Stephen R. Petteway

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein

Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.

A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrPSc) can serve as a marker for TSE infectivity.

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

A 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, Delta(K2-K5)Pg, was produced in the form of inclusion bodies at the yield of up to 200 mg/l in an Escherichia coli T7 expression system. Following protein refolding and purification on lysine-Sepharose, the conversion of the recombinant molecule Delta(K2-K5)Pg to the active enzyme mutant Delta(K2-K5)Pm by plasminogen activators was evaluated, and functional characteristics of the simplified plasmin were studied. Properties of Delta(K2-K5)Pg were similar to native, human plasma-derived plasminogen. Delta(K2-K5)Pg effectively bound epsilon-aminocaproic acid (K(d)=11.3+/-2.3 microM) and fibrin (C(50) approximately 0.3 microM). The plasminogen activators streptokinase, urokinase, and tissue plasminogen activator effectively converted the recombinant zymogen Delta(K2-K5)Pg to the active recombinant enzyme, Delta(K2-K5)Pm. Additionally, Delta[K2-K5]Pm was rapidly inhibited by alpha(2)-antiplasmin (1.1+/-0.1 x 10(7) M(-1) s(-1)) and alpha(2)-macroglobulin (7.6+/-0.6 x 10(5) M(-1) s(-1)). In an in-vitro model, Delta(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 alpha(2)-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. Delta[K2-K5]Pm) are warranted.

Pathogen safety of manufacturing processes for biological products: special emphasis on KOGENATE® Bayer

Summary.  Manufacturers of human therapeutic proteins derived from biological sources continuously strive to improve the pathogen safety profiles of these products. Efforts to improve pathogen safety margins for these biological products are directed towards several areas within the manufacturing processes including: (a) sourcing and screening of raw materials (b) determining the potential for manufacturing processes to reduce pathogen titres, and (c) incorporating methods designed specifically to remove or inactivate contaminating pathogens. Methods that could potentially reduce pathogen titres are a major focus for many manufacturers. In general, these methods are grouped into two categories, pathogen clearance and pathogen inactivation. Assessments are performed on small‐scale, laboratory simulations of the manufacturing process of interest that are spiked with a known amount of a selected pathogen. These studies provide estimates of the potential for a process step to remove or inactivate a particular pathogen. There are several pathogen clearance/inactivation methods that are inherent in manufacturing processes, however, some methods are intentionally incorporated into manufacturing for the sole purpose of reducing putative pathogen titres. Not only are well‐known pathogens such as viruses targeted, but also suspected pathogens such as those associated with the transmissible spongiform encephalopathies (TSEs). The production processes for the isolation of several biological products, including recombinant KOGENATE® Bayer (Kogenate®FS), have been evaluated for the ability to reduce pathogen titres and/or have been designed to incorporate methods for reducing potential pathogen safety risks. Several processing steps with the potential to reduce pathogen titres have been identified.

Safety evaluation of a recombinant plasmin derivative lacking kringles 2–5 and rt-PA in a rat model of transient ischemic stroke

BackgroundTissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat.MethodsMale spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm.ResultsThe main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2–3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats.ConclusionsThe superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.