Quantitative Risk assessment of product disulfide bond reduction in a recombinant protein manufacturing

Disulfide bond reduction occurred now and then during a recombinant protein manufacturing. Their reduction often led in the loss of batches. Various prevention measures were developed. Their implementation in a manufacturing inevitably increases its operation complexity and even might impact its product qualities and process performances. It is highly desirable to apply a reduction measure only if necessary and to apply no strong measure if a moderate one works. The reduction occurrence is determined both by the reduction sensitivity of a recombinant molecule and by the reduction power of harvested cell culture fluids. The reduction power of harvested cell culture fluids varies largely and depends on cells, lysis level, growth stage, and culture conditions. The method of the quantitative risk assessment of disulfide bond reduction was reported here. The quantitative assessment is realized by comparing the reduction sensitivity of a recombinant molecule with the reduction power of its harvested cell culture fluid.

Clinical Data on Transitory Teriparatide-related Hypercalcemia in Daily Practice Treatment for Severe Menopausal Osteoporosis

Teriparatide (TPT) (C181H291N55O51S2) represents a recombinant molecule acting as human osteoanabolic agent. Transitory hypercalcemia (TH) has been reported in osteoporotic patients treated with daily TPT. Our purpose is to analyze the biochemical features as well as clinical risk factors for fractures in patients with severe osteoporosis treated with TPT who developed TH during first 12 months. This is an observational real-life study. In patients with TH biochemical blood markers like osteocalcin, CrossLaps and P1NP assessed at one year have statistically significant higher values than in subjects associating serum calcium within normal limits under the drug.

NEW PROSPECTS IN PHARMACOLOGICAL TREATMENT OF ACUTE DECOMPENSATED HEART FAILURE. EXPERIENCE WITH SERELAXIN. A CLINICAL CASE

The article tells about new prospects in the treatment of acute heart failure with Serelaxin which is a recombinant molecule identical to the human peptide hormone - H2 relaxin. The author also shares personal experience on the use of the drug. Clinical trials of Serelaxin are reviewed. A clinical case of a prolonged, 48-hour intravenous infusion of Serelaxin to a patient with acute decompensated heart failure and results of the treatment are described. The hemodynamic parameters, safety profile and clinical efficacy in this patient during treatment with Serelaxin are evaluated.

Simplified recombinant plasmin: Production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin

SummaryA simplified and fully functional deletion mutant of plasminogen was created in which the middle portion of the molecule was removed, resulting in kringle 1 attachment to the serine protease domain. This recombinant plasminogen deletion mutant, Δ(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coliT7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Δ(K2-K5)Pg to the active enzyme mutant Δ(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Δ(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Δ(K2-K5)Pg effectively bound ε-aminocaproic acid (Kd = 1 1.3 ± 2.3 μ. M) and fibrin (C50 ≈ 0.3 μM).The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Δ(K2-K5)Pg to the active recombinant enzyme, Δ(K2-K5)Pm. Additionally, Δ[K2-K5]Pm was rapidly inhibited by α2-antiplas-min(1.1±0.1x107M−1s−1)andα2-macroglobulin(7.6±0.6x105 M−1s−1). In an in-vitro model, Δ(K2-K5)Pm demonstrated fibrinolytic potency comparable to human plasma-derived plasmin. Because of their unique biochemistry, including fibrin-binding properties and rapid inhibition by α2-antiplasmin, both native plasmin and a simplified deletion mutant of plasmin are potentially safe and effective direct thrombolytic agents for various thrombotic conditions. Further studies evaluating the in-vivo pharmacologic safety and clinical efficacy of this simplified plasmin (i.e. Δ[K2-K5]Pm) are warranted.

Interaction between Two Putative Glycosyltransferases Is Required for Glycosylation of a Serine-Rich Streptococcal Adhesin

ABSTRACT Fap1, a serine-rich glycoprotein, is essential for fimbrial biogenesis and biofilm formation of Streptococcus parasanguinis (formerly S. parasanguis). Fap1-like proteins are conserved in many streptococci and staphylococci and have been implicated in bacterial virulence. Fap1 contains two serine-rich repeat regions that are modified by O-linked glycosylation. A seven-gene cluster has been identified, and this cluster is implicated in Fap1 biogenesis. In this study, we investigated the initial step of Fap1 glycosylation by using a recombinant Fap1 as a model. This recombinant molecule has the same monosaccharide composition profile as the native Fap1 protein. Glycosyl linkage analyses indicated that N-acetylglucosamine (GlcNAc) is among the first group of sugar residues transferred to the Fap1 peptide. Two putative glycosyltransferases, Gtf1 and Gtf2, were essential for the glycosylation of Fap1 with GlcNAc-containing oligosaccharide(s) in both S. parasanguinis as well as in the Fap1 glycosylation system in Escherichia coli. Yeast two-hybrid analysis as well as in vitro and in vivo glutathione S-transferase pull-down assays demonstrated the two putative glycosyltransferases interacted with each other. The interaction domain was mapped to an N-terminal region of Gtf1 that was required for the Fap1 glycosylation. The data in this study suggested that the formation of the Gtf1 and Gtf2 complex was required for the initiation of the Fap1 glycosylation and that the N-terminal region of Gtf1 was necessary.

Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.