The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique feat... more The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique features that make it of special interest in relationship to cell replication. Principal products of the biosynthetic pathway, cholesterol, dolichyl phosphate and coenzyme Q, are essential, respectively, for plasma membrane stability and function, for arginine-linked glycoprotein synthesis and for hydrogen transport. Prenylation of important proteins such as lamin B1 and the ras oncogene protein2 may be required for their actions during cell replication, thus, together the products of this pathway influence cell structure and many cellular functions. Except for the conversion of cholesterol to other steroids in liver and endocrine cells, the polyprenyl products of the pathway do not seem to be degraded and, in replicating cells, their synthesis appears to be correlated with cell division3–5. The additional cholesterol needed for cell replication can be taken up from the extracellular medium if it is available6. But the requirement for dolichyl phosphate, coenzyme Q and for prenyl substitutuents on proteins, must be met by intracellular synthesis.
In the late 1970’s studies on the structures of peptides involved in fungi mating responses resul... more In the late 1970’s studies on the structures of peptides involved in fungi mating responses resulted in discovery of a novel post-translational modification; the covalent attachment of isoprene groups (Kamiya, et al., 1978). More recently, similar lipid modifications have been identified by the incorporation of an isoprene precursor, mevalonic acid into numerous proteins in a variety of higher eukaryotes (Schmidt, et al., 1984; Bruenger and Rilling, 1986; Maltese and Sheridan, 1987; Beck, et al., 1988). Most isoprenylated proteins contain a carboxy-terminal CaaX sequence consisting of a cysteine residue followed by two aliphatic amino acids and any amino acid. The isoprenyl groups are bound to the cysteine via a thioether linkage (Rilling, et al., 1989). In most proteins studied the three terminal residues are subsequently removed by proteolysis and the modified cysteine is carboxymethylated (Anderegg, et al., 1988). Chemical characterization shows that different isoprenoid species may be attached to different proteins; famesol (C15) and geranylgeraniol (C20) have been identified in covalent linkages to proteins (Maltese, 1990). The significance of these differences is not yet known but recent studies indicate that the precise carboxy-terminal sequence may define the type of polyprenyl group attached to cysteine.
Pda Journal of Pharmaceutical Science and Technology, Apr 18, 2016
A quantitative spectral method has been developed to precisely measure the color of protein solut... more A quantitative spectral method has been developed to precisely measure the color of protein solutions. In this method, a spectrophotometer is utilized for capturing the visible absorption spectrum of a protein solution, which can then be converted to color values (L*a*b*) which represent human perception of color in a quantitative three dimensional space. These quantitative values (L*a*b*) allow for calculating the best match of a sample's color to a European Pharmacopeia reference color solution. In order to qualify this instrument and assay for use in clinical quality control, a technical assessment was conducted to evaluate the assay suitability and precision. Setting acceptance criteria for this study required development and implementation of a unique statistical method for assessing precision in 3-dimensionsal space. Different instruments, cuvettes, protein solutions, and analysts were compared in this study. The instrument accuracy, repeatability, and assay precision were determined. The instrument and assay are found suitable for use in assessing color of drug substances and drug products and is comparable to the current European Pharmacopeia visual assessment method.
This study examined the feasibility of applying frequency-modulated spectroscopy (FMS) to test va... more This study examined the feasibility of applying frequency-modulated spectroscopy (FMS) to test vacuum seal integrity of lyophilized protein pharmaceuticals in glass vials. A lyophilized recombinant monoclonal antibody was used as an example to demonstrate that FMS is a non-destructive method that could accurately and quickly determine vial vacuum integrity within a pressure range of 0.04 to 0.5 atm. The coefficient of determination (R2) of a bench-top instrument was found to be >0.99. Only seconds were required to analyze each sample. The instrument sensitivity and specificity were 0.95 and >0.99, respectively, based on analysis of approximately 40,000 samples. Because of low energy input by the instrument, no adverse effect on the protein quality was found immediately after up to 1 h of continuous laser exposure. The laser-exposed samples had comparable stability to non-exposed control vials after 12 weeks of storage at 40 degrees C.
ABSTRACT Previous studies have established the importance of a complex, N-linked oligosaccharide ... more ABSTRACT Previous studies have established the importance of a complex, N-linked oligosaccharide chain, recognized by a monoclonal antibody (mAb 1223), in the formation of sea urchin embryonic skeletal components known as spicules. To further investigate the function of this epitope, mAb 1223 was added to primary mesenchyme (PM) cell cultures prior to spiculogenesis. The antibody did not inhibit cell migration, cell attachment, or synthesis of the filapodial networks upon which the spicules are deposited. However, it did block deposition of mineralized CaCO3 along these filapodia, strongly supporting the previously proposed role for the 1223 epitope in calcium accumulation and/or deposition. Previously the 1223 epitope has been most extensively studied in association with a mesenchyme-specific protein of 130 kDa (msp 130). It has now been established, by Western blot analysis of whole embryo and PM cell extracts using mAb 1223, that two other proteins of 205 and 250 kDa contain the 1223 epitope. A study of the developmental profiles of expression of these glycoproteins revealed that all three were first expressed just prior to spiculogenesis, consistent with a role for any or all of these proteins in this process. Additionally all three proteins incorporated ethanolamine, myristate, and palmitate, the precursors of the glycosylphosphatidylinositol (GPI) anchor. Further labeling studies revealed differences in the metabolic lability of the GPI anchor in the three proteins; pulse-chase studies demonstrated that the ethanolamine moiety was stable in msp 130, but was rapidly chased from the 205-kDa protein (T1/2 = 14 hr). Phosphatidylinositol-specific phospholipase C partially released (50%) msp 130 from the PM cell surface, whereas it had no effect on release of the 205- and 250-kDa proteins. Studies with 35SO4 labeling and PNGase F treatment directly established that all three proteins are sulfated, and that most of the sulfate is attached to the N-linked oligosaccharide chains. Thus, the three major mAb 1223-reactive glycoproteins in PM cells are also the three major proteins containing both sulfated N-linked oligosaccharide chains and GPI anchors. Further investigation of this intriguing correlation may help to define the precise function of the 1223 epitope in the process of spicule formation.
At present there is a rather clear picture of the basic steps involved in assembly of cell surfac... more At present there is a rather clear picture of the basic steps involved in assembly of cell surface glycoproteins containing N- and/or O-linked oligosaccharide chains. The earliest studies on the glycosylation of proteins utilized autoradiography and focused on the subcellular sites of protein synthesis and glycosylation. From these studies, it appeared that the assembly of the oligosaccharide chains of N-linked glycoproteins occurred in two phases: the initial phase was believed to take place in the endoplasmic reticulum, whereas the second occurred in the Golgi complex. These observations were followed by a series of important findings: the distinction between membrane-bound and free polysomes, the discovery of the signal peptide, the demonstration of the cotranslational glycosylation of ovalbumin, and the findings that, in accord with the signal hypothesis, newly translated and glycosylated proteins were sequestered within the lumen of the endoplasmic reticulum and that viral coat proteins were inserted with their carbohydrate chains facing the lumen. These findings were solidified by a number of in-depth biochemical studies on the mechanisms of assembly of the oligosaccharide chains of the glycoproteins. As a result of these studies one can draw the following general conclusions: (1) All eukaryotic cells are capable of the synthesis of glycoproteins that are destined to become components of the plasma membrane. In addition, many cell types commit a significant portion of their protein biosynthetic activity to the synthesis of secreted and/or lysosome- packaged glycoproteins. (2) The synthesis of membrane, secretory, or lysosomal glycoproteins is a highly segregated process that occurs with an intracellular membrane system composed of the endoplasmic reticulum, transfer vesicles, Golgi apparatus, and secretory vesicles. During their translation, glycosylation, and processing the glycoproteins are completely isolated from the cytoplasm and travel to the cell surface (in the case of membrane glycoproteins), to the extracellular environment (in the case of secretory glycoproteins), or the lysosomes (in the case of lysosomal enzymes) as part of, or within, these membrane compartments.
The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique feat... more The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique features that make it of special interest in relationship to cell replication. Principal products of the biosynthetic pathway, cholesterol, dolichyl phosphate and coenzyme Q, are essential, respectively, for plasma membrane stability and function, for arginine-linked glycoprotein synthesis and for hydrogen transport. Prenylation of important proteins such as lamin B1 and the ras oncogene protein2 may be required for their actions during cell replication, thus, together the products of this pathway influence cell structure and many cellular functions. Except for the conversion of cholesterol to other steroids in liver and endocrine cells, the polyprenyl products of the pathway do not seem to be degraded and, in replicating cells, their synthesis appears to be correlated with cell division3–5. The additional cholesterol needed for cell replication can be taken up from the extracellular medium if it is available6. But the requirement for dolichyl phosphate, coenzyme Q and for prenyl substitutuents on proteins, must be met by intracellular synthesis.
American Journal of Physiology-endocrinology and Metabolism, Dec 1, 1982
An assay system for measuring an active phosphate uptake in rat jejunal disks has been developed ... more An assay system for measuring an active phosphate uptake in rat jejunal disks has been developed and used to study the rat's response to 1,25-dihydroxyvitamin D3. The active component of phosphate uptake is stimulated by added KCl, fructose, sodium, and CaCl2, but not MgCl2. Half-saturation values obtained with this assay are 0.08 mM (1,25-dihydroxyvitamin D3-treated) and 0.11 mM (D-deficient). The maximal velocities are 9.6 nmol . cm-2 . min-1 (D-deficient). Using a 5-min preincubation and a 15-min incubation, a dose-response curve for 1,25-dihydroxyvitamin D3 shows 6 pmol to be the lowest dose to give a significant response and 60 pmol to be the level at which the maximal response is reached. Measurement of phosphate uptake versus time after dosing with 1,25-dihydroxyvitamin D3 (300 pmol) revealed a biphasic response with peaks at 8 and 36 h and a trough at 20 h.
PDA Journal of Pharmaceutical Science and Technology, 2020
Compendial testing methods are not required to be fully validated, but their suitability for test... more Compendial testing methods are not required to be fully validated, but their suitability for testing should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in the United States. ANVISA (Agência Nacional de Vigilância Sanitária) also requires that compendial analytical methods shall have their suitability demonstrated for the intended use by a partial validation study. Suitability verifications or partial validation can be divided into two major categories: visual and instrumental methods. For visual methods, the color and opalescence of interferences should be checked. If the color or clarity/opalescence of the sample is outside of the range of the Pharmacopeia standards/reference solutions, the validity of the test results should be evaluated. Specificity is usually waived because the methods are not specific to products, and accuracy/precision can be addressed by comparing re...
Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apop... more Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing hum...
The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique feat... more The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique features that make it of special interest in relationship to cell replication. Principal products of the biosynthetic pathway, cholesterol, dolichyl phosphate and coenzyme Q, are essential, respectively, for plasma membrane stability and function, for arginine-linked glycoprotein synthesis and for hydrogen transport. Prenylation of important proteins such as lamin B1 and the ras oncogene protein2 may be required for their actions during cell replication, thus, together the products of this pathway influence cell structure and many cellular functions. Except for the conversion of cholesterol to other steroids in liver and endocrine cells, the polyprenyl products of the pathway do not seem to be degraded and, in replicating cells, their synthesis appears to be correlated with cell division3–5. The additional cholesterol needed for cell replication can be taken up from the extracellular medium if it is available6. But the requirement for dolichyl phosphate, coenzyme Q and for prenyl substitutuents on proteins, must be met by intracellular synthesis.
In the late 1970’s studies on the structures of peptides involved in fungi mating responses resul... more In the late 1970’s studies on the structures of peptides involved in fungi mating responses resulted in discovery of a novel post-translational modification; the covalent attachment of isoprene groups (Kamiya, et al., 1978). More recently, similar lipid modifications have been identified by the incorporation of an isoprene precursor, mevalonic acid into numerous proteins in a variety of higher eukaryotes (Schmidt, et al., 1984; Bruenger and Rilling, 1986; Maltese and Sheridan, 1987; Beck, et al., 1988). Most isoprenylated proteins contain a carboxy-terminal CaaX sequence consisting of a cysteine residue followed by two aliphatic amino acids and any amino acid. The isoprenyl groups are bound to the cysteine via a thioether linkage (Rilling, et al., 1989). In most proteins studied the three terminal residues are subsequently removed by proteolysis and the modified cysteine is carboxymethylated (Anderegg, et al., 1988). Chemical characterization shows that different isoprenoid species may be attached to different proteins; famesol (C15) and geranylgeraniol (C20) have been identified in covalent linkages to proteins (Maltese, 1990). The significance of these differences is not yet known but recent studies indicate that the precise carboxy-terminal sequence may define the type of polyprenyl group attached to cysteine.
Pda Journal of Pharmaceutical Science and Technology, Apr 18, 2016
A quantitative spectral method has been developed to precisely measure the color of protein solut... more A quantitative spectral method has been developed to precisely measure the color of protein solutions. In this method, a spectrophotometer is utilized for capturing the visible absorption spectrum of a protein solution, which can then be converted to color values (L*a*b*) which represent human perception of color in a quantitative three dimensional space. These quantitative values (L*a*b*) allow for calculating the best match of a sample's color to a European Pharmacopeia reference color solution. In order to qualify this instrument and assay for use in clinical quality control, a technical assessment was conducted to evaluate the assay suitability and precision. Setting acceptance criteria for this study required development and implementation of a unique statistical method for assessing precision in 3-dimensionsal space. Different instruments, cuvettes, protein solutions, and analysts were compared in this study. The instrument accuracy, repeatability, and assay precision were determined. The instrument and assay are found suitable for use in assessing color of drug substances and drug products and is comparable to the current European Pharmacopeia visual assessment method.
This study examined the feasibility of applying frequency-modulated spectroscopy (FMS) to test va... more This study examined the feasibility of applying frequency-modulated spectroscopy (FMS) to test vacuum seal integrity of lyophilized protein pharmaceuticals in glass vials. A lyophilized recombinant monoclonal antibody was used as an example to demonstrate that FMS is a non-destructive method that could accurately and quickly determine vial vacuum integrity within a pressure range of 0.04 to 0.5 atm. The coefficient of determination (R2) of a bench-top instrument was found to be >0.99. Only seconds were required to analyze each sample. The instrument sensitivity and specificity were 0.95 and >0.99, respectively, based on analysis of approximately 40,000 samples. Because of low energy input by the instrument, no adverse effect on the protein quality was found immediately after up to 1 h of continuous laser exposure. The laser-exposed samples had comparable stability to non-exposed control vials after 12 weeks of storage at 40 degrees C.
ABSTRACT Previous studies have established the importance of a complex, N-linked oligosaccharide ... more ABSTRACT Previous studies have established the importance of a complex, N-linked oligosaccharide chain, recognized by a monoclonal antibody (mAb 1223), in the formation of sea urchin embryonic skeletal components known as spicules. To further investigate the function of this epitope, mAb 1223 was added to primary mesenchyme (PM) cell cultures prior to spiculogenesis. The antibody did not inhibit cell migration, cell attachment, or synthesis of the filapodial networks upon which the spicules are deposited. However, it did block deposition of mineralized CaCO3 along these filapodia, strongly supporting the previously proposed role for the 1223 epitope in calcium accumulation and/or deposition. Previously the 1223 epitope has been most extensively studied in association with a mesenchyme-specific protein of 130 kDa (msp 130). It has now been established, by Western blot analysis of whole embryo and PM cell extracts using mAb 1223, that two other proteins of 205 and 250 kDa contain the 1223 epitope. A study of the developmental profiles of expression of these glycoproteins revealed that all three were first expressed just prior to spiculogenesis, consistent with a role for any or all of these proteins in this process. Additionally all three proteins incorporated ethanolamine, myristate, and palmitate, the precursors of the glycosylphosphatidylinositol (GPI) anchor. Further labeling studies revealed differences in the metabolic lability of the GPI anchor in the three proteins; pulse-chase studies demonstrated that the ethanolamine moiety was stable in msp 130, but was rapidly chased from the 205-kDa protein (T1/2 = 14 hr). Phosphatidylinositol-specific phospholipase C partially released (50%) msp 130 from the PM cell surface, whereas it had no effect on release of the 205- and 250-kDa proteins. Studies with 35SO4 labeling and PNGase F treatment directly established that all three proteins are sulfated, and that most of the sulfate is attached to the N-linked oligosaccharide chains. Thus, the three major mAb 1223-reactive glycoproteins in PM cells are also the three major proteins containing both sulfated N-linked oligosaccharide chains and GPI anchors. Further investigation of this intriguing correlation may help to define the precise function of the 1223 epitope in the process of spicule formation.
At present there is a rather clear picture of the basic steps involved in assembly of cell surfac... more At present there is a rather clear picture of the basic steps involved in assembly of cell surface glycoproteins containing N- and/or O-linked oligosaccharide chains. The earliest studies on the glycosylation of proteins utilized autoradiography and focused on the subcellular sites of protein synthesis and glycosylation. From these studies, it appeared that the assembly of the oligosaccharide chains of N-linked glycoproteins occurred in two phases: the initial phase was believed to take place in the endoplasmic reticulum, whereas the second occurred in the Golgi complex. These observations were followed by a series of important findings: the distinction between membrane-bound and free polysomes, the discovery of the signal peptide, the demonstration of the cotranslational glycosylation of ovalbumin, and the findings that, in accord with the signal hypothesis, newly translated and glycosylated proteins were sequestered within the lumen of the endoplasmic reticulum and that viral coat proteins were inserted with their carbohydrate chains facing the lumen. These findings were solidified by a number of in-depth biochemical studies on the mechanisms of assembly of the oligosaccharide chains of the glycoproteins. As a result of these studies one can draw the following general conclusions: (1) All eukaryotic cells are capable of the synthesis of glycoproteins that are destined to become components of the plasma membrane. In addition, many cell types commit a significant portion of their protein biosynthetic activity to the synthesis of secreted and/or lysosome- packaged glycoproteins. (2) The synthesis of membrane, secretory, or lysosomal glycoproteins is a highly segregated process that occurs with an intracellular membrane system composed of the endoplasmic reticulum, transfer vesicles, Golgi apparatus, and secretory vesicles. During their translation, glycosylation, and processing the glycoproteins are completely isolated from the cytoplasm and travel to the cell surface (in the case of membrane glycoproteins), to the extracellular environment (in the case of secretory glycoproteins), or the lysosomes (in the case of lysosomal enzymes) as part of, or within, these membrane compartments.
The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique feat... more The branched pathway for the biosynthesis of polyisoprenoid compounds has a number of unique features that make it of special interest in relationship to cell replication. Principal products of the biosynthetic pathway, cholesterol, dolichyl phosphate and coenzyme Q, are essential, respectively, for plasma membrane stability and function, for arginine-linked glycoprotein synthesis and for hydrogen transport. Prenylation of important proteins such as lamin B1 and the ras oncogene protein2 may be required for their actions during cell replication, thus, together the products of this pathway influence cell structure and many cellular functions. Except for the conversion of cholesterol to other steroids in liver and endocrine cells, the polyprenyl products of the pathway do not seem to be degraded and, in replicating cells, their synthesis appears to be correlated with cell division3–5. The additional cholesterol needed for cell replication can be taken up from the extracellular medium if it is available6. But the requirement for dolichyl phosphate, coenzyme Q and for prenyl substitutuents on proteins, must be met by intracellular synthesis.
American Journal of Physiology-endocrinology and Metabolism, Dec 1, 1982
An assay system for measuring an active phosphate uptake in rat jejunal disks has been developed ... more An assay system for measuring an active phosphate uptake in rat jejunal disks has been developed and used to study the rat's response to 1,25-dihydroxyvitamin D3. The active component of phosphate uptake is stimulated by added KCl, fructose, sodium, and CaCl2, but not MgCl2. Half-saturation values obtained with this assay are 0.08 mM (1,25-dihydroxyvitamin D3-treated) and 0.11 mM (D-deficient). The maximal velocities are 9.6 nmol . cm-2 . min-1 (D-deficient). Using a 5-min preincubation and a 15-min incubation, a dose-response curve for 1,25-dihydroxyvitamin D3 shows 6 pmol to be the lowest dose to give a significant response and 60 pmol to be the level at which the maximal response is reached. Measurement of phosphate uptake versus time after dosing with 1,25-dihydroxyvitamin D3 (300 pmol) revealed a biphasic response with peaks at 8 and 36 h and a trough at 20 h.
PDA Journal of Pharmaceutical Science and Technology, 2020
Compendial testing methods are not required to be fully validated, but their suitability for test... more Compendial testing methods are not required to be fully validated, but their suitability for testing should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in the United States. ANVISA (Agência Nacional de Vigilância Sanitária) also requires that compendial analytical methods shall have their suitability demonstrated for the intended use by a partial validation study. Suitability verifications or partial validation can be divided into two major categories: visual and instrumental methods. For visual methods, the color and opalescence of interferences should be checked. If the color or clarity/opalescence of the sample is outside of the range of the Pharmacopeia standards/reference solutions, the validity of the test results should be evaluated. Specificity is usually waived because the methods are not specific to products, and accuracy/precision can be addressed by comparing re...
Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apop... more Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing hum...
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Papers by Bruce Kabakoff