Improved Expression of Recombinant Human Factor IX by Co-expression of GGCX, VKOR and Furin

Liu, Jianming; Jonebring, Anna; Hagström, Jonas; Nyström, Ann-Christin; Lövgren, Ann

doi:10.1007/s10930-014-9550-5

Cited by 19 publications

(22 citation statements)

References 19 publications

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“…The overall clinical experience of FVIII and FIX products with respect to HCPs and other process‐related impurities is instructive for several reasons. FVIII and FIX are complex proteins to express in mammalian cells and often they are expressed only at low levels, for example, 30 mg/L of culture . It is worth respecting the challenge this poses for the purification process.…”

Section: Host Cell Cytokine Impuritiesmentioning

confidence: 99%

“…Also, FVIII and FIX have a complex structure that requires numerous post‐translational modifications (PTMs). For example, factor IX requires γ‐carboxylation of up to 12 Glu residues in the N‐terminal Gla Domain and removal of the propeptide . These PTMs are required for functional activity but are inefficiently made in hamster cells.…”

Section: Host Cell Cytokine Impuritiesmentioning

confidence: 99%

“…These PTMs are required for functional activity but are inefficiently made in hamster cells. One strategy to boost expression level of active rFIX is to co‐express GGCX (gamma‐glutamyl carboxylase), VKOR (vitamin K epoxide reductase), and furin (a peptidase) . GGCX and VKOR are integral membrane proteins in the ER, but furin is associated with the Golgi membrane and can be produced in soluble form .…”

Section: Host Cell Cytokine Impuritiesmentioning

confidence: 99%

“…FVIII and FIX are complex proteins to express in mammalian cells and often they are expressed only at low levels, for example, 30 mg/L of culture. 55 It is worth respecting the challenge this poses for the purification process. Host cell protein impurities in vigorous fed batch cultures can run near 1 g/L, implying 97% of the starting material for purification may be host cell proteins and only 3% is the desired therapeutic.…”

Section: Hcps In Hemophilia Therapeuticsmentioning

confidence: 99%

“…For example, factor IX requires c-carboxylation of up to 12 Glu residues in the N-terminal Gla Domain and removal of the propeptide. 55 These PTMs are required for functional activity but are inefficiently made in hamster cells. One strategy to boost expression level of active rFIX is to co-express GGCX (gamma-glutamyl carboxylase), VKOR (vitamin K epoxide reductase), and furin (a peptidase).…”

Section: Hcps In Hemophilia Therapeuticsmentioning

confidence: 99%

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Experience with host cell protein impurities in biopharmaceuticals

Vanderlaan¹,

Zhu-Shimoni²,

Lin³

et al. 2018

Biotechnology Progress

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In the 40-year history of biopharmaceuticals, there have been a few cases where the final products contained residual host cell protein (HCP) impurities at levels high enough to be of concern. This article summarizes the industry experience in these cases where HCP impurities have been presented in public forums and/or published. Regulatory guidance on HCP impurities is limited to advising that products be as pure as practical, with no specified numerical limit because the risk associated with HCP exposure often depends on the clinical setting (route of administration, dose, indication, patient population) and the particular impurity. While the overall safety and purity track record of the industry is excellent, these examples illustrate several important lessons learned about the kinds of HCPs that co-purify with products (e.g., product homologs, and HCPs that react with product), and the kinds of clinical consequences of HCP impurities (e.g., direct biological activity, immunogenicity, adjuvant). The literature on industry experience with HCP impurities is scattered, and this review draws in to one reference documented examples where the data have been presented in meetings, patents, product inserts, or press releases, in addition to peer-reviewed journal articles. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:828-837, 2018.

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Section: Host Cell Cytokine Impuritiesmentioning

confidence: 99%

Section: Host Cell Cytokine Impuritiesmentioning

confidence: 99%

Section: Host Cell Cytokine Impuritiesmentioning

confidence: 99%

Section: Hcps In Hemophilia Therapeuticsmentioning

confidence: 99%

Section: Hcps In Hemophilia Therapeuticsmentioning

confidence: 99%

See 3 more Smart Citations

Experience with host cell protein impurities in biopharmaceuticals

Vanderlaan¹,

Zhu-Shimoni²,

Lin³

et al. 2018

Biotechnology Progress

View full text Add to dashboard Cite

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Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1

Pakdaman

Vatandoost

Bos

2019

Biotechnology Progress

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Replacement therapy with recombinant drugs is the main therapeutic strategy for hemophilia B patients. To reduce the production costs of recombinant coagulation factors, improvement of their expression and activity by enhancement of γ‐carboxylation might be of interest. The expression and functional activity of vitamin K‐dependent (VKD) coagulation proteins rely, in part, on the VKD process of γ‐carboxylation that is mediated by the enzymes γ‐carboxylase and vitamin K epoxide reductase (VKOR). Since the recombinant production of VKD proteins is hampered by the inefficiency of this enzymatic process, we specifically have examined the stable expression of functional blood coagulation factor IX (FIX) in HEK293 cells following transient overexpression of VKORC1 as an important part of VKOR component. Recombinant hFIX‐producing human embryonic kidney (HEK) cells were transfected to overexpress VKORC1. Following reverse transcription polymerase chain reaction (RT‐PCR) analysis, expression efficiency of the active hFIX was analyzed by performing enzyme‐linked immunosorbent assay and coagulation test. In addition, to quantify γ‐carboxylated recombinant FIX, the barium citrate method was used. Overexpression of VKORC1 in FIX‐producing HEK cells, resulting in a 3.2‐fold higher expression of functional FIX, which displayed a 1.4‐fold enhanced specific activity. Moreover, a 3.9‐fold enhanced recovery of fully γ‐carboxylated FIX following barium citrate adsorption was achieved. Collectively, these findings indicate that the overexpression of VKORC1 results in the production of higher levels of functional hFIX in HEK293 cells. The increase of the VKORC1 as a supplier of γ‐carboxylase seems to play a significant role in increasing the amount and efficiency of recombinant FIX production, thereby reducing the production costs.

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In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

et al. 2021

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Background and Aims: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids.Approach and Results: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and

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Improved Expression of Recombinant Human Factor IX by Co-expression of GGCX, VKOR and Furin

Cited by 19 publications

References 19 publications

Experience with host cell protein impurities in biopharmaceuticals

Experience with host cell protein impurities in biopharmaceuticals

Enhanced functional recombinant factor IX production by human embryonic kidney cells engineered to overexpress VKORC1

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

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