Induction conditions during fermentation:
Integrity or extent of “proteolytic nicking”
during fermentation
Ph
Temperature
Methanol feed rate
Potential CQAs and analytical method(s) for evaluation
Integrity (reduced and nonreduced)
Structure, conformation, or function
SDS-PAGE
Immunoblot with conformational mAbs
RP-HPLC
Plasmon surface resonance
Circular dichroism spectrum
Functional binding assay
Purity (reduced and nonreduced)
Posttranslational modifications
SDS-PAGE
N-linked glycosylation
RP-HPLC
O-linked glycosylation
Other
Solubility (SEC-HPLC)
N-terminal sequence
Aggregation
Peptide mapping (reduced and nonreduced)
MALS-SEC-HPLC
Sedimentation velocity or equilibrium
Stability
Mass
Impurity profile
Matrix-assisted laser desorption
ionization (MALDI) spectroscopy
Host cell protein content
Endotoxin
Electron spray ionization spectroscopy DNA content
MALS-SEC-HPLC, multiangle light scattering-SEC-HPLC; RP-HPLC, reverse-phase high-performance
liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC-HPLC,
size exclusion chromatography-HPLC.
AMA1, the impact of codon usage for generating a synthetic gene for expression in
P. pastoriswill be discussed, which lead to its successful cGMPpilot-scale production [30].
3.3.1 Production of Recombinant EBA-175 RII Protein
P. pastoris Expression of EBA-175 RII.
A comparison of the native EBA-175
RII gene sequence and the synthetic-mammalian-encoded EBA-175 RII gene sequence
is shown in Fig. 3.2. A total of 591 out of 1848 nucleotides (32.0%) were altered in the
synthetic RII gene. The result was that the A
T content of the RII gene changed from
þ
