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Biomedical subjects

W N Drohan

Publications and source records attributed to W N Drohan.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Rate limitations in posttranslational processing by the mammary gland of transgenic animals.

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

Amino Acid Sequence↗
PubMed1996

Activation of recombinant human protein C.

We have produced recombinant human Protein C (rHPC) in the milk of transgenic swine. After purification, we have analyzed the interaction of teh zymogen with Protac, thrombin/thrombomodulin and thrombin alone. The amidolytic and anticoagulant activities of rAPC after Protac activation were approximately 80% those of its human plasma counterpart. Upon the excision of the activation peptide by thrombin/thrombomodulin complex, both the natural and recombinant activation products had similar enzymatic and biological activities. This observation can be attributed to the difference in the mechanism of action between the two activators and structural differences between HPC and rHPC.

Animals↗
PubMed1996

Fibrin sealant: current and potential clinical applications.

There are few well-controlled studies of the clinical efficacy of fibrin sealant, defined by lives saved or reduced need for blood transfusions. Evaluation of fibrin sealant in trauma situations, e.g. liver laceration, has been difficult to perform. Only recently has fibrin sealant been actively promoted by US manufacturers as a commercially valuable alternative to the relatively inexpensive crude bovine thrombin and cryoprecipitate that are in current use. Regulatory agencies and manufacturers are aware that patients in the USA are receiving a suboptimal form of fibrin glue since cryoprecipitate is not virally inactivated and has a variable fibrinogen concentration. In addition, bovine thrombin is not regulated with respect to factor V content or any other impurities. During the past year regulatory agencies, together with manufacturers and clinicians, have begun to define clinically valid endpoints for efficacy of a commercially prepared fibrin sealant. These may include improvement in hemostasis compared with a placebo or agents considered to be 'standard of care'. Thus, the regulatory agencies may be willing to consider studies in animals that demonstrate efficacy as well as surrogate endpoints, such as reduced factor concentrate requirements in patients with severe hemophilia requiring dental extraction. As fibrin sealant becomes available in a liquid and potentially in a bandage form, it may also become an essential matrix for recombinant factors that can affect endothelial function.

Animals↗
PubMed1995

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Amino Acid Sequence↗
PubMed1995

Thermal stability and domain-domain interactions in natural and recombinant protein C.

Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.

Calcium↗
PubMed1995

Proteolytic processing of human protein C in swine mammary gland.

Maturation of human Protein C (HPC) precursor to a zymogen in the liver requires endoproteolytic cleavages after a basic dipeptide, Lys-2-Arg-1 in the propeptide and Lys156-Arg157 connecting the light and heavy chain. Recombinant human Protein C (rHPC) was expressed in the mammary gland of transgenic swine and its proteolytic processing was monitored. We found that about 10-20% of rHPC purified from the milk still retained the propeptide and 30-40% was in the single-chain form, indicating inefficient proteolytic cleavage. This demonstrates that endoprotease(s) of the swine mammary epithelial cells do not process fully the precursor of heterologous protein. rHPC was fractionated by anion exchange chromatography and polypeptides with novel N-termini at positions -1,152 and 157 were detected in addition to the known N-termini at residues -24, +1, and 158. Since rHPC was found to be stable both in milk and after purification, it is possible that these new cleavages on the amino side of arginine at dipeptide sites Lys-2-Arg-1, Lys151-Arg152, and Lys156-Arg157 could have occurred in the mammary gland. Thus, our results suggest that a portion of HPC precursor was proteolytically processed in swine mammary gland differently than those in other expression systems.

Amino Acid Sequence↗
PubMed1995

Large-scale production and properties of immunoaffinity-purified human activated protein C concentrate.

Activated protein C (APC) is a highly specific serine proteinase which functions as an important naturally occurring antithrombotic enzyme. APC also has anti-inflammatory properties. We have developed a large-scale process for the production of APC for therapeutic use starting with cryoprecipitate-poor human plasma. This report describes the process, its performance at the pilot plant scale, and the characteristics of immunoaffinity-purified human APC concentrate referred to as APC (human). The process consists of three chromatographic steps, an enzymatic conversion step, and incorporates a solvent/detergent treatment step for the inactivation of lipid-enveloped viruses. Solvent/detergent was shown to rapidly inactivate spiked HIV-1, as well as three marker viruses to nondetectable levels under process conditions. The immunoaffinity-purified protein C (PC) intermediate was enriched 13,600-fold over plasma and had a specific activity of 231 U/mg. The overall yield of the process following enzymatic conversion of the PC intermediate to APC and its processing by anion exchange chromatography was 36%. APC (human) was shown to be highly purified, functional and stable.

Antibodies, Monoclonal↗
PubMed1994

The porcine mammary gland as a bioreactor for complex proteins.

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Animals↗
PubMed1994

Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice.

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Inefficient processing of human protein C in the mouse mammary gland.

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2 kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3' flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7 mg ml-1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20-30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. The Km of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42-77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that gamma-carboxylation of rHPC is insufficient. These results show that proteolytic processing and gamma-carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.

Amino Acid Sequence↗
PubMed1994

Inactivation of lipid-enveloped and non-lipid-enveloped model viruses in normal human plasma by crosslinked starch-iodine.

BACKGROUND: The purpose of this study was to evaluate an iodine-based method of activating potentially harmful viruses that might be found in normal human plasma. STUDY DESIGN AND METHODS: A procedure has been developed for inactivating lipid-enveloped and non-lipid-enveloped model viruses in normal human plasma by using iodine complexed to crosslinked potato starch. The established conditions are an iodine concentration of 1.05 mg per mL and 60 minutes' incubation. RESULTS: Under these conditions, inactivation of more than 9 log10 of vesicular stomatitis virus, a lipid-enveloped virus, and more than 7 log10 of encephalomyocarditis virus, a non-lipid-enveloped virus, was achieved, with minimal losses of biologic activity of selected plasma proteins. Under these conditions, 70 percent of factor VIII activity, 77 percent of factor IX activity, and 100 percent of protein C activity in the plasma were retained. CONCLUSION: Crosslinked starch-iodine may be useful in the inactivation of viruses in single-donor plasma units and in pooled human plasma before fractionation.

Blood↗