Manufacturing issues with combining different antigens: a regulatory perspective.

The regulation of biological products is conducted within the framework Title 21 of the US Code of Federal Regulations (CFR). These regulations describe product and clinical testing requirements for drugs and biological products, as well as the requirements for licensure of such products. The requirements outlined in the CFR also apply to combination vaccines. In addition, the Center for Biologics Evaluation and Research has issued a Guidance to Industry document that discusses the manufacturing, testing, and clinical evaluation of combination vaccines. However, as the complexity of mixing the different antigens increases, the challenges associated with product development (e.g., demonstration of comparability of the components and lot consistency) require early interactions with the US Food and Drug Administration. The many areas of difficulty in the arena of combination vaccine development underscore the need for continued reevaluation of current guidance documents in addressing the increasing complexity of vaccines.

Cloning and expression of an A1 adenosine receptor from rat brain.

We have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist [3H]-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5'-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor. We conclude that we have cloned a cDNA encoding an A1 adenosine receptor linked to the inhibition of adenylyl cyclase activity.

Characterization of anti-peptide antibodies for the localization of D2 dopamine receptors in rat striatum.

Seven different peptides of 14-23 residues in length based on the predicted amino acid sequence of the cloned rat D2 receptor cDNA were used as immunogens to develop antibodies in rabbits. Two of these peptides were derived from the amino terminus and four were from the third cytoplasmic loop, including one to the splice variant insertion sequence and one to the carboxyl terminus of the receptor protein. These peptides were conjugated to bovine thyroglobulin prior to rabbit immunization. Antibody production was monitored by a solid-phase ELISA. With the exception of the carboxyl-terminal peptide, all of the peptide immunogens produced antiserum of high titer ranging from 1:10(4) to 1:10(6) on ELISA. Specificity of the reaction was demonstrated by the absence of a response in the preimmune serum and by the absence of cross-reactivity between the various antisera and the nonimmunization peptides. Moreover, preincubation of the antiserum with the immunization peptide, but not other peptides, blocked the subsequent ELISA reactions. Some of the antisera were additionally characterized by immunodot assays using solubilized rat striatal membranes blotted onto nitrocellulose. Positive reactions with antiserum dilutions of 1:500 were observed that were dependent on the presence and concentration of membrane protein and were not observed using preimmune serum. Additionally, immunofluorescent staining by the D2 receptor antiserum was observed on cells that had been transfected with the D2 receptor cDNA but not on untransfected cells. Immunoprecipitation of the photoaffinity-labelled and solubilized D2 receptor also suggested that the antisera were able to directly recognize the native receptor protein. Immunohistochemical localization of the D2 receptor in slices of fresh frozen and perfusion-fixed rat brain was performed using these antisera. Within the striatum, about 50% of the medium-sized neurons were labeled as well as large, putatively cholinergic interneurons.

Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation.

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotide-binding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the previously cloned catecholamine receptors. Northern blot analysis of various neural tissues revealed a transcript of approximately 4 kb that was predominantly located in the striatum with lesser amounts in the cortex and retina. In contrast, no mRNA was detected in the cerebellum, hippocampus, olfactory bulb, mesencephalon, or pituitary. In situ hybridization analysis also revealed a high abundance of mRNA in the striatum as well as in the olfactory tubercle. To establish the identity of this cDNA, we performed transient expression experiments in COS-7 cells. [3H]SCH-23390, a D1-selective radioligand, exhibited specific, saturable binding only in cells that were transfected with this cDNA. Competition binding analysis with a variety of dopaminergic ligands demonstrated a D1 dopaminergic pharmacology. In addition, dopamine as well as other D1-selective agonists stimulated cAMP accumulation in transfected COS-7 cells. We conclude that we have cloned a cDNA encoding the D1 dopamine receptor linked to the activation of adenylyl cyclase activity.

Multiple D2 dopamine receptors produced by alternative RNA splicing.

Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.

Stabilization of soluble active rat liver glucagon receptor.

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state.

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).

Human plasma lipid exchange protein(s): a method for separation of donor and acceptor lipoproteins by heparin-Sepharose chromatography.

The transfer or exchange of cholesteryl esters, triglycerides, and phospholipids between plasma very low (VLDL), low (LDL), and high (HDL) density lipoproteins is facilitated by specific lipid transfer proteins. The present report describes a method to separate donor and acceptor lipoprotein pools used in assays for lipid exchange activities. The method is based on the differential binding of lipoproteins to immobilized heparin. At 50 mM NaCl concentration, VLDL and LDL bind to heparin-Sepharose whereas greater than 85% of HDL is unretained; VLDL and LDL are than eluted with 300 mM NaCl, 2% sodium dodecyl sulfate with a recovery greater than 85%. The procedure is rapid and quantitative, as judged by a comparison to ultracentrifugation.