PB1107 - Specific Mutations in Blood Coagulation Factor VIII Have a Cumulative Effect for Increasing its Secretion

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Co-Author(s)

BS

Bo Shi

ORISE Fellow
U.S. Food and Drug Administration
Silver Spring, Maryland, United States

Co-Author(s)

Leonid Parunov, PhD

Staff Fellow
US Food and Drug Administration
Silver Spring, Maryland, United States

Co-Author(s)

PO

Philip Olivares

Staff Fellow
U.S. Food and Drug Administration
Silver Spring, Maryland, United States

Co-Author(s)

YL

Yideng Liang

Staff Scientist
US Food and Drug Administration
Silver Spring, Maryland, United States

Co-Author(s)

HC

Haarin Chun

Biologist
U.S. Food and Drug Administration
Silver Spring, Maryland, United States
SS

Svetlana A. Shestopal

Biologist
U.S. Food and Drug Administration
Silver Spring, Maryland, United States
AZ

Abu Hasanat Md Zulfiker

ORISE Fellow
U.S. Food and Drug Administration
Silver Spring, Maryland, United States

Presenting Author(s)

Andrey G. Sarafanov, PhD

Chemist
U.S. Food and Drug Administration
Silver Spring, Maryland, United States

Background: Production of recombinant blood coagulation factor VIII (FVIII), used for the therapy of its functional disorder (Hemophilia A), is known to be limited by low level of protein secretion. Previously, the mutation Phe309Ser was described to increase protein expression (Swaroop et al, 1997), as well as replacement of five specific amino acid residues (Ile86, Ala108, Gly132, Met147, and Leu152) within the same domain (A1) of FVIII into corresponding porcine residues (Cao et al, 2020). We aimed to investigate the effect of combining these mutations on FVIII expression.

Aims: To characterize protein expression for the five FVIII human to porcine replacements (x5) and the additional Phe309Ser mutation (x6) along with testing the transgene dose-dependence.

Methods: Lentivirus-based plasmid vectors with the x5 and x6 mutations within a codon optimized B domain-deleted FVIII (WT) gene were generated. Using a FVIII expression platform described previously (Shestopal et al, 2017), respective lentiviruses were transduced into Chinese Hamster Ovary cells with multiplicity of infection (MOI) 2, 4, or 8. Transgene copy number was tested by real-time (q) PCR, and media was tested for secreted FVIII by electro chemiluminescence assay (ECLA) using specific anti-FVIII antibodies.

Results: We observed a general positive correlation between increasing the number of introduced gene mutations, transgene copy number per cell, and protein secretion levels. For MOI=2, the difference in protein secretion levels between the x5 and x6 mutants was more pronounced; along with the MOI/transgene doubling, the secretion level doubled for each mutant, whereas increased only by ~60% for WT (Figure 1).

Conclusion(s): The six mutations of FVIII had cumulative effect on increasing protein secretion and were beneficial in converting the transgene number increase into additional protein secretion compared to non-mutated FVIII. The results can be useful for FVIII production for various applications.