Cloning, expression and purification method for the preparation of ranibizumab

1. A process for the preparation of Ranibizumab comprising steps of:

a) transforming a first host cell with a first vector comprising a first polynucleotide encoding a signal sequence of SEQ ID NO: 5 operably linked to a light chain of Ranibizumab of SEQ ID NO: 2, wherein the first polynucleotide is operably linked to an inducible promoter system;

b) transforming a second host cell with a second vector comprising a second polynucleotide encoding a signal sequence of SEQ ID NO: 6 operably linked to a heavy chain of Ranibizumab of SEQ ID NO: 4, for heavy chain of wherein the second polynucleotide is operably linked to an inducible promoter system;

c) separately culturing the first host cell and the second host cell in a growth medium;

d) expressing the light chain of Ranibizumab in the first host cell and the heavy chain of Ranibizumab in the second host cell as periplasmic inclusion bodies;

e) solubilizing the inclusion bodies; and

f) refolding in-vitro the solubilized light chain and heavy chain of Ranibizumab.

2. The process of claim 1 , wherein the signal sequence of SEQ ID NO: 5 in the expressing step directs the first host cell to transport the light chain of Ranibizumab to a periplasmic space of the first host cell.

3. The process of claim 1 , wherein the signal sequence of SEQ ID NO: 6 in the expressing step directs the second host cell to transport the heavy chain of Ranibizumab to a periplasmic space of the second host cell.

4. The process of claim 1 , wherein the first host cell in the culturing step is cultured to an OD600 of about 50.

5. The process of claim 1 , wherein the second host cell in the culturing step is cultured to an OD600 of about 100.

6. The process of claim 1 , wherein the first polynucleotide is nt 8 to nt 718 of SEQ ID NO: 1.

7. The process of claim 1 , wherein the second polynucleotide is nt 9 to nt 770 of SEQ ID NO: 3.