ABSTRACT
In response to concerns raised about the quality of parenteral vancomycin products, the U.S. Food and Drug Administration (FDA) is investigating the product quality of all FDA-approved parenteral vancomycin products available in the United States. Product quality was evaluated independently at two FDA Office of Testing and Research (FDA-OTR) sites. In the next phase of the investigation, being done in collaboration with the National Institute of Allergy and Infectious Diseases, the in vivo activity of these products will be evaluated in an appropriate animal model. This paper summarizes results of the FDA investigation completed thus far. One site used a validated ultrahigh-pressure liquid chromatography method (OTR-UPLC), and the second site used the high-performance liquid chromatography (HPLC) method for related substances provided in the British Pharmacopeia (BP) monograph for vancomycin intravenous infusion. Similar results were obtained by the two FDA-OTR laboratories using two different analytical methods. The products tested had 90 to 95% vancomycin B (active component of vancomycin) by the BP-HPLC method and 89 to 94% vancomycin by OTR-UPLC methods. Total impurities were 5 to 10% by BP-HPLC and 6 to 11% by OTR-UPLC methods. No single impurity was >2.0%, and the CDP-1 level was ≤ 2.0% across all products. Some variability in impurity profiles of the various products was observed. No adverse product quality issues were identified with the six U.S. vancomycin parenteral products. The quality parameters of all parenteral vancomycin products tested surpassed the United States Pharmacopeia acceptance criteria. Additional testing will characterize in vivo performance characteristics of these products.
Subject(s)
Vancomycin , Consumer Product Safety , United States , United States Food and Drug AdministrationABSTRACT
Decorin-binding lipoprotein, lpp-DBP, a bacterial surface adhesin, shows promise as a vaccine against Lyme disease. It is expressed in recombinant E. coli as an undesirable 20.5 KDa apoprotein that is subsequently lipidated in vivo to the desired 22 KDa lpp-DBP form. This study defines fermentation conditions for maximizing lpp-DBP yield. Super broth medium, a low post-induction temperature (30 degrees C), and a glucose feed based on dissolved oxygen resulted in high lpp-DBP yield and minimized apoprotein formation. Since cells lysed within 2-3 h after induction, the cell yield was maximized by growing cells to high cell density prior to induction. Compared to a glucose feed based on maintaining a constant fermentor glucose concentration (Glucose-Stat), feeding based on maintaining a constant dissolved oxygen level (DO-Stat) improved yields. Also, a dissolved oxygen level of 60% (air saturation) was best, as no product degradation was detected by Western blotting and SDS-PAGE. Acetic acid levels under both modes of glucose feed were sufficiently low, and no adverse growth effects were observed.
Subject(s)
Antigens, Bacterial/genetics , Escherichia coli/genetics , Lipoproteins/genetics , Lyme Disease/immunology , Bioreactors , Carbon , Culture Media , Fermentation , Glucose , Recombinant Proteins/genetics , TemperatureABSTRACT
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.
ABSTRACT
Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability to bind antigen in the presence of either CaCl2 or excess EDTA. Three stable clones were obtained which produced antibodies that bound to protein C only in the presence of EDTA. All three antibodies bound to the light chain of protein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized by a KD of 1.4 nM. In competition studies, it was found that the relative affinity of the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor IX or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately 60 microM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K0.5 of 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineated by immunoblotting and peptide studies and found to be present in the NH2-terminal 15 residues of protein C. Although residues corresponding to positions 10-13 of human protein C were necessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likely that Ca2+ occupation of a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NH2-terminal region of protein C which is favorable for antibody interaction, whereas Ca2+ binding to the low affinity site(s), known to be present in the gamma-carboxyglutamic acid domain, causes an unfavorable conformational change.
Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/immunology , Protein C/immunology , Animals , Antibody Affinity , Antibody Specificity , Binding, Competitive , Calcium Chloride/pharmacology , Edetic Acid/pharmacology , Factor IX/immunology , Factor X/immunology , Humans , Hybridomas/immunology , Immunoblotting , Male , Mice , Mice, Inbred BALB C , Protein Conformation/drug effects , Prothrombin/immunologyABSTRACT
The majority of methods used to prepare immunosorbents immobilize antibodies through their reactive amino acid residues. The bound antibody activity of these immunosorbents is low. Hydrazide-based matrices couple antibodies through carbohydrate chains frequently located in the Fc region. This paper reports a comparative study of the performance of immunosorbents prepared by cyanogen bromide or hydrazide immobilization methods. The experiments utilized murine monoclonal antibodies to the human plasma proteins Factor IX or Protein C. The antibodies were immobilized at low densities to beaded agarose matrices which had similar properties. The hydrazide immunosorbents had binding efficiencies which were lower (anti-Factor IX) or up to 1.6-fold higher (anti-Protein C) than comparable cyanogen bromide coupled gels. However, there was no improvement in performance due to lower recoveries of bound protein from the hydrazide gels. Control experiments demonstrated that oxidation of antibody which is required for its coupling to hydrazide gels had no effect on antibody binding to antigen. Our results indicate that, as with cyanogen bromide coupling methods, site-directed immobilization through carbohydrate residues results in a restricted ability to bind to antigen. Both monoclonals were found to contain carbohydrate in their Fab' regions through which coupling may have occurred. The frequency of carbohydrate in the Fab region and the ability to control glycosylation at these sites are factors which may impact the utility of carbohydrate-directed immobilization of antibodies.
Subject(s)
Antibodies, Monoclonal , Immunosorbents , Antigens/immunology , Carbohydrates/analysis , Cyanogen Bromide , Enzyme-Linked Immunosorbent Assay , Factor IX/analysis , Factor IX/immunology , Humans , Protein C/analysis , Protein C/immunologyABSTRACT
Synthetic polymers have long been used to modify various properties of proteins such as activity and solubility. Polyethylene glycol (PEG) has been widely used to form adducts with enzymes and antibodies. In this study, the polyoxazoline family of water-soluble polymers was used to synthesize adducts containing a synthetic peptide recognized by a monoclonal antibody (MAb) directed against human protein C (hPC). This is the first application of direct conjugation of unterminated or "living" polymer to a peptide. The avidity of the antibody for the various adducts was characterized with respect to size and hydrophilicity of methyl- and ethyl-substituted polyoxazoline polymers (POX). Avidity of the adducts was not found to be dependent upon the hydrophilicity and was slightly decreased due to polymer modification. The methyl-POX-peptide adducts were found to be highly water soluble, while the ethyl-POX-peptide adducts showed sporadic problems with aqueous solubility. Because the polymer-peptide adducts retained avidity for the antibody, polyoxazoline polymers may have potential application to protein-adduct chemistry.