Quantitative NAT for pathogen inactivation verification.

A novel Quantitative Nucleic Acid Test (Q-NAT) technology has been developed to demonstrate, quantify and verify pathogen inactivation by methods that break pathogen nucleic acids, specifically, gamma irradiation. The Q-NAT technology provides significant advantages in cost, efficiency and broad applicability compared with traditional methods for pathogen inactivation detection and quantification such as cell culture.

Gamma irradiation of intravenous immunoglobulin.

This paper describes gamma irradiation of a biotherapeutic product under conditions (the Clearant Process") that protect proteins and foster inactivation of viruses and other pathogens. The treated product was immunoglobulin paste from cold ethanol fractionation of human plasma, a process intermediate in the production of intravenous immunoglobulin (IGIV). The frozen paste was irradiated on dry ice to 45 kGy, conditions that inactivate > or = 4 log10 of non-enveloped viruses and > or = 6 log10 of enveloped viruses. When IGIV purified from the irradiated paste was characterized, no protein aggregation, fragmentation, oxidation or denaturation was detected and Fab functionality remained intact.

Inactivation of viral and prion pathogens by gamma-irradiation under conditions that maintain the integrity of human albumin.

BACKGROUND AND OBJECTIVES: The administration of therapeutic plasma protein concentrates has been associated with the real risk of transmitting viral diseases and the theoretical risks of prion transmission. Our objective was to determine if gamma-irradiation can inactivate viral or prion infectivity without damaging a protein biotherapeutically. MATERIALS AND METHODS: Human albumin 25% solution, spiked with four model viruses (including porcine parvovirus) or with brain homogenate from scrapie-infected hamsters, was gamma-irradiated at constant low-dose rates and assayed for viral and prion infectivity or for albumin integrity. RESULTS: At a radiation dose of 50 kGy, viruses were inactivated by >/= 3.2 to >/= 6.4 log10 and scrapie by an estimated 1.5 log10, whereas albumin was only moderately aggregated and fragmented. CONCLUSIONS: gamma-Irradiation can preferentially inactivate viral and prion pathogens without excessive damage to albumin structure.

Development of an animal model for assessment of the hemostatic efficacy of fibrin sealant in vascular surgery.

PURPOSE: Sustained hemostatic function of fibrin sealant (FS) is crucial when it is used in cardiovascular surgery. The purpose of this study was to develop a model that can determine the long-term hemostatic efficacy of tissue sealants in a vascular surgery. METHODS: To determine the ability of the model to detect differences in FS performance, various concentrations of FS were prepared and tested. Tensile strength of FS clots was determined in vitro using a tensiometer. Laparotomy was performed on 49 anesthetized rabbits, and a segment of the aorta was occluded, transected, and then sutured in an end-to-end fashion with four or eight interrupted 9-O sutures. The four-suture repair was covered with FS or placebo, and blood flow restored. Spilled blood was absorbed with gauze and weighed to estimate blood loss. Four weeks after surgery the animals were euthanized and the vessels recovered for histology. RESULTS: Average tensile strength of FS clots at 120, 90, and 60 mg/ml topical fibrinogen complex (TFC) concentration was 0.42 +/- 0.07 N, with no significant difference among them. The lowest TFC concentration, 30 mg/ml, produced weaker clots than either 120 or 90 mg/ml (P < 0.05). All rabbits with four-suture anastomoses that were treated with placebo bled to death after the vessel was unclamped (n = 6). Treatment of suture line with standard FS concentration (120 mg/ml TFC, n = 8) sealed the anastomosis and prevented blood loss. Hemostasis was sustained for 4 weeks, allowing vascular healing. All rabbits with the eight-suture anastomosis survived the operation but lost 42 +/- 9.2 ml blood (n = 5). Hemostatic efficacy of FS was unchanged when TFC was diluted to 90 mg/ml (n = 6) but further dilution to 60 mg/ml with water (n = 8) produced significantly less effective clots, with an average blood loss of 5.5 +/- 7.6 ml (P < 0.05) and two fatal clot failures postoperatively. When FS was diluted to 60 mg/ml TFC with a buffer, it maintained its hemostatic strength (n = 6). Further TFC dilution to 30 mg/ml led to consistent bleeding with an average blood loss of 35.3 +/- 10.3 ml (P < 0.001, n = 6). CONCLUSIONS: The four-suture anastomosis of rabbit aorta offers a consistent and reliable method for evaluating the short- and long-term hemostatic efficacy of FS products. This model is not only able to determine the functional differences in various concentrations of FS, but it is also sensitive to detect the subtle changes in FS preparation (e.g., medium composition) that is not detected by in vitro testing.

Metabolic and hemodynamic effects of CO2 pneumoperitoneum in a controlled hemorrhage model.

BACKGROUND: Intracavity infusion of fibrin sealant-based agents, as a novel modality to control internal bleeding, is associated with an increase of pneumoperitoneum (PP) pressure. The safe limit of such increase has not been well defined in hypovolemic subjects. The purpose of this study was to evaluate the hemodynamic and metabolic effects of increasing PP pressure and to define the limits of carbon dioxide (CO2) insufflation in a controlled hemorrhage rat model. METHODS: Ninety male rats (474 +/- 6 g, 37 degrees +/- 1 degrees C) were anesthetized, and mechanically ventilated. Animals were randomly distributed among 14 groups (n = 6-8) with an increasing amount of blood loss (0, 10, 15, and 17.5 mL/kg) and 15 minutes of CO2 insufflation at 0, 5, 10, and 15 mm Hg starting 15 minutes after hemorrhage, followed by desufflation. Mean arterial pressure (MAP), heart rate, and survival were recorded and arterial and venous blood samples were collected at baseline, at 15 minutes after hemorrhage, after insufflation, and after desufflation procedures to determine arterial blood gases and lactic acid levels. RESULTS: In nonhemorrhaged animals, increasing PP pressure up to 15 mm Hg produced only transient changes in MAP and no increase in lactate level. A moderate hemorrhage (10 mL/kg) limited the safe abdominal pressure to 10 mm Hg with metabolic changes that were restored 15 minutes after desufflation. Higher PP pressure (15 mm Hg) at this hemorrhage level produced a significant decline in MAP (42%, p < 0.001) and progressive metabolic acidosis with a 2.1-fold increase (p < 0.01) in lactate level. The more severe hemorrhage (15 mL/kg) further reduced the limits of PP pressure such that 10 and 15 mm Hg resulted in a progressive decline of blood pressures (52% and 54%, respectively; p < 0.001) and severe metabolic acidosis as manifested by 3.3- and 3.1-fold rises in lactate levels, respectively. In the most severe hemorrhaged animals (17.5 mL/kg), the 50% mortality was primarily determined by the severity of the blood loss and the additional PP at 5 mm Hg had no significant impact. CONCLUSION: The safe limit of PP pressurization with CO2 is dependent on the amount of blood loss. In this mechanically ventilated rat model, increasing the amount of blood loss from 0 to 15 mL/kg reduces the tolerable level of abdominal insufflation pressure from 15 mm Hg to 5 mm Hg. A 5-mm Hg PP pressure appears safe even in the most severely hemorrhaged animals.

Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels.

Colostrum and milk are natural vehicles for acquiring passive immunity and are valuable tools for decreasing neonatant mortality from diarrheal disease. The effects of recombinant human protein C (rhPC) expression levels on endogenous immunoglobulin and transferrin content of the milk of different lineages of transgenic pigs were studied. The levels of rhPC in the milk ranged from 40 to 1200 microg/ml. Transgenic pigs with rhPC expression levels less than 500 microg/ml had no significant differences in milk protein composition with respect to nontransgenic pigs. A line of transgenic pigs having rhPC expression levels of 960-1200 microg/ml had two- to three-fold higher IgG, IgM, and secretory IgA concentrations compared to other transgenic and nontransgenic pig groups (P < 0.05), and four- to five-fold higher transferrin levels than nontransgenic pigs (P < 0.05). Changes in milk protein composition were not associated with mastitis or other pathologic disruption of epithelial cell junctions as indicated by normal casein and albumin levels in milk. Since IgG, IgM, secretory IgA, and transferrin are transported into the milk by transcytosis, higher levels of these proteins indicate that transcyctosis in the mammary epithelial cell was likely upregulated in pigs having high rhPC expression levels. This study is the first that shows a statistically significant example that mammary tissue specific expression of a heterologous protein can enhance endogenous phenotypic characteristics of milk.

Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells.

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.

Assessment of topical hemostats in a renal hemorrhage model in heparinized rats.

Various topical hemostatic agents or devices have been employed to address the challenges associated with hemorrhage from parenchymal organs during surgery or trauma. Their relative efficacy, however, has not been assessed in a single animal model. The objective of this study was to develop a small animal renal hemorrhage model for comparing hemostatic efficacy of various topical agents, and then to compare fibrin sealant (FS) to an existing standard of care for topical hemostasis. A left heminephrectomy was performed in anesthetized adult male Sprague-Dawley rats. Animals were anticoagulated with 2000 IU/kg heparin IV and various topical hemostatic agents were applied to the injury. Treatment groups included FS applied as a spray; FS applied through a cannula; gelatin sponge (GS) soaked in 1000 IU/mL thrombin solution; GS soaked in 300 IU/mL thrombin; dry GS; and fibrinogen without thrombin applied as a spray. The main endpoints of the study were incidence of hemostasis, blood loss, acute survival trends, and maintenance of mean arterial pressure (MAP). Three treatment groups, the two FS groups and the GS soaked in 1000 IU/mL thrombin, afforded significant hemostasis compared to the controls (P < 0.01). Both FS groups had significantly less blood loss, longer survival times, and maintained higher MAPs than the GS-treated groups. Quantitative dose effects and functional deficiencies in topical hemostatic products could be assessed using this animal model. The study demonstrated that liquid FS was significantly more efficacious than a GS soaked in thrombin for abating hemorrhage from a renal excision in a heparinized rat.

Optimizing human demineralized bone matrix for clinical application.

The use of human demineralized bone matrix (DBM) powder in periodontal and orthopedic applications is limited by the variability in the osteoinductive or osteoconductive properties of the material. The goal of the present study was to establish simple in vitro and in vivo assays of DBM that would allow us to screen different lots of the material prior to testing in more rigorous animal models. The results demonstrate a wide variability in the performance of individual lots of DBM powder obtained from a single tissue bank. The studies also demonstrate that relatively simple screening can be used to establish the quality of the different lots, and that performance and ease of handling can be improved by using relatively small particle sizes delivered in a fibrin sealant matrix.

Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans.

BACKGROUND: Solid evidence from experimentally infected animals and fragmentary evidence from naturally infected humans indicate that blood may contain low levels of the infectious agent of Creutzfeldt-Jakob disease (CJD), yet blood components have never been identified as a cause of CJD in humans. STUDY DESIGN AND METHODS: Blood components and plasma fractions were prepared from the pooled blood of mice that had earlier been infected with a mouse-adapted strain of human transmissible spongiform encephalopathy (TSE). Infectivity bioassays were conducted in healthy mice, and the brains of all assay animals dying during the course of the experiments were examined for the presence of proteinase-resistant protein. RESULTS: Infectivity in the blood during the preclinical phase of disease occurred in the buffy coat at infectious unit (IU) levels between 6 and 12 per mL and was either absent or present in only trace amounts in plasma and plasma fractions. Infectivity rose sharply at the onset of clinical signs to levels of approximately 100 IU per mL of buffy coat, 20 IU per mL of plasma, 2 IU per mL of cryoprecipitate, and less than 1 IU per mL of fractions IV and V. Plasma infectivity was not eliminated by either white cell-reduction filtration or high-speed centrifugation. Approximately seven times more plasma and five times more buffy coat were needed to transmit disease by the intravenous route than by the intracerebral route. CONCLUSION: Epidemiologic evidence of the absence in humans of disease transmission from plasma components can probably be explained by 1) the absence of significant plasma infectivity until the onset of symptomatic disease, and comparatively low levels of infectivity during the symptomatic stage of disease; 2) the reduction of infectivity during plasma processing; and 3) the need for at least five to seven times more infectious agent to transmit disease by the intravenous than intracerebral route. These and other factors probably also account for the absence of transmission after the administration of whole blood or blood components.

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland.

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

Production of functional human alpha 1-antitrypsin by plant cell culture.

Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins.

Structural differences among monoclonal antibodies with distinct fine specificities and kinetic properties.

The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally defined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same Vk23 germ line gene, and all but HH8 use the same VH36-60 germ line gene. Of the three anti-HEL Abs, the sequences of HH26 variable regions are closest to those encoded by the respective germ line sequences. HH8 utilizes a different member of the VH36-60 gene family. Thus, the same Vk and VH genes, combined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different VH36-60 germ line genes are used to bind the same antigen (e.g. HH8 vs HH10 and HH26). While the affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, their affinities for mutated Ag vary over several orders of magnitude. Analyses of Fab binding kinetics with natural species variants and site-directed mutants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive association rate but more variable dissociation rates, and HH26 has variable association and dissociation rates. Only a few amino acid differences among the antibodies produce the observed differences in the robustness of the association and dissociation rates. Our results suggest that association and dissociation rates and mutation sensitivity of these rates may be independently modulated during antibody repertoire development.

The distribution of infectivity in blood components and plasma derivatives in experimental models of transmissible spongiform encephalopathy.

BACKGROUND: The administration of blood components from donors who subsequently develop Creutzfeldt-Jakob disease has raised the issue of blood as a possible vehicle for iatrogenic disease. STUDY DESIGN AND METHODS: We examined infectivity in blood components and Cohn plasma fractions in normal human blood that had been "spiked" with trypsinized cells from a scrapie-infected hamster brain, and in blood of clinically ill mice that had been inoculated with a mouse-adapted strain of human transmissible spongiform encephalopathy. Infectivity was assayed by intracerebral inoculation of the blood specimens into healthy animals. RESULTS: Most of the infectivity in spiked human blood was associated with cellular blood components; the smaller amount present in plasma, when fractionated, was found mainly in cryoprecipitate (the source of factor VIII) and fraction I+II+III (the source of fibrinogen and immunoglobulin); almost none was recovered in fraction IV (the source of vitamin-K-dependent proteins) and fraction V (the source of albumin). Mice infected with the human strain of spongiform encephalopathy had very low levels of endogenous infectivity in buffy coat, plasma, cryoprecipitate, and fraction I+II+III, and no detectable infectivity in fractions IV or V. CONCLUSION: Convergent results from exogenous spiking and endogenous infectivity experiments, in which decreasing levels of infectivity occurred in cellular blood components, plasma, and plasma fractions, suggest a potential but minimal risk of acquiring Creutzfeldt-Jakob disease from the administration of human plasma protein concentrates.

New methods for inactivation of lipid-enveloped and non-enveloped viruses.

Two new methods are described for inactivating lipid-enveloped and non-enveloped viruses in plasma-derived products such as coagulation factors and intravenous immunoglobulin (IGIV). Iodine/Sephadex delivers iodine to IGIV solutions in a slow, controlled way and allows for inactivation of > or = 4 logs of porcine parvovirus (PPV), a hardy non-enveloped virus, under conditions which do not measurably damage the structural or functional properties of the IGIV, and with essentially no iodination of the protein. All detectable enveloped and non-enveloped viruses were inactivated by this treatment. Gamma irradiation has been successfully used to inactivate viruses at the final vial stage in freeze-dried plasma proteins. Four logs of PPV were inactivated by irradiation in the presence of fibrinogen, factor VIII and alpha 1-proteinase inhibitor (API) at doses of 23, 28 and 30 kiloGray (kGy) respectively, while retaining 93% of fibrinogen solubility, 67% of factor VIII activity and over 80% of API activity. Bovine viral diarrhea virus (BVDV), a lipid-enveloped model for hepatitis C virus, was completely inactivated by radiation doses of 20-30 kGy in these products. Gamma irradiation was less effective in inactivating viruses in freeze-dried IGIV.

Novel delivery systems for coagulation proteins.

Long-term haemophilia prophylaxis with clotting factors administered by alternative delivery modes requires stable liquid formulations of these factors. We developed an aqueous-formulated human coagulation factor IX (hCFIX) with in vitro half-life (T 1/2) of 6 weeks at 37 degrees C and 18 months at 4 degrees C. Upon bolus subcutaneous (s.c.) injection in animals, hCFIX had a bioavailability of up to 16% compared to intravenous (i.v.) dose. When delivered by s.c. implanted pumps, hCFIX attained > 2% of normal human levels in the animal plasma. Hydrogels of hCFIX in a chitosan derivative, N,O-carboxymethyl chitosan (NOCC), released hCFIX slowly in vitro, and when injected s.c., gave prolonged plasma levels over those obtained by bolus i.v. or s.c. injection. Freeze-dried human coagulation factor VIII (hCFVIII) formulated in non-aqueous solvents had in vitro T 1/2 up to 80 days at 37 degrees C.

Haemophilia and advanced fibrin sealant technologies.

Fibrin sealant, which consists mainly of fibrinogen and thrombin, provides rapid haemostasis as well as tissue sealing and adhesion. Commercial, viral-inactivated products are available in Europe, Canada, and Japan. Liquid fibrin sealant (LFS) has been used clinically in haemophiliacs to perform dental procedures, orthopedic surgeries, non-orthopaedic surgeries, and circumcisions. LFS use is expected to increase as commercial products will soon be available in the US. Recombinant sources and transgenic animal bioreactor systems will replace plasma-derived products and become the predominant sources for this product in the next decade. Other areas of innovation include the development of fibrin sealant bandages or dressings, expandable foams, and spray powders which will provide the haemophiliac the ability to rapidly attain control of traumatic haemorrhages prior to hospital treatment with a significant reduction in the use of i.v. clotting factors. Fibrin sealant products have the potential to provide life-saving control of haemorrhage, reduction in factor dependency, lower viral exposure risk, and medical care cost reduction.

Preliminary study of biosensor optimization for the detection of protein C.

Methods to develop an immuno-optical biosensor for the detection and monitoring of Protein C (PC) concentrations are described. A tapered quartz fiber is enclosed in a glass tube (capacity approximately 300 microliters) and monoclonal antibody against PC (anti-PC) is immobilized on the surface of this fiber. PC within a sample, when injected into the chamber, will bind to the anti-PC in a specific reaction. The system is then probed with a fluorophore tagged secondary antibody against PC, also binding to PC in a specific reaction. Excitation light is applied through the fiber, and the amount of fluorescence is correlated with the PC concentration in the sample. This study offers encouraging results for the detection of PC deficiency in real-time.