Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product.

This report summarizes the identification of six cyclic imide [Asu] and two deamidation products from a sample of pramlintide final drug product that had been stressed at 40 degrees C for 45 days. The pramlintide degradation products were isolated by cation exchange high-performance liquid chromatography (HPLC) followed by reversed-phase HPLC. The isolated components were characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessary, by enzymatic (thermolysin) digestion followed by liquid chromatography/mass spectrometry (LC/MS) and sequence analysis. The isolated products were identified as [Asu14]-pramlintide, [Asu21]-pramlintide, [Asu22]-pramlintide, [Asu35]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-succinimide-pramlintide. Also identified were [Asp35]-pramlintide, the deamidation product of pramlintide at Asn35, and [Tyr37-OH]-pramlintide, the deamidation product of the pramlintide amidated C-terminal Tyr. Together these data support those presented earlier (C. Hekman et al., Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product. Pharm Res 1998;15:650-9) indicating that the primary mechanism of degradation for pramlintide in this pH 4.0 formulation is deamidation, with six of the eight possible deamidation sites observed to undergo deamidation. Gln-10 and Asn-31 are the only two residues subject to deamidation for which none is observed. The data indicate that the cyclic imide products account for approximately 20% of the total thermal degradation while the deamidation products account for 64%. The remaining degradation is due to peptide backbone hydrolysis.

Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product.

PURPOSE: This report summarizes the identification of nine deamidation and four hydrolysis products from a sample of pramlintide injection final drug product that was subjected to stress at 40 degrees C for 45 days. METHODS: The pramlintide degradation products were isolated by strong cation exchange HPLC followed by reversed-phase HPLC. Subsequent to isolation, the molecular weight of each component was determined by liquid chromatography-mass spectrometry (LC/MS). Further characterization was accomplished by amino acid sequence analysis and/ or enzymatic (thermolysin) digestion followed by LC/MS and sequence analysis. RESULTS: The isolated products were identified as [iso-Asp21]-pramlintide, [iso-Asp3]-pramlintide, and [iso-Asp22]-pramlintide, the deamidation products of pramlintide with rearrangement at Asn21, Asn3, and Asn22, respectively. Also found were [Asp/iso-Asp14]-pramlintide, and [Asp/iso-Asp35]-pramlintide, the deamidation products at Asn14, and Asn35, and [Asp21]-pramlintide together with [Asp22]-pramlintide. For the deamidations at the 14th and 35th residues, it could not be determined whether the substance corresponded to the Asp or the iso-Asp product. The [Asp21] and [Asp22] products could not be separated from each other chromatographically but were both identified in a single fraction. Two minor degradation products were also identified as deamidated species. However, the sites of deamidation remain unknown. Also identified were [1-18]-pramlintide, [1-19]-pramlintide, [19-37]-pramlintide, and [20-37]-pramlintide, the products of hydrolytic peptide backbone cleavage at amino acids His18/Ser19 and Ser19/Ser20, respectively. One other product was isolated and tentatively identified as a cyclic imide intermediate preceeding deamidation. CONCLUSIONS: The primary mode of thermally induced degradation for this peptide is deamidation. A second degradation mechanism is peptide backbone hydrolysis.

A spectrophotometric method for the quantitative determination of purity of diethylenetriaminepentaacetic anhydride.

A spectrophotometric method has been developed for the quantitation of purity of diethylenetriaminepentaacetic anhydride (DTPA-A). This spectrophotometric method is conducted by measuring the absorbance at 275 nm of a 1.5 mg/ml solution of DTPA-A in dry dimethylformamide (DMF). The extinction coefficient at 275 nm for DTPA-A in DMF has been determined to be 0.437 ml/cm mg. Quantitation is accomplished by calculation using Beer's law. The assay response is linear for DTPA-A concentrations ranging from 0.05 to 2.0 mg/ml DTPA-A. The assay is specific, precise, and sensitive with a detection limit of 0.015 mg/ml DTPA-A and a quantitation limit of 0.06 mg/ml DTPA-A. The mean recovery from the sample filters employed for sample preparation in this assay is 97.8% (n = 6).

Degradation of lyophilized and reconstituted MACROSCINT (DTPA-IgG): precipitation vs. glucosylation.

Diethylenetriaminepentaacetic anhydride (DTPA) conjugated to IgG (DTPA-IgG) and labeled with 111In is useful for detecting focal sites of infection and inflammation (R.H. Rubin, A.J. Fischman, R.J. Callahan, B. Khaw, F. Keech, M. Ahmad, R. Wilkinson and H.W. Strauss, 111In-labeled nonspecific immunoglobulin scanning in the detection of focal infection, N. Engl. J. Med., 321 (1989) 935-940). MACROSCINT contains DTPA-IgG formulated as a lyophile from a citrate buffer containing maltose. Exposure of both reconstituted and lyophilized MACROSCINT to intense light resulted in degradation primarily via formation of precipitating aggregates. However, lyophilized and reconstituted MACROSCINT responded differently to thermal stress. Reconstituted MACROSCINT subjected to thermal stress (65 degrees C) also degraded through formation of precipitating aggregates. In contrast, exposure of lyophilized MACROSCINT to thermal stress (65 degrees C) resulted primarily in an increase in the molecular size of the MACROSCINT DTPA-IgG monomer. This increase in molecular size was a function of both the moisture content in the vial and the amount of time for which the sample was stressed, but was not a function of the conjugation with DTPA. Monosaccharide analysis of the samples demonstrated that this increase in molecular size corresponded to an increase in the amount of glucose covalently attached to the IgG. These data suggest that the increase in molecular size as a function of thermal stress is due to the covalent attachment of maltose, which is a glucose disaccharide present in the lyophile as an excipient, to the IgG. This degradation pathway was only observed in the lyophile.

Mitochondrial ATP synthase complex. Membrane topography and stoichiometry of the F0 subunits.

The topography of the subunits of the membrane sector F0 of the ATP synthase complex in the bovine mitochondrial inner membrane was studied with the help of subunit-specific antibodies raised to the F0 subunits b, d, 6, F6, A6L, OSCP (oligomycin-sensitivity-conferring protein), and N,N' -dicyclohexylcarbodiimide (DCCD)-binding proteolipid and to the ATPase inhibitor protein (IF1) as an internal control. Exposure of F0 subunits in inverted and right-side-out inner membranes was investigated by direct antibody binding as well as by susceptibility of these subunits to degradation by various proteases as monitored by gel electrophoresis of the membrane digests and immunoblotting with the subunit-specific antibodies. Results show that subunits b, d, F6, A6L (including its C-terminal end) and OSCP were exposed on the matrix side. Sufficient masses of these subunits to recognize antibodies or undergo proteolysis were not exposed on the cytosolic side. This was also the case for subunit 6 and the DCCD-binding proteolipid on either side of the inner membrane. Quantitative immunoblotting in which bound radio-activity from [125I]protein A was employed to estimate the concentration of an antigen in a sample allowed the determination of the stoichiometry of several F0 subunits and IF1 relative to F1-ATPase. Results showed that per mol of F1 there are in bovine heart mitochondria 1 mol each of d, OSCP, and IF1, and 2 mol each of b and F6. Subunit 6 and the DCCD-binding proteolipid could not be quantitated, because the former transferred poorly to nitrocellulose and the latter's antibody did not bind [125I]protein A.

The F0 subunits of bovine mitochondrial ATP synthase complex: purification, antibody production, and interspecies cross-immunoreactivity.

The known subunits of the membrane sector F0 of the bovine mitochondrial ATP synthase complex are subunits b, d, 6, F6, OSCP (oligomycin sensitivity-conferring protein), the DCCD (dicyclohexylcarbodiimide) binding proteolipid, and A6L. The first six subunits were purified from SMP or preparations of the ATP synthase complex, and monospecific antibodies were raised against each. The antisera were shown to be competent for immuno-blotting, and each antiserum recognized a single polypeptide of the expected Mr in preparations of the ATP synthase complex. Immunoblots utilizing antibodies to OSCP and subunits d and 6, which exhibit the same Mr on dodecyl sulfate-polyacrylamide gels, showed clearly that these polypeptides are immunologically distinct. Immunological cross-reactivity was demonstrated between bovine, human, rat, Saccharomyces cerevisiae, Paracoccus denitrificans, and Escherichia coli for subunit 6; between bovine, human, and rat for subunits b, d, OSCP, and F6; and between bovine and rat for the DCCD binding proteolipid. Anti-subunit 6 antiserum, before or after immunopurification against the ATP synthase complex, recognized a single polypeptide in the bovine ATP synthase complex and S. cerevisiae mitochondria, but two polypeptides of different Mr in bovine SMP, human, and rat mitochondria, and Paracoccus and E. coli membranes.

Energy-induced modulation of the kinetics of oxidative phosphorylation and reverse electron transfer.

The kinetics of ATP synthesis by bovine heart submitochondrial particles (SMP) are modulated by the rate of energy production by the respiratory chain between two fixed limits characterized by apparent KmADP = 2-4 microM and Vmax approximately 200 nmol of ATP min-1 (mg of SMP protein)-1 at low energy levels and apparent KmADP = 120-160 microM and Vmax = 11,000 nmol of ATP min-1 (mg of SMP protein)-1 at high energy levels. These data indicate that KmADP and Vmax increase approximately 50-fold each; therefore, there is essentially no change in the catalytic efficiency of the ATP synthase complex in going from one extreme to the other. At intermediate rates of energy production, the kinetic data required introduction of a third, intermediate KmADP. A KmADP of 10-15 microM fitted all the data reported here and previously [Matsuno-Yagi, A., & Hatefi, Y. (1986) J. Biol. Chem. 261, 14031-14038]. However, this is not meant to suggest that there is a fixed intermediate KmADP, as the transition from one fixed limit to the other may be fluid or involve more than one intermediate state. In addition, it has been shown that kinetic plots of SMP-catalyzed and ATP-driven reverse electron transfer from succinate to NAD are curvilinear and resolvable into a minimum of two apparent KmNAD values of about 20-30 and 200-300 microM. These results have been discussed in relation to the three potentially active catalytic sites of F1-ATPase and the structure of the NADH:ubiquinone oxidoreductase complex, the curvilinear kinetics of ATP hydrolysis, and changes in KmADP and KmPi in photophosphorylation as affected by the duration and intensity of light.

Bovine plasminogen activator inhibitor 1: specificity determinations and comparison of the active, latent, and guanidine-activated forms.

The plasminogen activator inhibitor 1 (PAI-1) synthesized and released by cultured bovine aortic endothelial cells is present in conditioned medium in a latent form that can be activated by guanidine hydrochloride [Hekman, C. M., & Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587]. The purified, guanidine-activated PAI-1 was shown to inhibit both plasmin and trypsin in a dose- and time-dependent manner. Second-order rate constants for these interactions were calculated to be 6.6 X 10(5) and 7.0 X 10(6) M-1 s-1 for plasmin and trypsin, respectively. Experiments were conducted to compare the inherently active and the guanidine-activated forms of PAI-1. The two active forms had similar kinetic parameters for interaction with urokinase (Kd, 0.3 pM; kassoc, 1.5 X 10(8) M-1 s-1) and were both inactivated upon treatment with acid or base and by incubation at 37 degrees C. The latent form was relatively stable when incubated under similar conditions. The decrease in PAI-1 activity upon incubation at 37 degrees C was partially restored by a second treatment with guanidine hydrochloride. However, the degree of recovery decreased as a function of incubation time at 37 degrees C. These data suggest that active and guanidine-activated PAI-1 represent a single form of PAI-1. Incubation of this form at 37 degrees C yields two distinct populations of inactive PAI-1, one capable of reactivation and another that appears to be irreversibly inactivated.

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator.

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.

Mechanism of protein C-dependent clot lysis: role of plasminogen activator inhibitor.

The mechanism by which activated protein C stimulates fibrinolysis was studied in a simple radiolabeled clot lysis assay system containing purified tissue-type plasminogen activator, bovine endothelial plasminogen activator inhibitor (PAI), plasminogen, 125I-fibrinogen and thrombin. Fibrinolysis was greatly enhanced by the addition of purified bovine activated protein C; however, in the absence of PAI, activated protein C did not stimulate clot lysis, thus implicating this inhibitor in the mechanism. In clot lysis assay systems containing washed human platelets as a source of PAI, bovine-activated protein C-dependent fibrinolysis was associated with a marked decrease in PAI activity as detected using reverse fibrin autography. Bovine-activated protein C also decreased PAI activity of whole blood and of serum. In contrast to the bovine molecule, human-activated protein C was much less profibrinolytic in these clot lysis assay systems and much less potent in causing the neutralization of PAI. This species specificity of activated protein C in clot lysis assays reflect the known in vivo profibrinolytic species specificity. When purified bovine-activated protein C was mixed with purified PAI, complex formation was demonstrated using immunoblotting techniques after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These observations suggest that a major mechanism for bovine protein C-dependent fibrinolysis in in vitro clot lysis assays involves a direct neutralization of PAI by activated protein C.

Denaturant-induced stimulation of the beta-migrating plasminogen activator inhibitor in endothelial cells and serum.

Cultured bovine aortic endothelial cells and human serum contain plasminogen activator inhibitors (PAIs) that are immunologically related. In the present study, the electrophoretic mobilities, molecular weights (mol wt), and activities of these PAIs were compared. When fractionated by agarose zone electrophoresis, both PAIs migrated with beta mobility as compared with the mobilities of human plasma/serum proteins. Two-dimensional electrophoretic analysis, employing agarose zone electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension, indicated that these beta-PAIs comigrated, both having a mol wt of approximately 50,000. The activity of the PAI in endothelial cell-conditioned medium is enhanced severalfold by treatment with either sodium dodecyl sulfate or guanidine. In preliminary experiments, we were unable to stimulate the PAI activity of undiluted serum by similar treatments. However, the PAI activities in both diluted serum and gel-filtered or electrophoretically fractionated serum were enhanced by treatment with these denaturants. The gel filtration studies also revealed that serum contains multiple forms of the beta-PAI. These forms may represent polymeric PAI and/or complexes between the PAI and other serum components. These findings indicate that the primary PAIs in bovine endothelial cells and human serum are not only immunologically related but are also biochemically similar.

The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related.

Monospecific antiserum to an unusually stable Mr 50,000 plasminogen-activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells was employed in conjunction with reverse fibrin autography to determine whether human platelets, serum, and plasma contain immunologically related inhibitors. Reverse fibrin autography revealed the presence of a Mr 50,000 inhibitor in the platelet and serum samples but not in normal plasma. However, a Mr 50,000 inhibitor was detected in plasma obtained from individuals with increased PAI activity. In each case, treatment of the sample with the anti-inhibitor serum removed the Mr 50,000 inhibitor. The inhibitor present in each sample neutralized exogenously added tissue-type plasminogen activator in a rapid manner. Inhibition was associated with the formation of a NaDodSO4-resistant enzyme-inhibitor complex of Mr 120,000. Again, treatment of the samples with the anti-inhibitor serum removed both the inhibitory activity and the component in these samples that binds to tissue-type plasminogen activator. Thus, the rapidly acting PAI present in human platelets, serum, and patient plasma is immunologically related to the PAI synthesized by cultured bovine aortic endothelial cells. This molecule may be the physiologically relevant inhibitor of plasminogen activator in the vascular system and, as such, may serve an important role in regulating the initiation of vascular fibrinolysis.

Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants.

Conditioned medium from cultured bovine aortic endothelial cells contains an inactive plasminogen activator inhibitor (PAI). This latent PAI can be "activated" with denaturants. For example, less than 0.01 units/microliter of PAI activity was detected in untreated conditioned medium, but medium treated with sodium dodecyl sulfate (1.7 mM), guanidine HCl (4 M), urea (12 M) or KSCN (6 M) contained 0.9, 1.9, 0.8, and 0.5 units/microliter, respectively. This effect was dose-dependent with respect to the particular reagent used, and the same concentration of reagent which induced PAI activity also stimulated the ability of a component in conditioned medium to form sodium dodecyl sulfate-stable complexes with exogenously added plasminogen activators. Neither activity was stimulated by extensive dialysis or by treatment with NaCl (5 M), Na2SO4 (2.8 M), or dicetyl phosphate (0.1%). Analysis of treated and untreated conditioned medium by gel filtration revealed that the latent and active PAIs migrated with apparent Mr values of 30,000 and 50,000, respectively. Thus, "activation" is associated with an increase in the apparent Mr of the molecule. These observations suggest that activation does not result from the removal of either a small dialyzable component from the medium, or of a large Mr component that is bound to the latent PAI. Other possible mechanisms of activation are discussed. We recently isolated an active PAI from bovine endothelial cells (van Mourik, J.A., Lawrence, D.A., and Loskutoff, D.J. (1984) J. Biol. Chem. 259, 14914-14921). Monospecific antiserum to this active PAI selectivity immunoprecipitated the latent PAI from conditioned medium. These results indicate that the two PAIs are immunologically related and suggest that the latent form is converted into the active form by the sodium dodecyl sulfate present during the purification.