Lentiviral vector platform for production of bioengineered recombinant coagulation factor VIII

Abstract

Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.

Figure 1

Design of ET-801i and the lentiviral production platform. (a) Design of ET-801i. The percent amino acid identities to B-domain-deleted recombinant human fVIII (h-fVIII) are 100, 90, and 88, respectively for constructs 2, 3, and 4. FVIII domain boundaries were defined using the human fVIII amino acid sequence numbering as follows: residues 1–372 (A1), 373–740 (A2), 1,649–1,689 (ap), 1,690–2,019 (A3), 2,020–2,172 (C1), and 2,173–2,332 (C2). (b) Schematic representation of the ET-801i transgene in the LentiMax expression vector. ET-801i was cloned into the LentiMax expression vector and recombinant lentivirus vector encoding the ET-801i transgene under the control of the elongation factor 1-α (EF-1α) promoter was generated using the LentiMax production system. cPPT, central polypurine tract; CTS, central termination sequence; LTR, long-terminal repeat; RRE, Rev-responsive element; WPRE, woodchuck hepatitis post-transcriptional regulatory element.

Figure 2

ET-801i production following lentiviral transduction. (a) Serial transduction of baby hamster kidney–derived (BHK-M) cells using ET-801i lentivirus was performed at a multiplicity of infection (MOI) of 5 for transductions 1–3, MOI of 10 for transduction 4, and MOI of 20 thereafter. After each transduction, fVIII production was assessed using a one-stage coagulation assay (gray bars) and proviral copy number determined by quantitative PCR (black bars). Lenti-GFP, a LentiMax lentiviral vector expressing the green fluorescent reporter protein under the control of the elongation factor-1α (EF-1α) promoter, was used as a negative control. (b) ET-801i expression from individual BHK-M clones was studied by limiting dilution of the mixed cell population post-transduction and determination of fVIII production by one-stage coagulation assay. Error bars represent one sample standard deviation. GFP, green fluorescent protein.

Figure 3

Clonal analysis of BHK-M cells expressing ET-801i. A total of 26 clones were analyzed for transgene copy number and transcript levels. (a) FVIII activity versus mRNA transcript level, (b) fVIII activity versus transgene copy number, and (c) transgene copy number versus RNA transcript levels are plotted.

Figure 4

Pilot-scale ET-801i production run. Clone 3–10 cells were cultured in 500 cm² flasks under serum-free conditions for 8 days. Media was collected daily (hatched bars) and fVIII activity in the bulk supernatant was measured. Gray bars represent fVIII activity as determined by one-stage coagulation assay and black circles represent the activation quotient as determined by a two-stage coagulation assay.

Figure 5

Analysis of purified ET-801i. (a) FVIII-containing cell culture supernatant was loaded onto an SP-Sepharose Fast Flow column and ET-801i was eluted using a linear 0.18–0.7 mol/l NaCl gradient. Fractions collected were assayed for A₂₈₀, fVIII activity, AQ, and conductivity. (b) Two µg of human fVIII and ET-801i were subjected to 4–15% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and visualized by silver staining. Where indicated, samples were treated with 100 nmol/l porcine thrombin ± PNGase F endoglycosidase for 5 minutes before analysis to demonstrate proteolytic activation and the presence of N-linked glycan modifications, respectively. BDD, B-domain-deleted; HC, heavy chain; LC, light chain; SC, single chain.

Figure 6

In vivo efficacy of ET-801i. Hemophilia A mice were infused with either saline, or ET-801i at a dose of 290 units/kg. Following administration of saline or fVIII, the mice were subjected to a hemostatic challenge of 4 mm tail transection. Total blood loss was determined over a 40-minute period.