The manufacturing process for B-domain deleted recombinant factor VIII

Eriksson, Rolf; Fenge, Christel; Lindner-Olsson, Elisabeth; Ljungqvist, Charlotta; Rosenquist, Johan; Smeds, Anna‐Lisa; Östlin, A; Charlebois, Timothy S.; Leonard, Mark; Kelley, Brian; Ljungqvist, Anders

doi:10.1016/s0037-1963(01)90105-2

Cited by 35 publications

(10 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Helixate NexGen ® are produced introducing human FVIII genes into baby hamster kidney (BHK‐21) cell line while Advate ® and Refacto ® derived from Chinese hamster ovary (CHO) cells 12–14. An inoculum, meeting strict criteria of viability, sterility and growth rate, is then fermented in a bioreactor using a batch‐refeed process.…”

Section: Introductionmentioning

confidence: 99%

Recombinant clotting factor VIII concentrates: Heterogeneity and high‐purity evaluation

et al. 2010

View full text Add to dashboard Cite

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate, Helixate NexGen and Refacto), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refacto showed glutathione S-transferase and beta-lactamase, whereas Advate apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants.

show abstract

Section: Introductionmentioning

confidence: 99%

Recombinant clotting factor VIII concentrates: Heterogeneity and high‐purity evaluation

et al. 2010

View full text Add to dashboard Cite

show abstract

“…The mAb-Seph column poorly retained model viruses, with LRVs ranging from 3.8 for PV to ≥7.9 for infectious bovine rhinotracheitis virus (bovine herpesvirus 1). The manufacturing process included three other chromatography steps using Seph-based resins (CEC, AEC, HIC) [232], not tested for viral clearance but expected to provide additional virus removal. Note, however, that the anti-FVIII mouse mAb (8A4) ligand was replaced later by a synthetic peptide (TN8.2) to avoid the use of animal-derived materials in the manufacturing process of ReFacto -leading to the third-generation product Xyntha™/ReFacto AF (albumin free) (Pfizer).…”

Section: Mab Ligands: Anti-fviii Immunoaffinity Chromatography (Iac)mentioning

confidence: 99%

Polysaccharide-based chromatographic adsorbents for virus purification and viral clearance

Junter

Lebrun

2020

Journal of Pharmaceutical Analysis

View full text Add to dashboard Cite

“…, 1988). Estudos utilizando esse tipo de construção, isto é, com o domínio B parcial ou completamente deletado para a expressão direta de moléculas de FVIII totalmente funcionais estão disponíveis na literatura (ERIKSON et al, 2001;HERLITSCHKA et al, 1998;PICANÇO et al, 2007;PIPE, 2008). Picanço et al (2007) relatou que a expressão da proteína chega a ser de 2 a 10 vezes maior quando comparado à expressão da molécula de FVIII intacta.…”

Section: Fator VIII -A Estrutura Da Proteínaunclassified

“…O tratamento por reposição do fator VIII começou a ser aplicado em meados da década de 1960, quando se desenvolveu o crioprecipitado (concentrado precipitado a frio do fator VIII), que possibilitou o isolamento desta proteína do plasma de pessoas sadias. No entanto, nas décadas subsequentes, a obtenção do fator a partir do plasma humano começou a trazer riscos significativos de transmissão do vírus HIV e das hepatites B e C. Além disso, a disponibilidade do fator VIII era limitada e insuficiente para todos os pacientes (ERIKSON et al, 2001).…”

Section: Hemofilia Aunclassified

“…De acordo com a literatura (ERIKSON et al, 2001;SOUKHAREV et al, 2002), o ReFacto ® , da Wyeth/Genetics Institute, é produzido em células CHO adaptadas ao cultivo em suspensão e, diferentemente dos produtos da Bayer e da Baxter, é um FVIII B-deletado e apresenta eficácia similar a outros produtos recombinantes e derivados de plasma sanguíneo.…”

Section: Produção De Fator VIII Em Células Animaisunclassified

See 1 more Smart Citation

Desenvolvimento de processo de produção de fator VIII recombinante em biorreator.

Andrade¹

View full text Add to dashboard Cite

The deficiency of activity of the coagulation fator VIII (FVIII) is associated to a cronical disease, heamophilia A, which is controlled by the reposition of this protein in the blood. Nowadays, in Brazil, all the factor VIII consumed is produced abroad and has blood plasma origin. However, the recombinant factor VIII (rFVIII) is a safer regarding contamination risks. Aiming to obtain the proper activity and to avoid immunogenic reactions, it is desirable to produce a rFVIII in post-translation modifications similar to those observed at the normal protein. The objective of this work was to establish a process for rFVIII production in a bench bioreactor, bubble-free, using two human cell lines transfected by Hemocentro de Ribeirão Preto -rHeLa e rSKHep. When cultured in DMEM medium with 10% of fetal bovine serum (FBS), these cell lines showed similar behaviors regarding cell growth and metabolism. Both had an average cell production (X) of 1,47x10 6 cells/mL (CV=4%) and average specific cell growth rate ( X,MÁX ) of 0,0284 h -1 (CV=2%). Results for Y X/GLC and Y LAC/GLC were also similar, with average values of 3,65x10 8 cell/g (CV=14%) and 0,734 g/g (CV=2%), respectively. The main difference was observed in the Y NH4/X value that was 4,15 times higher in the rHeLa cell line than in SKHep. The rHeLa cells were easily adapted to suspension and to serum-free, animal-component-free and chemically defined media. Preliminary studies to evaluate the best conditions to cell growth and metabolism were developed using this cell line. were developed. However, analytical techniques implemented during the project showed that rHeLa cells had lost its ability to produce the rFVIII. The rSKHep cell line couldn't be successfully adapted to suspension growth or to serum free media, so it was necessary to adopt microcarrier (mic). The studies to idenitify the best conditions for the culture system (3 gmic/L and 3 cells/mic) were performed using previous results from the rHeLa studies. Studies of rFVIII stability showed that it degradates 65% when exposed to 37 °C for 48 hours. Therefore, the perfusion mode, with and internal spinfilter, was an alternative to produce continuously a protein which activity wouldn't be seriously affected due to its low exposure time to high temperatures. The culture in perfusion mode was started with 3 cells/mic and it was used 3 g mic/L. The perfusion mode started before the interruption of the exponential growth phase, with a hydraulic residence time of 24 hours and independent glucose and glutamine feeds aiming to maintain the concentrations of these substrates at 1 and 0,35 g/L, respectively. The controlled variables (cell concentration, glucose and glutamine) were kept constant for three hydraulic residence times. The maximum rFVIII concentration obtained was similar to the one from the batch culture. However, the total amount of rFVIII produced at the perfusion mode (16,156.27 UI) was 5,5 times higher than in batch mode, resulting in a productivity of 2,5 times higher.

show abstract

The manufacturing process for B-domain deleted recombinant factor VIII

Cited by 35 publications

References 2 publications

Recombinant clotting factor VIII concentrates: Heterogeneity and high‐purity evaluation

Recombinant clotting factor VIII concentrates: Heterogeneity and high‐purity evaluation

Polysaccharide-based chromatographic adsorbents for virus purification and viral clearance

Desenvolvimento de processo de produção de fator VIII recombinante em biorreator.

Contact Info

Product

Resources

About