Increased branching and sialylation of N-linked glycans correlate with an improved pharmacokinetic profile for BAY 81-8973 compared with other full-length rFVIII products.

Background: BAY 81-8973 (Kovaltry) is an unmodified full-length recombinant factor VIII (rFVIII) for treatment of hemophilia A. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns of BAY 81-8973 and two other rFVIII (sucrose-formulated rFVIII [rFVIII-FS; Kogenate FS]) and antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM; Advate) and their pharmacokinetic (PK) characteristics.

BAY 81-8973, a full-length recombinant factor VIII: manufacturing processes and product characteristics.

BAY 81-8973 (Kovaltry , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human factor VIII (FVIII) approved for prophylaxis and on-demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81-8973 manufacturing process is based on the process used for sucrose-formulated recombinant FVIII (rFVIII-FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal- and human-derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81-8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII-FS cell line, a change that improved cell line robustness and productivity.

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line.

Pharmacokinetic properties of BAY 81-8973, a full-length recombinant factor VIII.

Introduction: BAY 81-8973 is a full-length recombinant factor VIII (FVIII) with the same primary amino acid sequence as sucrose-formulated recombinant FVIII (rFVIII-FS) but is produced with advanced manufacturing technologies.

Aim: To analyse the pharmacokinetics (PK) of BAY 81-8973 after single and multiple dosing across different age and ethnic groups in the LEOPOLD clinical trial programme.

Methods: The LEOPOLD trials enrolled patients with severe haemophilia A aged 12-65 years (LEOPOLD I and II) or ≤12 years (LEOPOLD Kids) with ≥150 (LEOPOLD I and II) or ≥50 (LEOPOLD Kids) exposure days to any FVIII product and no history of FVIII inhibitors.

A new large-scale manufacturing platform for complex biopharmaceuticals.

Complex biopharmaceuticals, such as recombinant blood coagulation factors, are addressing critical medical needs and represent a growing multibillion-dollar market. For commercial manufacturing of such, sometimes inherently unstable, molecules it is important to minimize product residence time in non-ideal milieu in order to obtain acceptable yields and consistently high product quality. Continuous perfusion cell culture allows minimization of residence time in the bioreactor, but also brings unique challenges in product recovery, which requires innovative solutions.

Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems.

Plants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell-based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies.

Plant-produced idiotype vaccines for the treatment of non-Hodgkin's lymphoma: safety and immunogenicity in a phase I clinical study.

Plant-made vaccines have been the subject of intense interest because they can be produced economically in large scale without the use of animal-derived components. Plant-made therapeutic vaccines against challenging chronic diseases, such as cancer, have received little research attention, and no previous human clinical trials have been conducted in this vaccine category. We document the feasibility of using a plant viral expression system to produce personalized (patient-specific) recombinant idiotype vaccines against follicular B cell lymphoma and the results of administering these vaccines to lymphoma patients in a phase I safety and immunogenicity clinical trial.

Wolman disease/cholesteryl ester storage disease: efficacy of plant-produced human lysosomal acid lipase in mice.

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Genetic LAL mutations lead to Wolman disease (WD) and cholesteryl ester storage disease (CESD). An LAL-null (lal(-/-)) mouse model resembles human WD/CESD with storage of CEs and TGs in multiple organs.

Making an ally from an enemy: plant virology and the new agriculture.

Historically, the study of plant viruses has contributed greatly to the elucidation of eukaryotic biology. Recently, concurrent with the development of viruses into expression vectors, the biotechnology industry has developed an increasing number of disease therapies utilizing recombinant proteins. Plant virus vectors are viewed as a viable option for recombinant protein production.

Synthetic melanin suppresses production of proinflammatory cytokines.

An overproduction of proinflammatory cytokines mediates the damaging sequelae of inflammation in pathologic conditions such as rheumatoid arthritis, graft-vs-host reaction, cachexia, and sepsis syndrome. We examined the cytokine regulatory activity of synthetic melanin, exemplified by biosynthetic l-glycine-l-tyrosine-based polymer (ME-1) and chemosynthetic dihydroxyphenylalanine-based polymer (MC-1). At nontoxic concentrations, both compounds effectively (>/=60%) and reversibly suppressed the production of tumor necrosis factor (TNF), even when applied after stimulation of human peripheral blood monocytes with lipopolysaccharide (LPS).

Melanin production in Escherichia coli from a cloned tyrosinase gene.

We have cloned and functionally expressed a tyrosinase gene from Streptomyces antibioticus in Escherichia coli under the control of an inducible bacteriophage T7 promoter. Recombinant E. coli cells containing the induced tyrosinase gene produced melanin pigments on agar plates and in liquid culture when supplemented with copper and tyrosine.

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba.

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities.

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid.

A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5' ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3' ends.

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient.

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr.

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba.

The cytoplasmic male sterility (CMS) trait of at least one line of Vicia faba plants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the "447" male sterile line of V.