Coagulation Factor IX for Hemophilia B Therapy

Орлова, Н. А.; Ковнир, С. В.; Vorobiev, I. I.; Gabibov, A. G.

doi:10.32607/actanaturae.10645

Cited by 11 publications

(22 citation statements)

References 51 publications

(55 reference statements)

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“…One of the key quality attributes of rFIX is g-carboxylation of the GLA domain [7,21,68]. FIX's GLA domain contains 12 Glu residues which are essentially completely modified to gcarboxyglutamic acid in plasma derived FIX (pdFIX) (Fig.…”

Section: Predicting G-carboxylation Levels In Purified Rfix By Analyzmentioning

confidence: 99%

“…Mutations in the X-linked genes encoding factors VIII and IX lead to bleeding disorders called Hemophilia A and B, respectively [6]. Hemophilia B impacts ~ 1 in 25,000 male births, and is characterized by spontaneous bleeding and an inability to clot [6][7][8]. Factor IX (FIX) is a key player in the coagulation cascade, and FIX deficiency severely impacts the quality of life of affected individuals.…”

Section: Introductionmentioning

confidence: 99%

“…These rFIX are produced in mammalian expression systems to ensure native post-translational modifications (PTMs), which are required for rFIX activity, stability, and serum half-life [1,9]. rFIX industrial production is typically 100-1000 fold less for the same cell culture volume than monoclonal antibodies, indicating the presence of substantial biosynthetic bottlenecks [7,10]. Two well-known biosynthetic bottlenecks in rFIX are proteolysis of the propeptide and the gcarboxylation of the GLA domain ( [11,12], and reviewed in [10]).…”

Section: Introductionmentioning

confidence: 99%

“…Measuring PTMs on purified biopharmaceutical proteins is critical, given the key role of PTMs in determining protein stability, half-life, function, and immunogenicity [7,[37][38][39]. PTMs are routinely measured after product purification as part of the quality control process during manufacture.…”

Section: Introductionmentioning

confidence: 99%

“…The AP contains two N-glycans at N 157 and N 167 , four O-glycans at T 159 , T 169 , T 172 , and T 179 , sulfation of Y 155 , and phosphorylation of S 158 and T 159 . The serine protease domain (orange) contains one Nlinked glycan at N 258 .FIX is a highly post-translationally modified glycoprotein [7,14] (Fig. 1).…”

mentioning

confidence: 99%

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Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Zacchi

Recinos

Pegg

et al. 2020

Preprint

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Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete g-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX posttranslational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, g-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses. Figure 1: Coagulation Factor IX (FIX) structural domains and post-translational modifications (PTMs). Schematic of mature FIX with previously described PTMs in plasma derived FIX and/or recombinant FIX (figure modified from [13]). The GLA domain (red) contains 12 potential sites of g-carboxylation on E 7-40 and one O-glycan at T 38/39 . The EGF-like 1 domain (pink) contains two O-glycans on S 53 and S 61 , b-hydroxylation of D 64 , and phosphorylation of S 68 . The EGF-like 2 domain (violet) has no known PTMs. The short domain (white) linking the EGFlike 2 domain with the activation peptide (AP, green) contains one O-glycan at S 141 . The AP contains two N-glycans at N 157 and N 167 , four O-glycans at T 159 , T 169 , T 172 , and T 179 , sulfation of Y 155 , and phosphorylation of S 158 and T 159 . The serine protease domain (orange) contains one Nlinked glycan at N 258 .FIX is a highly post-translationally modified glycoprotein [7,14] (Fig. 1). Immature FIX is composed of six structural domains: an N-terminal propeptide, the GLA domain, two consecutive EGF-like domains followed by a short linker, the activation peptide (AP), and the C-terminal serine protease domain (Fig. 1). Most described PTMs are on or near the GLA, the EGF-like 1, and the AP domains. The propeptide and the AP are removed through proteolysis events, and both proteolysis are required for function [1,9,15]. FIX's propeptide is cleaved by the trans-Golgi protease PACE/Furin during transit through the secretory pathway [16,17]. FIX's AP is removed in the blood by Factor XIa as part of the c...

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Section: Predicting G-carboxylation Levels In Purified Rfix By Analyzmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Zacchi

Recinos

Pegg

et al. 2020

Preprint

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Industrial production of clotting factors: Challenges of expression, and choice of host cells

Kumar

2015

Biotechnology Journal

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The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored.

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Proteome Multimarker Panel With Multiple Reaction Monitoring–Mass Spectrometry for Early Detection of Hepatocellular Carcinoma

Yeo

Kim²,

Kim

et al. 2020

Hepatol Commun

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There is an urgent need for new biomarkers that address the shortcomings of current screening methods which fail to detect a large proportion of cases with hepatocellular carcinoma (HCC) at early stage. To develop a robust, multiple‐biomarker panel based on multiple reaction monitoring–mass spectrometry with high performance in detecting early‐stage HCC within at‐risk populations. In the discovery set, 150 samples were analyzed to identify candidate biomarkers. The resulting list of candidates was tested in the training set (713 samples) to establish a multimarker panel, which was evaluated in the validation set (305 samples). We identified 385 serum HCC biomarker candidates in the discovery set and developed a multimarker panel consisting of 28 peptides that best differentiated HCC from controls. The area under the receiver operating characteristic curve of multimarker panel was significantly higher than alpha‐fetoprotein (AFP) in the training (0.976 vs. 0.804; P < 0.001) and validation (0.898 vs. 0.778; P < 0.001) sets. In the validation set, this multimarker panel, compared with AFP, showed significantly greater sensitivity (81.1% vs. 26.8%; P < 0.001) and lower specificity (84.8% vs. 98.8%; P < 0.001) in detecting HCC cases. Combining AFP with the multimarker panel did not significantly improve the area under the receiver operating characteristic curve compared with the panel alone in the training (0.981 vs. 0.976; P = 0.37) and validation set (0.906 vs. 0.898; P = 0.75). Conclusion: The multiple reaction monitoring–mass spectrometry multimarker panel consisting of 28 peptides discriminates HCC cases from at‐risk controls with high performance and may have potential for clinical application in HCC surveillance.

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Coagulation Factor IX for Hemophilia B Therapy

Cited by 11 publications

References 51 publications

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Industrial production of clotting factors: Challenges of expression, and choice of host cells

Proteome Multimarker Panel With Multiple Reaction Monitoring–Mass Spectrometry for Early Detection of Hepatocellular Carcinoma

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