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.

Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups.

The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethyl-maleic anhydride prior to processing. Immobilon AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected epsilon-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzene sulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 microliter) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.

Current progress in the production of recombinant human fibrinogen in the milk of transgenic animals.

The mammary gland of transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein. While the mammary gland appears to be able to secrete fully assembled recombinant human fibrinogen (rhfib) at 0.1 to 5 g/l levels, some unassembled rhfib chains are also secreted. Presently, the relationship between unassembled rhfib and the coordinated translation of each nascent rhfib polypeptide in the mammary epithelia is unknown. The secretion of fully and partially assembled rhfib is widely variable among mammalian cell lines and where previously no cell line has been shown to secrete beta chain alone. We have observed that mammary epithelia can secrete B beta chain into milk as well as immature forms of other recombinant proteins, suggesting it likely uses a different secretory pathway than does the liver. This difference in secretory behavior is possibly due to the natural design of milk, where the precise regulation of post translational modifications and intracellular pools of nascent polypeptides needed to achieve fibrinogen assembly may be less important to fulfill the nutritional function of most milk proteins.

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.

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.

Zn(2+)-selective purification of recombinant proteins from the milk of transgenic animals.

The milk of transgenic livestock is becoming a viable, large-scale source of post-translationally complex, recombinant therapeutic proteins. Recombinant vitamin K-dependent proteins such as human protein C (rhPC) and Factor IX can be produced in milk. However, rate limitations in post-translational modification such as intrachain proteolytic cleavage and gamma-carboxylation occur in the mammary gland. Thus, most desirable recombinant products often exist as sub-populations in milk because the mammary gland tends to secrete incompletely processed polypeptides. In general, a nonaffinity purification strategy by which to purify mature recombinant proteins from milk is desirable. Zn2+ is used to selectively modify ion-exchange adsorption behavior of endogenous and recombinant milk proteins through conformational changes which cause aggregation and or precipitation. Zn(2+)-selective precipitation of milk and recombinant proteins results in the purification of active rhPC at high yield from the milk of transgenic pigs using expanded bed chromatography. This method selects for rhPC which is both heterodimeric and properly gamma-carboxylated. Due to the homology of milk proteins among different species, this same Zn(2+)-selective precipitation strategy is useful for developing purification methods for other recombinant proteins from the milk of transgenic livestock.

Ovulation rate, zygote recovery and follicular populations in FSH-superovulated goats treated with PGF(2alpha) and/or GnRH.

Follicular development and ovulation were examined in superovulated Nubian and Nubian-cross dairy goats following prostaglandin F(2alpha) (PGF(2alpha)) and/or gonadotropin releasing hormone (GnRH) treatment. Estrus was synchronized with Synchromate-B((R)) implants. Superovulation was induced with follicle stimulating hormone (FSH) and augmented with GnRH and/or PGF(2alpha). The PGF(2alpha) treatment was administered on Day 2 of superovulation. Implants were removed from all goats on Day 3 of superovulation. The GnRH treatment was administered 24 h after implant removal. All does were exposed to fertile males for 48 h at the time of GnRH injection. Surgical embryo recovery and ovarian response evaluation were conducted 64 to 78.5 h after implant removal. The number of ovulations was higher with GnRH treatment (18.5 +/- 7; x +/- SEM) than that in the controls (5.3 +/- 4.1; P < 0.05). There were fewer follicles in the GnRH-treated does than in the untreated does (10.9 +/- 2.9 vs 22.1 +/- 3.2; P < 0.05). The number of follicles smaller than 4 mm in diameter (5.8 +/- 0.8) did not differ between treatments. The GnRH-treated does had fewer 4- to 8-mm follicles (4.2 +/- 2.0 vs 9.1 +/- 1.6; P < 0.05) and fewer follicles larger than 8 mm (0.7 +/- 1.4 vs 7.3 +/- 1.6; P < 0.01) than the controls. Predicted times for 1- and 2-cell embryo recoveries were 68.5 and 73.7 h following implant removal, respectively. This study demonstrates that GnRH is an effective supplement used with FSH superovulation regimens in dairy goats. Moreover, GnRH provides for enhanced early embryo collection for DNA microinjection studies.

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer.

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.

Effect of culture conditions, donor age, and injection site on in vitro development of DNA microinjected porcine zygotes.

A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37 degrees C or 39 degrees C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. Mean percentages developing to the expanded or hatched blastocyst stage in modified NCSU-23 (42.9) and modified NCSU-37 (40.1) did not differ, but development was greater than that for zygotes cultured in CZB (8.8; P < .05). Development was greater at 39 degrees C (P < .05) than at 37 degrees C (36.5 vs 24.6%). Microinjection of DNA decreased development (P < .05) from that of noninjected controls (18.1 vs 43.1%). Zygotes from postpubertal gilts had a higher percentage (68.0) of expanded and hatched blastocysts than zygotes from prepubertal donors (29.0; P < .05). No development difference was found between DNA injection into the pronucleus (23.1%) or cytoplasm (17.4%), but development was less than for control embryos (64.9%; P < .05). DNA microinjected porcine zygotes can be successfully cultured to the expanded blastocyst stage in modified NCSU-23 and modified NCSU-37 media at 39 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro development of zygotes from prepubertal gilts after microinjection of DNA.

The effect of pronuclear microinjection of DNA and culture in excised mouse oviducts on the development of porcine zygotes was assessed in this study. Precocious ovulation was induced in prepubertal gilts with pregnant mare's serum gonadotrophin and hCG. Zygotes received either pronuclear microinjection of buffer alone, buffer containing a DNA construct, or no microinjection. Zygotes were cultured in vitro in either modified Krebs-Ringer bicarbonate medium (KRB) for 144 h or in mouse oviduct (MO) explant culture with KRB for 48, 72, 96, or 120 h. Pronuclear microinjection of DNA resulted in a lower (P less than .05) cleavage index (CI) than did buffer or no microinjection (CI 2.16 +/- .10 vs 2.80 +/- .13 and 2.93 +/- .10). The CI loss was greatest for DNA-injected zygotes at the two-cell stage of development. Coculture of zygotes in MO resulted in a higher CI (P less than .01) than did culture in KRB. Culture in MO for 72 h was the most beneficial system compared with MO for 48, 96, or 120 h (P less than .05; CI 3.25 +/- .12 vs 2.66 +/- .18, 2.79 +/- .14, and 2.40 +/- .14, respectively). Microinjection of DNA, not merely the mechanical procedure, was detrimental to early zygote development and may be the cause of low pregnancy rates.

Scale-up of microbubble dispersion generator for aerobic fermentation.

A laboratory-scale microbubble dispersion (MBD) generator was shown to improve oxygen transfer to aerobic microorganisms when coupled to the conventional air-sparger. However, the process was not demonstrated on a large scale to prove its practical application. We investigated the scale-up of a spinning-disk MBD generator for the aerobic fermentation of Saccharomyces cerevisiae (baker's yeast). A 1-L spinning-disk MBD generator was used to supply air for 1- and 50-L working volume fermentation of baker's yeast. For the two levels investigated, the MBD generator maintained an adequate supply of surfactant-stabilized air microbubbles to the microorganisms at a relatively low agitation rate (150 rpm). There was a significant improvement in oxygen transfer to the microorganism relative to the conventional sparger. The volumetric mass transfer coefficient, kLa, for the MBD system at 150 rpm was 765 h(-1) compared to 937 h(-1) for the conventional sparger at 500 rpm. It is plausible to surmise that fermentation using larger working volumes may further improve the kLa values and the dissolved oxygen (DO) levels because of longer hold-up times and, consequently, improve cell growth. There was no statistically significant difference between the cell mass yield on substrate (0.43 g/g) under the MBD regime at an agitation rate of 150 rpm and that achieved for the conventional air-sparged system (0.53 g/g) at an agitation rate of 500 rpm. The total power consumption per unit volume of broth in the 50-L conventional air-sparged system was threefold that for the MBD unit for a similar product yield. Practical application of the MBD technology can be expected to reduce power consumption and therefore operating costs for aerobic fermentation.

Functional factor VIII made with von Willebrand factor at high levels in transgenic milk.

BACKGROUND: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. OBJECTIVES: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. METHODS: Transgenic mice were made with a mammary-specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, western blot, and one-stage and two-stage clotting assays. The hemostatic activity of immunoaffinity-enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. RESULTS AND CONCLUSIONS: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single-chain molecule that retained high specific activity, similar to therapeutic-grade FVIII. 226/N6 had > 450-fold higher IU mL(-1) than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle for the study of expression of 226/N6 and perhaps other single-chain bioengineered rFVIIIs in the milk of transgenic livestock.

An o-toluidine method for detection of carbohydrates in protein hydrolysates.

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.

Effect of antibody orientation on immunosorbent performance.

The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab functionality upon immobilization.

Transgenic animals as drug factories: a new source of recombinant protein therapeutics.

The utility of transgenic animal bioreactors for the production of complex therapeutic proteins is based on lower production costs, higher production capacities and safer, pathogen free products. Until gene therapy becomes broadly efficacious, transgenic-derived therapeutics are the most attractive alternative for prophylactic, replacement therapy in genetic disorders, such as haemophilia. Many other disease states need short-term treatment of significant amounts of recombinant proteins that could be made amply available from transgenic animal sources. In addition, transgenic animals will provide an ideal expression system for the production of a portfolio of alternative therapeutics for patient populations developing inhibiting antibodies, for enhanced bioactivity, or for increased plasma clearance times. The FDA approval of a transgenic-derived therapeutic is still pending, but a review of Phase I & II data from antithrombin III from goat milk is encouraging, and companies are continuing to add potential therapeutics to their product pipeline.

The use of Fab-masking antigens to enhance the activity of immobilized antibodies.

This study demonstrates that masking the Feb regions of a monoclonal antibody (Mab) with synthetic antigens prior to covalent immobilization efficiency. Water-soluble adducts of poly(2-methyloxazoline) polymers and a synthetic-peptide epitope for the Mab were constructed. These synthetic antigens are referred to as Fab-masking antigents (FMAs). The antibody used in this study is a Ca(2+)-dependent murine monoclonal lgG directed against the plasma protein, human protein C (hPC). The FMAs were pre-equilibrated with Mab in the presence of calcium prior to immobilization and were then removed by EDTA, which destabilized the FMA-Mab complexes. The antigen binding efficiency and accessibility of the Fab domain of the immobilized antibody was significantly increased for Mab immobilized in the presence of FMA relative to those Mab immobilized without FMA. The increase in binding efficiency was most pronounced for the largest FMA employed. No appreciable differences were detected in the avidity of hPC-Mab complexes formed by immunosorbents produced by either masked or unmasked antibody. These results provide evidence that orientation may play an important role in the binding activity of immobilized antibodies.