Humanization

Phagemid Construction

Large-scale

Humanization

Light chain Heavy chain

A4.6.1

(murine anti-VEGF antibody)

Y0317

(anti-VEGF antibody Fab fragment)

humanized

Phagemid construction

• phoA alkaline phosphatase promoter

• stII periplasm secretion signal

• geneIII coat proteins of M13 bacteriophage

Light chain

Heavy chain

Periplasm

12~15 nm

Gram negative bacteria cell wall

• Contains only 4% of the total cell protein

• Easy purification by osmotic shock

• Oxidizing environment facilitates the formation

of disulfide bond

periplasm

E. coli strain engineering

• Overexpress chaperone Protein folding– GroEL+GroES– KJE complex (DnaK+DnaJ & GrpE)

• Overexpress disulfide bond enzyme Forming disulfide bond– DsbA

• Lower protease Inhibit protein degradation

Alkaline phosphatase promoter

Growth time

Cell number

Fast growth

High phosphate

Low phosphate

InductionHigh phosphate medium

Low expression (cell growth)

NO Need Inducer!!

Low phosphate medium

High expression (induction)