1) Aspartame degradation kinetics depend on factors like pH, temperature, buffer type and concentration, and water activity. Higher temperatures, pH, buffer concentrations and water activities increase degradation rates.
2) The activation energies for aspartame degradation decrease with increasing pH or moisture content. Phosphate buffer significantly enhances degradation more than citrate buffer.
3) In solid systems, degradation rates increase with higher initial buffer concentrations and water activities. However, the glass transition temperature does not influence degradation rates as much as water activity.
This chapter reviews the oxidation of methionine residues in model peptides. It discusses how neighboring amino acids can influence the oxidation pattern of methionine. For example, when a hydroxyl radical attacks Thr-Met, the neighboring threonine residue is cleaved. It also notes that the oxidation of peptides and proteins is a complex process that depends on the nature of the oxidizing species and the peptide/protein sequence and structure. Oxidation can lead to chain reactions as oxidation products themselves can initiate further oxidation remote from the initial attack site.
A Novel Statistical Method for Thermostable Proteins Discrimination IJORCS
This document summarizes a novel statistical method for discriminating between thermostable proteins. The method uses amino acid frequency, dipeptide frequency, and physical-chemical features extracted from protein sequences to classify proteins as mesophilic, thermophilic, or hyper-thermophilic. The researchers tested the effect of each set of features individually and combined on classification accuracy. Their results show the proposed method using all feature types together achieved the best performance, correctly classifying over 90% of proteins on average.
The document examines the proteomic response of Saccharomyces cerevisiae to hydrogen peroxide induced oxidative stress. Twelve protein spots were analyzed using mass spectrometry, with one spot identified as the protein Sah1. Sah1 is involved in the transmethylation metabolic pathway, which produces glutathione to relieve oxidative stress. Exposure to hydrogen peroxide increased the levels of Sah1, likely to increase glutathione production and combat the reactive oxygen species. Proteomics allows the study of entire protein networks and interactions, helping uncover proteins' functions during stress responses.
Stability studies of proteins and peptides.SULABH910
This document discusses stability studies of proteins and peptides. It covers both chemical and physical degradation mechanisms and factors that influence degradation rates. Chemical degradation includes deamidation, racemization, hydrolysis, disulfide formation, oxidation, and cross-linking. Physical degradation involves changes in structure like denaturation, aggregation, adsorption, and precipitation. Degradation rates depend on factors like pH, temperature, moisture content, and excipients. Kinetics are often first-order and follow Arrhenius behavior, allowing prediction of long-term stability from accelerated studies. Understanding degradation mechanisms is key to developing stable protein and peptide drug formulations.
Introduction
Classification
Therapeutic values of peptidomimetics
Design of peptidomimetics by manipulation of amino acids
Modification of peptide backbone
Chemistry of prostaglandins, leukotrienes and thromboxanes
This chapter discusses the application of light scattering techniques to analyze the solution behavior of protein pharmaceuticals. It provides examples of using light scattering to characterize proteins and protein complexes, detect soluble aggregate formation, and elucidate protein-ligand interactions. The chapter also describes the theoretical background and instrumentation for light scattering measurements and analysis. It presents applications of light scattering including analyzing self-associating protein systems, selecting optimal solvent conditions, and studying the kinetics of molecular interactions.
A biopharmaceutical, also known as a biological medical product, biological, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semi synthesized from biological sources are called protein drugs.
Irreversible protein aggregation is problematic in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage processes. The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.
Chemical protein engineering synthetic and semisyntheticAli Hatami
This document summarizes various methods for chemically synthesizing and modifying peptides and proteins. It discusses solid phase peptide synthesis, native chemical ligation using peptide thioesters, and fragment condensation strategies. It also covers chemoselective ligations using oxime and hydrazone bonds and decarboxylative amide formation. Additionally, the document outlines chemical modifications like PEGylation, phosphorylation, and backbone modifications. Finally, it examines enzyme-mediated ligation techniques like sortase and biotin ligase that can link proteins and peptides in a sequence-specific manner.
This chapter reviews the oxidation of methionine residues in model peptides. It discusses how neighboring amino acids can influence the oxidation pattern of methionine. For example, when a hydroxyl radical attacks Thr-Met, the neighboring threonine residue is cleaved. It also notes that the oxidation of peptides and proteins is a complex process that depends on the nature of the oxidizing species and the peptide/protein sequence and structure. Oxidation can lead to chain reactions as oxidation products themselves can initiate further oxidation remote from the initial attack site.
A Novel Statistical Method for Thermostable Proteins Discrimination IJORCS
This document summarizes a novel statistical method for discriminating between thermostable proteins. The method uses amino acid frequency, dipeptide frequency, and physical-chemical features extracted from protein sequences to classify proteins as mesophilic, thermophilic, or hyper-thermophilic. The researchers tested the effect of each set of features individually and combined on classification accuracy. Their results show the proposed method using all feature types together achieved the best performance, correctly classifying over 90% of proteins on average.
The document examines the proteomic response of Saccharomyces cerevisiae to hydrogen peroxide induced oxidative stress. Twelve protein spots were analyzed using mass spectrometry, with one spot identified as the protein Sah1. Sah1 is involved in the transmethylation metabolic pathway, which produces glutathione to relieve oxidative stress. Exposure to hydrogen peroxide increased the levels of Sah1, likely to increase glutathione production and combat the reactive oxygen species. Proteomics allows the study of entire protein networks and interactions, helping uncover proteins' functions during stress responses.
Stability studies of proteins and peptides.SULABH910
This document discusses stability studies of proteins and peptides. It covers both chemical and physical degradation mechanisms and factors that influence degradation rates. Chemical degradation includes deamidation, racemization, hydrolysis, disulfide formation, oxidation, and cross-linking. Physical degradation involves changes in structure like denaturation, aggregation, adsorption, and precipitation. Degradation rates depend on factors like pH, temperature, moisture content, and excipients. Kinetics are often first-order and follow Arrhenius behavior, allowing prediction of long-term stability from accelerated studies. Understanding degradation mechanisms is key to developing stable protein and peptide drug formulations.
Introduction
Classification
Therapeutic values of peptidomimetics
Design of peptidomimetics by manipulation of amino acids
Modification of peptide backbone
Chemistry of prostaglandins, leukotrienes and thromboxanes
This chapter discusses the application of light scattering techniques to analyze the solution behavior of protein pharmaceuticals. It provides examples of using light scattering to characterize proteins and protein complexes, detect soluble aggregate formation, and elucidate protein-ligand interactions. The chapter also describes the theoretical background and instrumentation for light scattering measurements and analysis. It presents applications of light scattering including analyzing self-associating protein systems, selecting optimal solvent conditions, and studying the kinetics of molecular interactions.
A biopharmaceutical, also known as a biological medical product, biological, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semi synthesized from biological sources are called protein drugs.
Irreversible protein aggregation is problematic in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage processes. The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.
Chemical protein engineering synthetic and semisyntheticAli Hatami
This document summarizes various methods for chemically synthesizing and modifying peptides and proteins. It discusses solid phase peptide synthesis, native chemical ligation using peptide thioesters, and fragment condensation strategies. It also covers chemoselective ligations using oxime and hydrazone bonds and decarboxylative amide formation. Additionally, the document outlines chemical modifications like PEGylation, phosphorylation, and backbone modifications. Finally, it examines enzyme-mediated ligation techniques like sortase and biotin ligase that can link proteins and peptides in a sequence-specific manner.
Solution And Solid Phase Synthesis Publicationadotse
This document describes the solution- and solid-phase synthesis of peptide-substituted thiazolidinediones as potential ligands for peroxisome proliferator-activated receptors (PPARs). Initial studies focused on the low-yielding solution-phase synthesis of two target compounds. Improved yields were obtained using solid-phase synthesis and protecting the thiazolidinedione nitrogen. A small library of nine resin-bound peptide-substituted thiazolidinediones was then synthesized to examine structural features that facilitate PPAR binding and identify new PPAR activators/inhibitors.
Gawrisch Insights From Biophysical Studies On The Role Of Pufa For Function O...lab13unisa
This document summarizes recent insights into the role of polyunsaturated fatty acids, specifically docosahexaenoic acid (DHA), for the function of G-protein coupled membrane receptors like rhodopsin. It discusses how DHA chains have high conformational flexibility and tend to be located near lipid-water interfaces, altering the properties of lipid bilayers. It also presents evidence that DHA interacts weakly and nonspecifically with some sites on rhodopsin, and that certain photointermediates of rhodopsin have stronger interactions with DHA.
This document describes a new method for selective N-methylation of peptide backbone amides on resin using the Mitsunobu reaction. The key aspects are:
1) N-trifluoroacetamide (Tfa) was used as the protecting group on resin-bound peptides, which can generate a nucleophilic anion for methylation via Mitsunobu conditions.
2) Tfa-protected peptides on resin underwent efficient (80-99%) and selective N-methylation of the backbone amide using the Mitsunobu reaction with triphenylphosphine, methanol and diisopropyl azodicarboxylate.
3) Unlike other reports, the T
Characterization of mg state of mb in presence of peg 10 (z a parray) originalZahoor Parray
This document summarizes a study that characterized the intermediate state of myoglobin (Mb) in the presence of polyethylene glycol 10 (PEG 10) under physiological conditions. The researchers found that PEG 10 perturbed the tertiary structure of Mb but did not significantly change its secondary structure. PEG 10 was found to induce a molten globule state in Mb, where the intermediate state had hydrophobic patches and a larger hydrodynamic volume than the native protein. Isothermal titration calorimetry showed strong binding between Mb and PEG 10 at physiological pH. The researchers hypothesize that PEG 10 induces a molten globule conformation in Mb by interacting with its heme group. They conclude that protein-crowder interactions need careful
This document describes research on triplex formation by oligodeoxynucleotides (ODNs) containing 5-methyldeoxycytidine conjugated to spermine (5-Me-dC-N4-(spermine)). The key findings are:
1) ODNs containing 5-Me-dC-N4-(spermine) form stable triplexes at physiological pH (pH 7.3), unlike unmodified ODNs which only form triplexes under acidic conditions.
2) The triplex stability for 5-Me-dC-N4-(spermine) ODNs decreases with decreasing pH, in contrast to unmodified ODNs whose stability increases under acidic conditions
This document discusses peptides containing β-amino acid patterns and their applications in medicinal chemistry. It begins with an introduction to β-amino acids and their classification. Various methods for synthesizing β-amino acids are then reviewed, including metal catalysis, organocatalysis, and common reaction types. Applications discussed include using β-amino acid peptides for membrane targeting, mimicking protein functions like receptor binding, and as ligands for receptors involved in conditions like diabetes, cancer, and inflammation. The document concludes by noting the challenges and successes in developing bioactive molecules using β-amino acid peptide scaffolds.
A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 μM in cellular phosphorylation assays (IC50 0.47–0.69 μM) and are highly selective against a small kinase panel. Structure-activity relationship studies identified substitutions on the aniline ring and benzoxazepine core that improved potency, with meta-bromo and bis-methoxy substitutions yielding the most active compounds. These tricyclic azepine derivatives represent a new scaffold for EGFR kinase inhibitors with desired selectivity.
This document outlines the goals and key concepts regarding protein structure. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary. Methods for determining protein structure are also covered, including protein purification techniques like chromatography, electrophoresis, and centrifugation. Protein sequencing methods such as Edman degradation are also summarized. The document provides an overview of protein structure and analysis.
Peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect.
Peptidomimetics are designed to circumvent some of the problems associated with a natural peptide for example
Stability against proteolysis (duration of activity)
Poor bioavailability.
Receptor selectivity or potency (often can be substantially improved).
This document discusses protein structure and analysis techniques. It describes the four levels of protein structure: primary, secondary, tertiary, and quaternary. Common secondary structures like alpha helices and beta sheets are formed by hydrogen bonding patterns in the protein backbone. Tertiary structure refers to a protein's three-dimensional shape, while quaternary structure involves multiple polypeptide subunits. Several techniques for analyzing proteins are also outlined, including electrophoresis, chromatography, and spectroscopy.
This document provides an overview of peptidomimetics. It begins with the evolution of peptidomimetics and how they were developed to overcome limitations of peptides as drugs. It then covers classification of peptidomimetics, design strategies like modifying amino acids and imposing structural constraints, and examples of peptidomimetic drugs that inhibit enzymes like ACE, thrombin, and HIV protease. The document concludes by stating peptidomimetics are an important area of drug design for developing small molecule mimics of peptide functions.
This document discusses various analytical techniques used to evaluate protein and peptide drug formulations, including stability testing, bioassays, UV spectroscopy, Bradford assay, differential scanning calorimetry, chromatography, and electrophoresis. Stability testing evaluates how environmental factors affect the quality of a drug over time. Bioassays assess potency by monitoring the in vitro or in vivo pharmacological response to the protein. UV spectroscopy, Bradford assay, and electrophoresis can be used to detect and quantify the amount of protein present in a sample. Chromatography and differential scanning calorimetry provide information about stability and conformational changes of proteins.
This document provides an overview of enzymology. It defines enzymes as biological catalysts that accelerate chemical reactions without being consumed. Enzymes are classified based on the types of reactions they catalyze, such as oxidation-reduction or hydrolysis. The active site of an enzyme is where substrate binding occurs. The Michaelis-Menten equation and Lineweaver-Burk plots are used to determine kinetics parameters like Vmax and Km. Enzymes can be inhibited by inhibitors that reduce reaction rates. There are different types of inhibition depending on the binding site. The document also lists examples of enzymes used for diagnostic testing and as therapeutic agents.
1) The study examines how long- and short-range electrostatic interactions affect the rheology and protein-protein interactions (PPI) of highly concentrated monoclonal antibody (mAb) solutions.
2) At high concentrations, both long- and short-range interactions contribute significantly to PPI, whereas at low concentrations only long-range interactions are important.
3) The study uses high frequency rheology, dynamic light scattering, circular dichroism, and zeta potential measurements to characterize PPI over a range of pH and ionic strengths, and develops a 3D computer model of the mAb to study charge distribution.
This document discusses protein structure and folding. It begins by explaining that proteins fold into specific three-dimensional structures determined by their amino acid sequences. It then describes how proteins can become denatured by factors like heat or chemicals, losing their structure and function. However, some proteins can renature and refold into their original conformation. The classic example discussed is ribonuclease, which spontaneously refolds into its active form after denaturation. The document concludes by examining models of the protein folding process, which involves hierarchical formation of secondary and tertiary structure to minimize free energy and arrive at the native conformation.
This document discusses assembly-assisted peptide ligation in native conditions. It proposes that peptide fragments can assemble into protein-like structures through non-covalent interactions in native conditions, bringing peptide termini into close proximity. This proximity could allow the termini to ligate without the need for cysteine residues, as seen in native chemical ligation. The document outlines an experiment to test this hypothesis using two fragments of a zinc finger protein, which would assemble due to zinc ion binding and potentially ligate using the coupling reagent PMSF in native conditions.
Year 12 biology early comm presentation intro onlyRachelCaico
This document contains information for students taking an early commencement biology course, including:
1. An outline of the contents of the course handbook, which includes the textbook, worksheets, presentations, and assessment information.
2. Details of the two biology exams for Units 3 and 4, which will assess students' understanding of key knowledge and skills through a series of questions.
3. An overview of the two areas of study in Unit 3 - Molecules of Life and Detecting & Responding. It lists the key topics to be covered and outcomes to be assessed for each area of study.
4. Details of the school-assessed coursework and examination that make up the 17
This document discusses monoclonal antibodies (mAbs), how they differ from traditional molecules, how they are constructed, and how they degrade. It explains that mAbs are much larger and more complex than traditional drugs. The structure of mAbs, including the variable regions that provide antigen binding and the constant regions, is important to their mechanism of action. Degradation can occur through chemical processes like oxidation, deamidation, and hydrolysis, as well as physical processes like fragmentation, denaturation, and aggregation. Factors that can drive degradation include formulation, storage conditions, manufacturing processes, and surface interactions. Examples of degradation include bevacizumab aggregation due to silicon, surfactant, agitation, storage devices, and
This document summarizes the synthesis of novel 3'-C-methylene thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates and phosphonamidites for modifying the backbone of oligonucleotides. The key intermediates 3'-C-iodomethyl nucleosides were reacted with in situ generated bis(trimethylsilyl)phosphite (BTSP) using modified Arbuzov reaction conditions to directly obtain the desired 5'-O-DMT and MMT protected 3'-C-methylene modified H-phosphonates without removing protecting groups. Some phosphonates were further converted to corresponding phosphonamidite monomers through a one-pot multi
This study compared the efficiency of 8 elutable affinity tags for purifying proteins from E. coli, yeast, Drosophila, and HeLa extracts. The tags included 2 protein tags (GST and MBP) and 6 peptide tags. Results showed the tags differed substantially in purity, yield, and cost. The HIS tag provided good yields but only moderate purity from E. coli extracts and poorer purification from other extracts. The Strep II tag appeared to be an excellent candidate overall due to producing high purity material in good yields at a moderate cost. The choice of tag depends on experimental requirements around yield, purity and cost.
The document discusses the growth of electronic data and its impact on e-discovery in litigation. It notes that e-discovery cases and sanctions are on the rise as data volumes grow exponentially. Various judges weigh in on parties' obligations around e-discovery and the consequences for failing to meet those obligations. The use of cloud computing and managed services is presented as a way for law firms to more efficiently handle e-discovery. The document concludes by emphasizing the need for a coordinated approach and proper resources to successfully manage e-discovery.
Solution And Solid Phase Synthesis Publicationadotse
This document describes the solution- and solid-phase synthesis of peptide-substituted thiazolidinediones as potential ligands for peroxisome proliferator-activated receptors (PPARs). Initial studies focused on the low-yielding solution-phase synthesis of two target compounds. Improved yields were obtained using solid-phase synthesis and protecting the thiazolidinedione nitrogen. A small library of nine resin-bound peptide-substituted thiazolidinediones was then synthesized to examine structural features that facilitate PPAR binding and identify new PPAR activators/inhibitors.
Gawrisch Insights From Biophysical Studies On The Role Of Pufa For Function O...lab13unisa
This document summarizes recent insights into the role of polyunsaturated fatty acids, specifically docosahexaenoic acid (DHA), for the function of G-protein coupled membrane receptors like rhodopsin. It discusses how DHA chains have high conformational flexibility and tend to be located near lipid-water interfaces, altering the properties of lipid bilayers. It also presents evidence that DHA interacts weakly and nonspecifically with some sites on rhodopsin, and that certain photointermediates of rhodopsin have stronger interactions with DHA.
This document describes a new method for selective N-methylation of peptide backbone amides on resin using the Mitsunobu reaction. The key aspects are:
1) N-trifluoroacetamide (Tfa) was used as the protecting group on resin-bound peptides, which can generate a nucleophilic anion for methylation via Mitsunobu conditions.
2) Tfa-protected peptides on resin underwent efficient (80-99%) and selective N-methylation of the backbone amide using the Mitsunobu reaction with triphenylphosphine, methanol and diisopropyl azodicarboxylate.
3) Unlike other reports, the T
Characterization of mg state of mb in presence of peg 10 (z a parray) originalZahoor Parray
This document summarizes a study that characterized the intermediate state of myoglobin (Mb) in the presence of polyethylene glycol 10 (PEG 10) under physiological conditions. The researchers found that PEG 10 perturbed the tertiary structure of Mb but did not significantly change its secondary structure. PEG 10 was found to induce a molten globule state in Mb, where the intermediate state had hydrophobic patches and a larger hydrodynamic volume than the native protein. Isothermal titration calorimetry showed strong binding between Mb and PEG 10 at physiological pH. The researchers hypothesize that PEG 10 induces a molten globule conformation in Mb by interacting with its heme group. They conclude that protein-crowder interactions need careful
This document describes research on triplex formation by oligodeoxynucleotides (ODNs) containing 5-methyldeoxycytidine conjugated to spermine (5-Me-dC-N4-(spermine)). The key findings are:
1) ODNs containing 5-Me-dC-N4-(spermine) form stable triplexes at physiological pH (pH 7.3), unlike unmodified ODNs which only form triplexes under acidic conditions.
2) The triplex stability for 5-Me-dC-N4-(spermine) ODNs decreases with decreasing pH, in contrast to unmodified ODNs whose stability increases under acidic conditions
This document discusses peptides containing β-amino acid patterns and their applications in medicinal chemistry. It begins with an introduction to β-amino acids and their classification. Various methods for synthesizing β-amino acids are then reviewed, including metal catalysis, organocatalysis, and common reaction types. Applications discussed include using β-amino acid peptides for membrane targeting, mimicking protein functions like receptor binding, and as ligands for receptors involved in conditions like diabetes, cancer, and inflammation. The document concludes by noting the challenges and successes in developing bioactive molecules using β-amino acid peptide scaffolds.
A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 μM in cellular phosphorylation assays (IC50 0.47–0.69 μM) and are highly selective against a small kinase panel. Structure-activity relationship studies identified substitutions on the aniline ring and benzoxazepine core that improved potency, with meta-bromo and bis-methoxy substitutions yielding the most active compounds. These tricyclic azepine derivatives represent a new scaffold for EGFR kinase inhibitors with desired selectivity.
This document outlines the goals and key concepts regarding protein structure. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary. Methods for determining protein structure are also covered, including protein purification techniques like chromatography, electrophoresis, and centrifugation. Protein sequencing methods such as Edman degradation are also summarized. The document provides an overview of protein structure and analysis.
Peptidomimetics are compounds whose essential elements (pharmacophore) mimic a natural peptide or protein in 3D space and which retain the ability to interact with the biological target and produce the same biological effect.
Peptidomimetics are designed to circumvent some of the problems associated with a natural peptide for example
Stability against proteolysis (duration of activity)
Poor bioavailability.
Receptor selectivity or potency (often can be substantially improved).
This document discusses protein structure and analysis techniques. It describes the four levels of protein structure: primary, secondary, tertiary, and quaternary. Common secondary structures like alpha helices and beta sheets are formed by hydrogen bonding patterns in the protein backbone. Tertiary structure refers to a protein's three-dimensional shape, while quaternary structure involves multiple polypeptide subunits. Several techniques for analyzing proteins are also outlined, including electrophoresis, chromatography, and spectroscopy.
This document provides an overview of peptidomimetics. It begins with the evolution of peptidomimetics and how they were developed to overcome limitations of peptides as drugs. It then covers classification of peptidomimetics, design strategies like modifying amino acids and imposing structural constraints, and examples of peptidomimetic drugs that inhibit enzymes like ACE, thrombin, and HIV protease. The document concludes by stating peptidomimetics are an important area of drug design for developing small molecule mimics of peptide functions.
This document discusses various analytical techniques used to evaluate protein and peptide drug formulations, including stability testing, bioassays, UV spectroscopy, Bradford assay, differential scanning calorimetry, chromatography, and electrophoresis. Stability testing evaluates how environmental factors affect the quality of a drug over time. Bioassays assess potency by monitoring the in vitro or in vivo pharmacological response to the protein. UV spectroscopy, Bradford assay, and electrophoresis can be used to detect and quantify the amount of protein present in a sample. Chromatography and differential scanning calorimetry provide information about stability and conformational changes of proteins.
This document provides an overview of enzymology. It defines enzymes as biological catalysts that accelerate chemical reactions without being consumed. Enzymes are classified based on the types of reactions they catalyze, such as oxidation-reduction or hydrolysis. The active site of an enzyme is where substrate binding occurs. The Michaelis-Menten equation and Lineweaver-Burk plots are used to determine kinetics parameters like Vmax and Km. Enzymes can be inhibited by inhibitors that reduce reaction rates. There are different types of inhibition depending on the binding site. The document also lists examples of enzymes used for diagnostic testing and as therapeutic agents.
1) The study examines how long- and short-range electrostatic interactions affect the rheology and protein-protein interactions (PPI) of highly concentrated monoclonal antibody (mAb) solutions.
2) At high concentrations, both long- and short-range interactions contribute significantly to PPI, whereas at low concentrations only long-range interactions are important.
3) The study uses high frequency rheology, dynamic light scattering, circular dichroism, and zeta potential measurements to characterize PPI over a range of pH and ionic strengths, and develops a 3D computer model of the mAb to study charge distribution.
This document discusses protein structure and folding. It begins by explaining that proteins fold into specific three-dimensional structures determined by their amino acid sequences. It then describes how proteins can become denatured by factors like heat or chemicals, losing their structure and function. However, some proteins can renature and refold into their original conformation. The classic example discussed is ribonuclease, which spontaneously refolds into its active form after denaturation. The document concludes by examining models of the protein folding process, which involves hierarchical formation of secondary and tertiary structure to minimize free energy and arrive at the native conformation.
This document discusses assembly-assisted peptide ligation in native conditions. It proposes that peptide fragments can assemble into protein-like structures through non-covalent interactions in native conditions, bringing peptide termini into close proximity. This proximity could allow the termini to ligate without the need for cysteine residues, as seen in native chemical ligation. The document outlines an experiment to test this hypothesis using two fragments of a zinc finger protein, which would assemble due to zinc ion binding and potentially ligate using the coupling reagent PMSF in native conditions.
Year 12 biology early comm presentation intro onlyRachelCaico
This document contains information for students taking an early commencement biology course, including:
1. An outline of the contents of the course handbook, which includes the textbook, worksheets, presentations, and assessment information.
2. Details of the two biology exams for Units 3 and 4, which will assess students' understanding of key knowledge and skills through a series of questions.
3. An overview of the two areas of study in Unit 3 - Molecules of Life and Detecting & Responding. It lists the key topics to be covered and outcomes to be assessed for each area of study.
4. Details of the school-assessed coursework and examination that make up the 17
This document discusses monoclonal antibodies (mAbs), how they differ from traditional molecules, how they are constructed, and how they degrade. It explains that mAbs are much larger and more complex than traditional drugs. The structure of mAbs, including the variable regions that provide antigen binding and the constant regions, is important to their mechanism of action. Degradation can occur through chemical processes like oxidation, deamidation, and hydrolysis, as well as physical processes like fragmentation, denaturation, and aggregation. Factors that can drive degradation include formulation, storage conditions, manufacturing processes, and surface interactions. Examples of degradation include bevacizumab aggregation due to silicon, surfactant, agitation, storage devices, and
This document summarizes the synthesis of novel 3'-C-methylene thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates and phosphonamidites for modifying the backbone of oligonucleotides. The key intermediates 3'-C-iodomethyl nucleosides were reacted with in situ generated bis(trimethylsilyl)phosphite (BTSP) using modified Arbuzov reaction conditions to directly obtain the desired 5'-O-DMT and MMT protected 3'-C-methylene modified H-phosphonates without removing protecting groups. Some phosphonates were further converted to corresponding phosphonamidite monomers through a one-pot multi
This study compared the efficiency of 8 elutable affinity tags for purifying proteins from E. coli, yeast, Drosophila, and HeLa extracts. The tags included 2 protein tags (GST and MBP) and 6 peptide tags. Results showed the tags differed substantially in purity, yield, and cost. The HIS tag provided good yields but only moderate purity from E. coli extracts and poorer purification from other extracts. The Strep II tag appeared to be an excellent candidate overall due to producing high purity material in good yields at a moderate cost. The choice of tag depends on experimental requirements around yield, purity and cost.
The document discusses the growth of electronic data and its impact on e-discovery in litigation. It notes that e-discovery cases and sanctions are on the rise as data volumes grow exponentially. Various judges weigh in on parties' obligations around e-discovery and the consequences for failing to meet those obligations. The use of cloud computing and managed services is presented as a way for law firms to more efficiently handle e-discovery. The document concludes by emphasizing the need for a coordinated approach and proper resources to successfully manage e-discovery.
This document outlines a journey involving several stages: a call, crossing a threshold, facing challenges, descending into an abyss, undergoing a transformation, receiving a revelation, making an atonement, and returning. The stages represent an experience of personal growth and change.
Infochimps is an organization that supports open data by allowing anyone to post datasets under open licenses for free, forever. It aims to increase access to structured data by hosting thousands of datasets contributed by the online community across many topics and formats. People can search the datasets by tags and also sell their own datasets on the site for a fee, while choosing the license and pricing.
Este documento describe los ríos de España. Define un río como una corriente de agua que fluye desde las montañas hasta el mar u otro río. Explica los elementos clave de un río como su curso, caudal y régimen. Luego detalla las tres principales vertientes hidrográficas de España - Cantábrica, Mediterránea y Atlántica - y sus ríos más importantes. Finalmente, se enfoca en los ríos de Asturias, destacando el Navia, Nalón, Sella y Deva.
1) Polymeric excipients like PEG can stabilize proteins against denaturation during freezing by increasing the transfer free energy of the protein. However, these same polymers can induce phase separation in aqueous solutions.
2) During lyophilization, the concentrating effects of freezing can cause formulations to enter the two-phase region, resulting in liquid/liquid phase separation. This subjects the protein to potential partitioning between phases with different compositions.
3) Experimental studies on hemoglobin lyophilized in PEG/dextran mixtures provide evidence that phase separation during lyophilization can damage protein structure in the dried state.
1) G.N. Ramachandran created the Ramachandran plot in 1963, which is an essential tool for understanding protein structure. The plot analyzes allowed regions of phi and psi dihedral angles in peptide units.
2) Protein stability refers to a protein maintaining its native folded conformation rather than becoming denatured. Stability depends on a balance of forces and is important for protein function.
3) Factors that influence protein stability include pH, ligand binding, disulfide bonds, and interactions within the protein and between the protein and solvent. Chaperone proteins and proteases also help maintain stability in cells.
Practical consideration of protien and peptidesSuchandra03
This document summarizes a seminar on practical considerations for protein and peptide drug delivery. It begins with an introduction discussing the two main pathways of protein delivery research: non-invasive delivery methods and increasing drug half-life. It then covers the major barriers to protein delivery such as poor permeability, degradation, and stability issues. The document reviews protein structure, classification, properties, degradation pathways, and the rationale for protein drug delivery. It discusses various delivery strategies including chemical modifications, formulation vehicles, advancement like molecular conjugation and encapsulation, and considers toxicity, commercialization factors, and concludes discussing the challenges of oral protein delivery.
This document discusses protein and peptide drug delivery systems. It begins by defining proteins and peptides, and describing their structures. It then discusses various challenges in delivering protein and peptide drugs, such as stability issues like denaturation, aggregation, oxidation, and proteolysis. It also categorizes different drug delivery routes like parenteral, pulmonary, transdermal, and oral. Finally, it provides examples of marketed protein and peptide drug formulations and discusses strategies to improve stability and delivery of these drugs.
Protein and-peptide-drug-delivery-systemsGaurav Kr
The document discusses protein and peptide drug delivery systems. It begins by defining proteins and peptides, noting that proteins are molecules composed of over 50 amino acids, while peptides are molecules composed of less than 50 amino acids. It then discusses how scientific advances in molecular and cell biology have led to the development of recombinant DNA and hybridoma technology to produce protein products. The document provides examples of marketed protein and peptide drugs and discusses challenges with delivering these drugs orally due to their large molecular size and susceptibility to enzymatic degradation. It explores approaches to protein and peptide delivery including non-parenteral systemic delivery methods and various considerations for developing delivery systems for these pharmaceuticals.
By Saransh Jain Drug Delivery System (Evaluation Parameter of Drug Deliver...saranshneri
This document discusses various evaluation parameters for protein drug delivery systems, including stability testing, bioassays, UV spectroscopy, Bradford assay, differential scanning calorimetry, chromatography, and electrophoresis. Stability testing evaluates how well a formulation maintains quality over time and under various storage conditions. Bioassays, which can be in vitro or in vivo, assess the potency and pharmacological response to the protein. UV spectroscopy, Bradford assay, and chromatography can be used to analyze and detect proteins, while differential scanning calorimetry studies protein conformation and stability. Electrophoresis determines protein molecular weight.
This document summarizes a presentation on protein and peptide drug delivery systems. It discusses the importance of pre-formulation and formulation considerations for proteins and peptides, including toxicity, immunogenicity, and stability from regulatory perspectives. The key points covered include the definition of proteins and peptides, common formulation excipients to prevent denaturation like serum albumin and amino acids, and analytical techniques used for characterization like UV spectroscopy, electrophoresis and liquid chromatography. Maximizing oral absorption through chemical modifications like hydrophobization is also summarized.
1. Protein purification involves isolating one or a few proteins from a complex mixture through a series of extraction, precipitation, ultracentrifugation, and chromatographic steps.
2. Maintaining the proper buffering environment is essential to prevent changes in pH that could denature proteins. Buffers should be selected so their pH is within 1 unit of the protein's pKa.
3. When proteins are purified, they should be stored with additives at the appropriate pH and temperature depending on the protein's stability requirements for downstream applications. Storage conditions need to be optimized for each protein.
PEPTIDOMIMETICS/SAGAR SHARMA/DEPARTMENT OF PHARMACEUTICAL SCIENCESSagarMudgil1
Peptidomimetics are compounds that mimic natural peptides and proteins in 3D space. They are created by modifying the side chain or backbone of peptides. Peptidomimetics are designed to be highly selective, bioavailable, and metabolically stable.
Variation in trypsin inhibitor activity (TIA) and protein solubility within commercial soybean meals (SBM) is believed to affect animal performance. The objective of this research was to investigate the proteolytic effect of purified pancreatic trypsin/chymotrypsin and a purified protease from Nocardiopsis prasina on 9 commercial SBM samples varying in chemical composition, TIA and protein solubility characteristics. SBM was incubated with or without protease (pH 7, 3 hours, 40°C) and the supernatants were analyzed by SDS-page and for level of free soluble amino ends using the o-phthal-dialdehyde method. SDS-page analysis showed differences in the amount and composition of the soluble protein fraction of the SBM. The proteolytic efficiency of the lower dosages of pancreatic protease varied extensively between SBM batches whereas at the highest dose the efficiency was comparable for all SBM. The difference could not be explained by differences in protein solubility and TIA values as isolated variables, but the data strongly suggest that the hydrolysis of soya protein by pancreatic proteases depends on the commercial batch of SBM used. Incubation with N. prasina protease showed similar response for the different SBM, the main difference being a somewhat lower effect at high protease dose for one of the SBM batches, which might be in part explained by its distinct lower protein solubility
Protein-protein interactions (PPIs) are important for many cellular functions. There are two main types of PPIs - transient interactions which are brief, and stable interactions which form multiprotein complexes. Crosslinking can capture both transient and stable PPIs by covalently binding interacting proteins. In vivo crosslinking studies PPIs in their native environment while in vitro crosslinking allows better reaction control. Pull-down assays use affinity purification to isolate stable protein complexes and identify binding partners of a bait protein. SDS-PAGE is commonly used to separate and visualize proteins isolated by techniques like pull-down.
Protein and peptides drug delivery systemsVINOTH R
This document provides an overview of protein and peptide drug delivery. It discusses the structure of proteins and peptides, which consist of amino acids linked by peptide bonds. Various drug delivery techniques for proteins are described, including polymer, liposome, hydrogel, and emulsion-based systems. Challenges in delivering proteins include instability, degradation, and poor absorption. The document also discusses pumps, barriers to delivery, manufacturing, stability testing, instability issues, and some marketed protein and peptide drugs.
This document discusses stability problems and prevention strategies for proteins and peptides used in drug delivery systems. It describes how protein structure, including primary, secondary, tertiary, and quaternary levels can impact stability. Physical stability problems like denaturation from changes in solvent, pH, temperature and adsorption are explained. Chemical stability issues such as deamidation, oxidation, and reduction are also outlined. Methods to prevent various stability problems involving controlling solvents, pH, temperature, and use of stabilizing agents are presented.
This document provides an overview of the schedule and topics for a course on metabolic networks. The course covers various topics related to metabolism, including enzyme assays, mass spectrometry techniques, primary metabolism, glycogen metabolism, metabolic networks in humans, flux analysis, and biochemical databases. Homework discussions are scheduled every other Friday. Guest lecturers will discuss biochemical databases and tools for modeling metabolism on the last two class meetings. The course includes no class on Veteran's Day and Thanksgiving.
It will be helpful for students who is having very less time to prepare for enzyme immobilization and coimmobilization of an enzyme. This presentation have covered various methods and its induatrial applicability.
This document summarizes the design and testing of a bisubstrate inhibitor for the insulin receptor tyrosine kinase (IRK). Researchers designed a compound that links ATPγS to a peptide substrate analog via a two-carbon spacer, setting the distance between the nucleophilic atom and phosphoryl group to mimic a dissociative transition state as suggested by previous studies of IRK's mechanism. Testing found this compound to be a potent and selective competitive inhibitor of IRK, with a Ki of 370 nM, making it the most potent inhibitor reported for this important signaling enzyme. A crystal structure confirmed the inhibitor bound as designed and validated the mechanism-inspired approach.
This document provides an overview of proteomics and protein analysis techniques. It discusses how the proteome represents all proteins expressed by a genome and how protein expression changes with health, disease, and toxicity. It also describes seven attributes of proteins, including identity, quantity, post-translational modifications, structure, interactions, spatial relationships, and function. Common techniques for protein analysis are also summarized, such as chromatography, mass spectrometry, crystallization, and sequencing. Chromatography methods separate proteins based on properties like size, charge, hydrophobicity, and specific binding interactions.
Protein-protein interactions (PPIs) occur when two or more proteins bind together to carry out biological functions. PPIs are important for most cellular processes and determining disease pathways. Experimental techniques like yeast two-hybrid systems are used to detect PPIs on a large scale. Studying PPIs provides insight into protein functions and disease mechanisms, aiding drug design and development.
This document is the user manual for the VP-DSC MicroCalorimeter. It provides specifications for the instrument, safety information, and instructions for operation. Key details include:
- The VP-DSC allows for high sensitivity measurement of heat capacity, binding thermodynamics, and kinetics.
- Safety precautions must be followed when using hazardous or volatile solutions in the tantalum cells.
- VPViewer software interfaces with Origin for instrument control and real-time data display.
- Sections provide tutorials for common experiments, calibration procedures, troubleshooting tips, and maintenance instructions.
1. The study characterized the aggregation of recombinant human Interleukin-1 receptor type II (rhuIL-1RII) using differential scanning calorimetry (DSC) and size exclusion chromatography (SEC).
2. A scan-rate dependence in the DSC experiment and a break from linearity in initial aggregation rates near the melting temperature (Tm) suggested that protein unfolding significantly contributes to the aggregation reaction pathway.
3. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from the irreversibly denatured aggregation process by simulating how unfolding properties could predict aggregation rates at different temperatures above and below the Tm.
This document reviews lyophilization (freeze-drying) as a method for developing solid protein pharmaceuticals. Lyophilization generates stresses that can denature proteins, including low temperature stress, freezing stresses from increased concentration and ice formation, and drying stress from removing the hydration shell. Several studies are discussed that demonstrate denaturation of specific proteins from these stresses during lyophilization and storage. The review discusses excipient protection of proteins, lyophilization cycle design, and formulation strategies to increase stability of solid protein pharmaceuticals and overcome instability issues.
This document summarizes the calculation of translational friction and intrinsic viscosity for four globular proteins (ribonuclease A, lysozyme, myoglobin, and chymotrypsinogen A) using their detailed atomic structures. The inclusion of a 0.9 Angstrom thick hydration shell around each protein allows the calculated translational friction and intrinsic viscosity to match experimental measurements. This hydration shell thickness corresponds to a hydration level of 0.3-0.4 grams of water per gram of protein, consistent with measurements from other techniques. Using detailed protein structures thus allows hydrodynamic measurements to support a unified picture of protein hydration, in contrast to earlier models that treated proteins as ellipsoids and found widely varying hydr
This document summarizes a study that investigated how different salts screen charge interactions in proteins. Specifically, it examined the effects of NaCl, guanidinium chloride, and guanidinium thiocyanate on the stability of wild-type E. coli thioredoxin and a variant. The results suggest that more denaturing salts like guanidinium chloride are more efficient at screening charge interactions than NaCl. This efficiency correlates with the salts' position in the Hofmeister series and ability to accumulate on protein surfaces. An electrostatic model was used to estimate contributions of charge interactions to stability.
Proton euilibria in minor groove of dnamganguly123
1) The document describes an experiment testing the prediction that regions of increased hydrogen ion density exist in the grooves of DNA. Probes with variable linker lengths and a proton-sensitive carboxyl group were attached to DNA in the minor groove.
2) The apparent pKa values of the carboxyl groups were higher than in free solution, increasing with shorter linker lengths. This agrees with calculations showing higher hydrogen ion density in the grooves.
3) The experiment provides experimental evidence supporting the theoretical prediction of acidic domains with elevated hydrogen ion density in the DNA minor groove.
The document describes an experiment measuring the static light scattering of concentrated protein solutions as a function of concentration. Specifically, it measured bovine serum albumin, ovalbumin, ovomucoid, and mixtures of these proteins up to 125 g/L, as well as chymotrypsin A at different pH levels up to 70 g/L. The measured scattering was quantitatively accounted for by an effective hard particle model, in which each protein is represented as a hard sphere and interactions are treated as hard particle repulsions and association equilibria.
This document summarizes a study that investigated the effects of two disaccharides (trehalose and sucrose) and trimethylamine N-oxide (TMAO) on amyloid-beta (Aβ) aggregation and interaction with lipid membranes. The key findings were:
1) In the absence of lipid vesicles, trehalose and sucrose delayed Aβ aggregation as measured by Thioflavin T fluorescence, but TMAO did not affect aggregation.
2) In the presence of lipid vesicles, all three osmolytes (trehalose, sucrose, TMAO) significantly attenuated dye leakage from the vesicles induced by Aβ aggregates.
3) Hydrogen exchange mass spectrometry (HX-MS) and
The document discusses several phase diagrams generated using different experimental data visualization techniques including:
1) A phase diagram of ricin toxin A-chain created using fluorescence and circular dichroism spectroscopic data showing four protein states.
2) An empirical phase diagram of the respiratory syncytial virus determined from multiple biophysical measurements across a pH range.
3) Phase diagrams of various non-viral gene delivery vehicles and proteins mapped against pH and temperature.
Basic fibroblast growth factor (bFGF) is being investigated for its ability to accelerate wound healing. Sulfated compounds like heparin enhance the stability of bFGF against thermal denaturation. To assess the effect on bFGF shelf life, formulations containing these excipients were incubated and analyzed. In the presence of sulfated compounds, precipitates formed that dissociated back to multimers with native structure, whereas without them precipitates were unfolded protein. Disulfide-linked multimers also increased in solution with sulfated compounds. Heparin stabilized bFGF structure and prevented rearrangement of disulfide bonds, indirectly promoting multimerization. However, loss of soluble bFGF monomer still
This document summarizes the use of differential scanning calorimetry (DSC) to optimize an antibody manufacturing process. DSC was used to screen conditions for a viral inactivation step and identify increased pH storage conditions for maximum stability. Low pH treatment reduced thermal stability, indicating structure loss. DSC provided insights into instability causes and process improvements, demonstrating its role in biotherapeutic development.
This document describes a study on controlled intracranial delivery of antibodies in rats. The researchers developed polymer matrices and microspheres for long-term antibody release directly in the brain. They implanted polymer discs containing IgG antibodies in rat brains and measured IgG concentrations at the implantation site and other brain regions over 28 days, finding highest levels with the polymer implants. The polymer provided sustained antibody levels beyond what was achieved with direct injection.
This document summarizes a study that measured the enthalpy change (ΔH) associated with the α-helix to random coil transition of an alanine peptide in water using calorimetry. The researchers synthesized a 50-residue peptide containing primarily alanine residues and determined its ΔH to be between 0.9-1.3 kcal/mol per residue, providing a basic parameter for predicting thermal unfolding of peptide helices. Circular dichroism spectra and melting curves confirmed the peptide adopted an α-helical structure at low temperatures and underwent a reversible helix-coil transition. The ΔH value suggests the peptide backbone, rather than side chains, makes the dominant contribution to helix stability.
Ion water interaction biophysical journalmganguly123
The document discusses how the charge density of ions affects their strength of hydration and interactions in biological structures. It finds that small, highly charged ions (kosmotropes) strongly bind water molecules, while large monovalent ions of low charge density (chaotropes) weakly bind water. Crystalline salts dissolve exothermically only when one ion is a kosmotrope and the other is a chaotrope. This suggests kosmotropes and chaotropes preferentially form ion pairs in solution. The major intracellular ions—phosphate and carboxylate anions and potassium/arginine cations—behave as kosmotropes and chaotropes, respectively, allowing them
This document presents a study using differential scanning calorimetry (DSC) to examine the thermal stability of S-protein and its complexes with S-peptide at pH 7.0. DSC measurements showed that S-protein denatures through a reversible two-state transition with a denaturation temperature between 38.5-40.0°C and enthalpy of 165-180 kJ/mol, demonstrating its lower stability without S-peptide. A two-dimensional nonlinear regression analysis of excess heat capacity curves at varying temperatures and S-peptide concentrations was used to determine the binding thermodynamic parameters, yielding values of Kb = 1.10 × 106 M-1, ΔbH = -185 kJ
The document summarizes the origin of photosensitivity in a monoclonal immunoglobulin G (IgG). UV irradiation of the monoclonal IgG causes a 70% decrease in intrinsic fluorescence and the appearance of new fluorescence, suggesting photooxidation of one or two tryptophan residues. This leads to extensive quenching of the protein's fluorescence through nonradiative energy transfer, even though few residues are directly oxidized. The photooxidation products, N-formylkynurenine and kynurenine, absorb light above 300 nm and contribute to changes in the UV-visible absorption spectrum with irradiation.
This study examines the effect of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of lipid bilayer membranes composed of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE), and dimyristoylphosphatidylglycerol (DMPG) using differential scanning calorimetry. The results show that GS interacts more strongly with anionic DMPG bilayers than with zwitterionic DMPC or DMPE bilayers, reducing the temperature and cooperativity of DMPG's phase transition to a greater degree. In contrast, GS has little effect on DMP
1) Circular dichroism arises from the differential absorption of left and right circularly polarized light by chiral molecules.
2) CD spectra are more sensitive to conformational changes in proteins and nucleic acids than absorption spectra.
3) CD spectra can provide information about secondary structure in proteins.
11 20-09 mbsb single molecule techniques.pptmganguly123
This document contains over 100 images from various scientific sources related to topics in optics, microscopy, fluorescence, plasmonics, and single molecule techniques. The images depict experimental setups, microscopy images, diagrams, graphs, and other figures illustrating different scientific phenomena and techniques. Captions provide brief descriptions and citations for the source of each image.
The document discusses the benefits of meditation for reducing stress and anxiety. Regular meditation practice can help calm the mind and body by lowering heart rate and blood pressure. Studies have shown that meditating for just 10-20 minutes per day can have significant positive impacts on both mental and physical health.
2. 68 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
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Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
3. 3. B E L L Aspartame Stability in Solids and Solutions 69
Aspartame Degradation Kinetics
The degradation pathways of aspartame change as a function of pH. At pH values
less than 5.2, aspartame forms a-aspartylphenylalanine (a-AP) via hydrolysis of the
methyl ester group, diketopiperazine (DKP) via cyclization, phenylalanine methyl
ester (PMe) via hydrolysis of the peptide bond, and ^-aspartame (0-APM) via
structural rearrangement through a cyclic intermediate (1-3). At these low pH
values, 0-aspartylphenylalanine (0-AP) is also produced from ester hydrolysis of
/3-aspartame and the rearrangement of a-AP (2). The aspartame degradation
pathways at pH < 5.2 are shown in Figure 1. At pH > 5.2, aspartame degrades
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into DKP and a-AP without the formation of the 0-isomers (1-3).
Based on the degradation products formed from aspartame at pH 7, the rate
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
expression for the loss of aspartame can be written as
-d[APM|/dt = k DKP [ A P M ] + k . [APM]
a AP (1)
where k is the net rate constant for diketopiperazine formation, k . is the net
D K P a AP
rate constant for ester hydrolysis and consequent a-aspartylphenylalanine formation,
and [APM] is the concentration of aspartame. For the acid-base catalyzed
degradation of aspartame, each net rate constant, k , can be expressed in general
x
terms as follows:
+
k = k + k [ H ] + k f O H ] + k [BH] + k [B]
x G H OH BH B (2)
where k is the rate constant for the uncatalyzed reaction, k is the rate constant for
G H
catalysis by hydronium ions, k is the rate constant for catalysis by hydroxyl ions,
OH
k is the rate constant for buffer-mediated acid catalysis, and k is the rate constant
BH B
for buffer-mediated base catalysis (4). In buffered solutions, the concentrations of
the buffering components as well as the pH remain unchanged during the course of
the reaction, which allows for the combination of rate constants to yield the pseudo
first order expression
-d|APM]/dt = k [APM]
obs (3)
where k is the observed first order rate constant. The pseudo first order
obs
degradation model for aspartame in solutions and solids has been verified
experimentally (1,3,5-11). Based on the above studies, the factors which affect
aspartame stability in solutions and solids can be addressed.
Temperature
The effect of temperature on aspartame degradation has been evaluated in solutions
and solid systems (1,6-9). Higher temperatures enhance peptide degradation rates,
as expressed by the Arrhenius equation. The sensitivity of the reaction to
temperature is given by the activation energy.
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
4. 70 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
In solution, the activation energy for aspartame degradation decreased
significantly with increasing pH (8-9). For example, Bell and Labuza found the
activation energy changed from 19.7+1.4 kcal/mol at pH 3 to 15.2+0.8 kcal/mol
at pH 7 in 0.1 M phosphate buffer (8). As mentioned previously, the pathways by
which aspartame degrades change as a function of pH. Different pathways would
each have different activation energies and the contribution from each pathway
would differ with pH. Thus, an influence of pH on the activation energy for
aspartame degradation would be expected.
In solid systems, the activation energies for aspartame degradation decreased
with increasing moisture content (8). For example at pH 5, the activation energies
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for aspartame degradation decreased from 27.1 + 1.1 kcal/mol at 6% moisture to
24.5 + 1.3 kcal/mol at 14% moisture to 17.9+1.5 kcal/mol in solution (8). The
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
decrease in activation energy with increasing moisture content has been explained
using principles of enthalpy-entropy compensation (12-13). Assuming the
degradation of aspartame requires the formation of an activated complex, then as
moisture content increases, the entropy (AS) increases while the free energy (AG)
decreases, resulting in a decrease in enthalpy (AH) and thus a lower activation
energy (13). A detailed discussion of enthalpy-entropy compensation as applied to
aspartame degradation can be found elsewhere (13).
The higher activation energy for reactions in low moisture solids can have
definite implications with respect to product stability. If a product at 6% moisture
(E = 27 kcal/mol) and as a solution (E = 18 kcal/mol) both have shelf lives of 12
a a
months at 22°C, the low moisture system would have a longer shelf life than the high
moisture product at refrigerated temperatures. However, under product abuse at
32°C, the low moisture system will lose shelf life prior to the high moisture system
(2.7 months compared to 4.4 months). Low moisture products are usually more
stable than their high moisture counterparts, which would not be the case for this
product at 32°C.
Buffer Type and Concentration
In addition to temperature, the buffer type and concentration also influence the
stability of aspartame in solutions as shown in Figure 2. Increasing the buffer
concentration increased the rate of aspartame degradation (9,11). The buffer
concentration logically influences the degradation rate of acid-base catalyzed
reactions by increasing each net rate constant, k , as shown by equation 2. At pH
x
7 and phosphate buffer concentrations of 0.01 and 0.1 M , catalysis by the buffer
components was responsible for approximately 87.3 and 98.5% of the degradation
rate constant, respectively (77). Similar trends were also observed at pH 3 (77).
Numerous other pharmaceuticals (e.g. ampicillin, chlorampenicol) are also sensitive
to buffer concentration (14).
In solutions, phosphate buffer significantly enhanced the degradation of
aspartame as compared to citrate buffer (77). The Bronsted equation,
log(k ) = log(C) + (z)log(K )
obs d (4)
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
5. 3. B E L L Aspartame Stability in Solids and Solutions 71
relates the rate constant (k ) of an acid-base catalyzed reaction, such as aspartame
obs
degradation, to the buffer ionization constant (K ); C and z in equation 4 are
d
constants (4). Figure 3 shows the Bronsted plot for aspartame degradation in 0.1 M
buffer solutions at pH 7 and 25°C. Except for aspartame degradation in phosphate
buffer, the log of the degradation rate constant changed linearly with the log of the
buffer ionization constant (77). The degradation in phosphate buffer is faster than
expected based on the buffer ionization constant alone, indicating that a factor other
than the buffer ionization constant is enhancing the reaction. The effect of buffer
type on aspartame degradation was explained by the ability of the small phosphate
anion to simultaneously accept and donate the protons (i.e., bifunctional catalytic
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activity) required for the conversion of aspartame into diketopiperazine (77). The
exchange of protons between citrate anions and aspartame is believed to occur less
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
rapidly due to partial steric inhibition (77).
The effect of buffer on aspartame stability is not limited to solutions. Figure
4 shows the effect of the initial phosphate buffer concentration (i.e., prior to
lyophilization and rehydration) on aspartame degradation in a solid system at pH 5
containing 6% moisture. The degradation rate in the low moisture system increased
as buffer concentration increased (8,15). Products containing components
susceptible to catalysis by buffer salts should be formulated at the lowest buffer
concentration possible to minimize degradation rates and enhance shelf life.
Water Activity and Glass Transition
Water has many potential effects on the degradation of peptides. The influence of
moisture on the folding and stability of polypeptides has been documented (16-18).
This discussion will focus on the chemical potential of water (i.e., water activity)
and the plasticization effect of water on amorphous solids. Water activity has been
shown to correlate with reaction rates (79). In addition, the plasticization ability of
water may convert amorphous glassy systems into amorphous rubbery systems upon
moisture sorption, leading to enhanced mobility and potentially chemical reactivity
as well (20).
Figure 5 shows the effect of water activity on the degradation of aspartame
at pH 5 and 30°C. A 0.1 increase in water activity decreases the shelf life by
approximately 40 to 50% (10,13,21). Moisture transfer into pharmaceutical solids
during storage, which increases the water activity of the system, can enhance
degradation rates (14).
The glass transition may influence chemical reactions if the mechanism has
a mobility-dependent step. As mentioned, moisture sorption can convert a glassy
matrix into a rubbery matrix of increased mobility, which may result in increased
reaction rates. Using polyvinylpyrrolidone of different molecular weights, the
influence of the glass transition on aspartame degradation was evaluated at constant
temperature (10). Figure 6 shows that aspartame degradation at pH 7 is not
influenced by the glass transition, but rather by the water activity. If mobility was
rate-limiting, a dramatic increase in the rate constant would have occurred at the
glass transition temperature (T-T =0). However, rate constants at constant water
g
activity and different glass transition temperatures were similar whereas the rate
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
6. 72 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
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Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
0.0 0.1 0.2 0.3 0.4 0.5 0.6
Buffer Concentration (M)
Figure 2. Effect of buffer type and concentration on the rate constant of
aspartame degradation in solution at pH 7 and 25°C (Adapted from ref. 11).
Figure 3. Bronsted plot of aspartame degradation in 0.1 M buffer at pH 7 and
25°C (Reproduced with permission from ref. 11. Copyright 1995 ACS).
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
7. BELL Aspartame Stability in Solids and Solutions
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Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
v 0.2 M
20 -j , , , , ,
0 100 200 300 400 500 600
Time (days)
Figure 4. Aspartame degradation in a 6% moisture solid system at pH 5 and
30°C as influenced by initial phosphate buffer concentration (Adapted from ref.
8 and 15).
3 0 0 -, ,
1 0 H 1 1 1 1 1 1
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Water Activity
Figure 5. Relationship between aspartame half-life at 30°C and water activity
in a lyophilized solid containing 0.1 M phosphate buffer at pH 5 prior to
lyophilization (Adapted from ref. 8 and 15).
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
8. 74 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
0 14
Glassy State Rubbery State
0 12
1
"O
0 10
•+->
c
o
-t—'
(f)
c
0 08
0 06
i
o
o
i
Downloaded by UNIV OF PITTSBURGH on August 19, 2010 | http://pubs.acs.org
<D 0 04
-*-> O ow 0 . 3 3
w
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
o
on 0 0 2 • a w 0.54
v a w 0.76
0 00
-60 -40 -20 0 20 40 60
T-T
Figure 6. Aspartame degradation rate constants as influenced by the distance
from the glass transition temperature in solids containing 0.1 M phosphate
buffer at pH 7 and 25°C (Reproduced with permission from ref. 10. Copyright
1994 ACS).
i 1 1 1 1 r
1 2 3 4 5 6 7 8
Initial S o l u t i o n pH
Figure 7. pH-rate profile for aspartame degradation in 0.1 M phosphate buffer
at 30°C (Reproduced with permission from ref. 3. Copyright 1991 Cryo-
Letters).
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
9. 3. B E L L Aspartame Stability in Solids and Solutions 75
constants at different water activities and similar glass transition temperatures were
significantly different (10). The amount of free volume in the solid system is
sufficient for the localized conformational flexibility necessary for aspartame to
cyclize into D K P . Long range molecular mobility (e.g., diffusion) required for
other types of reactions would probably be more dependent on whether the system
was in a glassy or rubbery state.
PH
The impact of pH on aspartame degradation has also been examined in liquid and
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solid systems. Aspartame is most stable between pH 3.5 and 5, with increased acid
hydrolysis at lower pH values and increased base catalysis at higher pH values
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
(1,3,5-6,8-9,15,22). Figure 7 shows the U-shaped pH-rate profiles for aspartame
degradation in a high moisture semi-solid gel (92% moisture) and a low moisture
solid (6% moisture). In the low moisture system, rate constants are generally lower,
as expected based on the previous water activity discussion. However, the profile
for the low moisture system also has a broader minima plateau than that of the high
moisture system and a lower slope on the base-catalyzed portion of the profile
(3,15).
The difference between the two profiles can be explained by evaluating the
pH of the low moisture system. The degradation products formed in the high
moisture gel and at 6% moisture are listed as a function of the initial solution pH in
Table I. In the high moisture gel, the 0-isomers are formed at pH values of 5 and
lower while at 6% moisture the 0-isomers are also formed at higher pH values (e.g.,
initial solution pH of 6.5) where these products are not expected to be formed. One
explanation for the appearance of 0-isomers at the higher pH values in the 6%
moisture system could be that the pH of this system decreased upon lyophilization
Table I. Aspartame Degradation Products Formed at 30°C
Initial Semi-solid Gel Solid
Solution pH (92% moisture) (6% moisture)
2.6 DKP, a-AP, 0-AP, 0-APM, PMe DKP, a-AP, 0-APM
3.0 DKP, a-AP, 0-AP, 0-APM, PMe DKP, a-AP, 0-AP, 0-APM
4.4 DKP, a-AP, /3-AP, 0-APM DKP, a-AP, 0-AP, 0-APM
5.0 DKP, a-AP, 0-AP, 0-APM DKP, a-AP, 0-AP, 0-APM
5.5 DKP, a-AP DKP, a-AP, 0-AP, 0-APM
6.0 DKP, a-AP DKP, a-AP, 0-AP, 0-APM
6.5 DKP, a-AP DKP, a-AP, 0-APM
Adapted from ref. 3 and 15.
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
10. 76 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
and rehydration (3,15). Similarly, Table II shows the DKP:a-AP concentration ratio
as a function of the initial solution pH. These ratios from the 6% moisture system
at higher pH values are similar to the ratios from the high moisture system at lower
pH values, which suggests that the pH decreased by about 2 units in the low
moisture system (3,15). This lowering of pH may be attributable to selective
precipitation of buffer salts (i.e., solubility limitations) and the increased
concentration of protons in the limited aqueous phase of the low moisture solid
(3,23-25).
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Table II. DKP:a-AP Ratio from Aspartame Degradation at 30°C
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
Initial Semi-solid Gel Solid
Solution pH (92% Moisture) (6% Moisture)
a
2.0 0.2 N.D.
2.6 0.5 0.2
3.0 1.0 0.3
4.4 2.0 0.3
5.0 3.0 0.6
5.5 5.0 0.6
6.0 22.0 0.7
6.5 75.0 3.0
a
N . D . = not determined; Adapted from ref. 3 and 15.
Excipient Interactions
Most of the studies examining interactions between aspartame and other substances
have focused on carbonyl compounds. In the presence of glucose, aspartame has
been shown to participate in the Maillard reaction at elevated temperatures (26-27).
It has also been suggested that other carbonyls (e.g., vanillin) react similarly with
aspartame in solution (28). In a 0.1 M phosphate buffer solution at pH 5 and 30°C,
0.3 mM aspartame degraded faster in the presence of 0.8 mM glucose than in a non-
glucose containing solution (Bell, L . N . , University of Minnesota, unpublished data).
In reduced-moisture solid systems, the addition of glucose alone did not enhance the
loss of aspartame (21). However, reduced-moisture solid systems containing both
glucose and emulsified oil did display faster aspartame degradation rates as
compared to similar systems of aspartame alone or aspartame with glucose (21).
While the mechanism by which oil enhanced the degradation of aspartame is
unknown, the oil may have provided an additional reaction medium or the oil and
water may have formed a reactive interface (21).
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
11. 3. B E L L Aspartame Stability in Solids and Solutions 77
A limited number of studies have examined the interaction of aspartame with
substances other than buffer salts or carbonyls. At 30°C, aspartame in 0.1 M
phosphate buffer solutions at pH 3, 5, and 7 (8) had degradation rate constants
similar to the same solution formed as an agar/microcrystalline cellulose gel (3),
which suggests these carbohydrate polymers are inert with respect to aspartame
stability. The addition of calcium caseinate to 0.01 M phosphate buffer at pH 7 and
80°C decreased the aspartame degradation rate; the mechanism for this protective
effect is unknown (29). Aspartame in solution is also stabilized by complexation
with 0-cyclodextrin (30). It is clearly worthwhile to understand the effect excipients
have on peptide stability during product formulation.
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Summary
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch003
The formulation of peptide products is challenging and requires a thorough
understanding of the factors which influence peptide stability as well as how stability
differs in solids and liquids. Aspartame stability data can provide some useful
information regarding problems that may arise during formulation. Due to different
activation energies at different pH values and moisture contents, the activation
energy for the reaction in the specific formulation should be used to accurately
predict product shelf life. Buffer salts catalyze the degradation of aspartame; the
potential effects of buffer type and concentration need to be determined to minimize
degradation rates and enhance shelf life. Water activity correlates with aspartame
stability. However, depending on the mechanism of degradation and the extent of
movement required for degradation, glass transition may influence the degradation
of larger peptides. Understanding whether water activity or glass transition
influences chemical reactions is necessary for optimizing solid state peptide stability.
Changes in pH upon lyophilization not only change the rate of aspartame degradation
but the mechanism by which it degrades. Peptide-based products should therefore
be formulated such that the final pH is optimized rather than the initial solution pH
of the system. Carbonyl compounds, due to the Maillard reaction, can enhance
peptide degradation, depending upon the moisture content of the system. However,
caseinates and cyclodextrins can reduce A P M degradation. Thus, the effect of
excipients on peptide stability must also be evaluated. By keeping these factors in
mind during formulation, the pharmaceutical scientist can develop peptide products
of optimum stability.
Acknowledgments
This work is supported by project ALA-13-005 of the Alabama Agricultural
Experiment Station and is published as AAES Journal No. 10-965200.
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