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.

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.

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.

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.

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.

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.

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.

Gel compression considerations for chromatography scale-up for protein C purification.

This work is to establish theoretical and experimental relationships for the scale-up of Immobilized Metal Affinity Chromatography (IMAC) and Immuno Affinity Chromatography for the low cost production of large quantities of Protein C. The external customer requirements for this project have been established for Protein C deficient people with the goal of providing prophylactic patient treatment. Deep vein thrombosis is the major symptom for protein C deficiency creating the potential problem of embolism transport to important organs, such as, lung and brain. Gel matrices for protein C separation are being analyzed to determine the relationship between the material properties of the gel and the column collapse characteristics. The fluid flow rate and pressure drop is being examined to see how they influence column stability. Gel packing analysis includes two considerations; one is bulk compression due to flow rate, and the second is gel particle deformation due to fluid flow and pressure drop. Based on the assumption of creeping flow, Darcy's law is being applied to characterize the flow through the gel particles. Biot's mathematical description of three-dimensional consolidation in porous media is being used to develop a set of system equations. Finite difference methods are being utilized to obtain the equation solutions. In addition, special programs such as finite element approaches, ABAQUS, will be studied to determine their application to this particular problem. Experimental studies are being performed to determine flow rate and pressure drop correlation for the chromatographic columns with appropriate gels. Void fraction is being measured using pulse testing to allow Reynolds number calculations. Experimental yield stress is being measured to compare with the theoretical calculations. Total Quality Management (TQM) tools have been utilized to optimize this work. For instance, the "Scatter Diagram" has been used to evaluate and select the appropriate gels and operating conditions via Taguchi techniques. Targeting customer requirements under the structure of TQM represents a novel approach to graduate student research in an academic institution which is designed to simulate an industrial environment.

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.

Transgenic pigs produce functional human factor VIII in milk.

Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Hemostatic efficacy of a fibrin sealant-based topical agent in a femoral artery injury model: a randomized, blinded, placebo-controlled study.

PURPOSE: The efficacy of currently available topical hemostatic agents requires the formation of fibrin generated from circulating blood. Fibrin sealant, which is prepared from high concentrations of thrombin and fibrinogen, has been used in liquid form to promote hemostasis during vascular surgery. In a blinded, randomized, placebo-controlled fashion, we evaluated a dry dressing of purified, viral-inactivated human fibrinogen and human thrombin in a large animal model of arterial injury. METHODS: Dressings were prepared by application of a layer of lyophilized human fibrin sealant or immunoglobulin G (IgG, control) to a silicone backing material. Six anesthetized female Yorkshire pigs (16 to 27 kg) received bilateral, 4 mm longitudinal femoral arteriotomies after surgical exposure of the arteries. The arteriotomies were not closed. In each animal a fibrin sealant dressing was applied to one artery and a control dressing to the other. Each dressing was secured on the arteriotomy by a mechanical device. After application of the dressings, blood flow was restored to each limb for 1 hour. The compressive device was released for 5 seconds at intervals of 15 minutes to assess hemostasis. Blood flow was measured distal to each arteriotomy with a dual-channel flowmeter to adjust equal bilateral compression. RESULTS: Blood loss (mean +/- SEM) was significantly less from the arteriotomy treated with the fibrin-based dressing compared with the control dressing (4.9 +/- 4.0 ml versus 82.3 +/- 11.1 ml; p = 0.0005). Complete hemostasis was achieved at the first 15-minute interval in five of six arteriotomies treated with fibrin sealant and in none of the six control arteriotomies during 1 hour of assessment (p = 0.03). Blood flow through each femoral artery at baseline was the same in both treatment and control arteries (fibrin sealant, 114.2 +/- 17.4 ml/min; control, 106.7 +/- 16.5 ml/min; p = 0.24) and was not significantly different throughout the experiment. CONCLUSIONS: Fibrin-based dressings provide effective hemostasis in a large animal model of arterial injury. Further development of these dressings will address optimal formulation and configuration for clinical use. Our results suggest that fibrin-based dressings will be effective in promotion of hemostasis in arterial bleeding, without compromising blood flow.

Implications of new dry fibrin sealant technology for trauma surgery.

Trauma patients have been bleeding to death for thousands of years. The methods used to control hemorrhage (tourniquets, pressure, bandages, and ligatures) have not changed for 2000 years. Technology now exists to amplify the normal clotting system with human proteins, thus providing almost instant hemorrhage control in the face of bleeding. The increasing body of clinical and animal research and safety data regarding new fibrin sealant technologies is compelling. When combined with the evolving concepts of extended trauma resuscitation, acceptance of this technology will finally add a new method of rapid, easy hemostasis to the armamentarium of the surgeon faced with an unstable hemorrhaging patient. Several important issues remain unresolved, such as optimal thrombin and fibrinogen content, amount of material required for hemostasis, long-term effects, distribution of breakdown products, and role of recombinant proteins. These issues are under active investigation. Despite these unanswered questions, the field of absorbable, off-the-shelf, rapidly active hemostatic agents that do not require refrigeration is an exciting area that should yield significant improvements in the care of injured patients.

The past, present and future of transgenic bioreactors.

Hybrid genes can control the tissue-specific synthesis of human proteins in transgenic animals. Thus, it is now possible to produce proteins of biomedical value in the body fluids or cells of transgenic livestock. In fact, the first transgenically produced protein, antithrombin III, is now in clinical trials and others will soon follow.

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C.

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100-1800 micrograms ml-1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100-400 micrograms ml-1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein.