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Formulation and evaluation of protein drug delivery system.

The document addresses the formulation and evaluation of protein and peptide drug delivery systems, discussing the barriers and methodologies for effective delivery. It covers various components such as protein concentration, buffer systems, polymers, stabilizers, and preservatives that ensure the stability and efficacy of protein-based therapeutics. Evaluation methods like stability testing, bioassays, UV spectroscopy, and electrophoresis are also detailed to assess the performance and integrity of these drug delivery systems.

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FORMULATION AND EVALUATION OF Protein and peptide DRUG delivery SYSTEM 1 Presented by : Khaja Anees Ahmed M-PHARM 1st SEM Dept Of Pharmaceutics HKE’S MTRIPS Gulbarga
Previously Asked Questions 1. Discuss barriers associated with protein and peptides delivery.(7.5m) 2. Write in detail delivery system of protein and other macromolecules.(7.5m) 3. Explain evaluation methods for delivery systems of protein..(7.5m) 4. Explain the barriers of protein delivery systems. Write briefly on formulation of protein (7.5m) 2
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INTRODUCTION: • The proteins are relatively large molecules with complex structure. • Proteins have high molecular weight. • It acts as Hormones, Enzymes, Structural elements and Immunoglobulins. • Protein are highly purified. • Proteins are heat-unstable molecules with a high tendency to aggregate. • Chemical reactions such as oxidation, deamination, proteolysis, racemization, isomerization, disulfide exchange, photolysis gives rise to chemical instability. 4
Formulation : Drug and excipients Uses examples Protein concentration At High concentration – aggregation & ppt . At Low concentration - loss of protein . • Insulin • Vasopressin tannate Buffers It regulate pH Stabilize & Destabilize the protein So selection of buffers is very important • Phosphate • Acetate • Citrate Selection of solvent system The solvent which are likely to stabilize the proteins are used such as polyhydric alcohols • Glycerol • Erythritol • Sorbitol Polymers It should be * biodegradable * biocompatible * non toxic * easily available and cheap NP: collagen , haemoglobin and gelatin SP : PLA & PGA Stabilizers Types:Serum albumin,amino acid , surfactant, chelating agent , Anti oxident etc Glycine, poloxamer 188 , polysorbate , EDTA Preservatives Prevents microbial growth most widely used are : • Phenol 0.3-0.5% • Cholobutanol 0.3-0.5% 5
Protein concentration • Protein concentration refers to the amount of protein present in a given volume or mass of a solution. • It’s typically measured in units like grams per litre (g/L) or milligrams per millilitre (mg/mL). • The optimal protein concentration refers to the concentration at which a protein exhibits its desired characteristics or performs most effectively in a given application. • example: • Too high a concentration of insulin could lead to aggregation or precipitation, reducing its effectiveness . • Conversely, too low a concentration may not provide a therapeutic effect. 6
• In the formulation of mAbs for intravenous administration. • The protein concentration typically ranges from 5 to 200 mg/mL, depending on the specific therapeutic indication, dosing regimen, and formulation requirements. 7
Buffers • Buffers play a crucial role in protein drug delivery systems by maintaining the pH within a specific range. • This ensures the stability and activity of the protein drug throughout formulation, storage, and administration. • Additionally, buffers help prevent protein denaturation, aggregation, and degradation, thereby preserving the efficacy of the drug. • By providing a stable pH environment, buffers contribute to the overall safety and effectiveness of protein-based therapeutics. • Examples: phosphate buffer, acetate buffer, citrate buffer and carbonate buffer. 8
• Phosphate buffer : To prepare a 0.1 M phosphate buffer at pH 7.4, you would mix monosodium phosphate 34.0(NaH2PO4) and disodium phosphate 58.4(Na2HPO4) in specific proportions. • Acetate buffer : To prepare a 0.1 M acetate buffer at pH 4.75, you would mix acetic acid 99% purity (CH3COOH) and sodium acetate27.6 (CH3 COONa) in specific proportions. • Citrate buffer : To prepare a 0.1 M citrate buffer at pH 6.0, you would mix citric acid21.01 (C6H8O7) and tri sodium citrate 289.41(Na3C6H5O7) in specific proportions. 9
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Selection of solvent system • Solvent Systems due to its significant impact on the stability, solubility, and efficacy of the protein drug. 1.Solubility: The solvent system should be capable of solubilizing the protein drug at the desired concentration without causing denaturation or aggregation. • Different solvent systems have varying abilities to dissolve proteins, and the selection depends on the specific characteristics of the protein. 2.Stability: The solvent system should maintain the stability of the protein drug by minimizing interactions that could lead to denaturation, aggregation, or degradation. 11
3.Compatibility: The solvent system should be compatible with other formulation components, such as buffers, excipients, and delivery vehicles. • Compatibility issues can arise if the solvent interacts with these components, affecting the overall stability and efficacy of the formulation. 4. Biocompatibility: In some cases, the solvent system itself may be part of the delivery vehicle or carrier system administered to the patient. • In such cases, biocompatibility is essential to ensure the safety of the formulation upon administration. 12
• Examples: • Aqueous solvent: Water is often the primary solvent due to its compatibility with proteins • Organic solvent :Organic solvents like ethanol or acetone can be used to solubilize hydrophobic components in the formulation. • Co – solvent : Co-solvents such as glycerol or propylene glycol are often employed to enhance protein stability and solubility. 13
Polymers • Polymers play a significant role in protein drug delivery systems by providing structural support, controlling drug release kinetics, and enhancing stability. 1. Encapsulation: Polymers can encapsulate protein drugs, protecting them from degradation and enzymatic activity in the body. • This encapsulation can be achieved through various techniques such as microencapsulation, nanoparticle formation, or polymeric matrix systems. 14
2.Stabilization : Certain polymer can stabilize protein drugs by shielding from harsh environmental conditions such as pH changes, temperature, fluctuations and proteolytic enzymes . • This stabilization helps Preserve the integrity and activity of the protein drug during storage and administration. 3. Biodegradability and Biocompatibility : Many polymers used in protein drug delivery systems are biodegradable and biocompatible , minimizing the risk of adverse drug reactions and allow for safe degradation . • Examples of polymers used in protein drug delivery systems include Poly lactic co -glycolic acid (PLGA), polyethylene glycol (PEG) , chitosan, alginate etc. 15
Stabilizer: Stabilizers play a critical role in protein drug delivery systems by helping to maintain the stability and integrity of the protein drug throughout formulation, storage, and administration. 1. Amino acid Glycine is most commonly used stabilizer Mechanism of action of amino acid as stabilizer by one of the following 1. Reduce surface adsorption 2. Inhibit aggregate formation 3. Stabilise protein against heat denaturation. Examples : Glycine ,lysine and arginine . 16
2. Surfactant • They cause denaturation of protein by hydrophobic desorption. • Optimal concentration of surfactant for stabilization should be greater than CMC • Ionic surfactant are more effective than non ionic surfactant. • Example: Polysorbate20 polysorbate80 and poloxamer 188. 3. Polyhydric alcohol and carbohydrates • Many therapeutic protein and peptides are derived from blood such as immuno globin, coagulation factor, For viral destruction pasteurization at 60°c for 10 hrs is needed for chemical stability. • Long chain of polyhydric alcohol are more effective as stabilizer eg: xylitol, Sorbitol • Carbohydrates eg: mannose, glucose, ribose 17
4. Anti –oxidant • Anti–oxidants are the compound which inhibit the oxidation reaction • Eg: Thioacetic , Triethanolamine. 5. Chelating agent • Some anions and cations will directly bind to proteins. • For example divalent cation directly bind protein which lower the solubility • Removal of these ion by chelating agent such as EDTA may help to solubility . 6. Miscellaneous Certain enzyme can be stabilized by compounds having similar structure Eg: glucose stabilize glycomase and asparagine stabilize asparaginase. 18
Preservatives • Preservatives are included in protein drug delivery systems to prevent microbial contamination and maintain the stability of the formulation during storage and use. 1. Benzyl Alcohol: Benzyl alcohol is a widely used preservative in protein drug formulations due to its broad spectrum antimicrobial activity. • It is effective against bacteria, fungi, and some viruses at low concentrations. • However, benzyl alcohol can be cytotoxic at higher concentrations, so its use must be carefully controlled, especially in formulations intended for injection. 19
2. Benzalkonium Chloride: Benzalkonium chloride is a quaternary ammonium compound with antimicrobial properties. • It is used in some protein drug formulations as a preservative, particularly in ophthalmic preparations. • However, its use can be limited by its potential to cause irritation or allergic reactions. 4. Parabens: Parabens, such as methyl paraben and propyl paraben, are commonly used preservatives in pharmaceutical formulations due to their antimicrobial properties. • However, their use in protein drug formulations can be limited by concerns about potential interactions with proteins . 20
EVALUATION OF PROTEIN AND PEPTIDE DRUG DELIVERY SYSTEM 21
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STABILITY TESTING : Stability testing of protein drug delivery systems is crucial to ensure the integrity, efficacy, and safety of the product throughout its shelf-life. • This testing involves evaluating the physical, chemical, and biological stability of the system under various storage conditions and stresses. 1. Accelerated Stability Studies: • Accelerated stability studies of protein drug delivery systems involve subjecting the systems to conditions that mimic long-term storage or harsh environmental factors, such as high temperature, humidity, and light exposure. • These studies help assess the physical and chemical stability of the protein- based drug formulations over time, aiding in the determination of shelf life and storage conditions. 23
2.Compatibility Studies: Evaluate the compatibility of the protein drug with excipients, packaging materials, and delivery device components to identify any interactions that could affect stability. • Assess changes in protein structure, activity, and degradation products upon exposure to these materials. 3.Container Closure Integrity Testing: Conduct container closure integrity testing to ensure that the packaging maintains the integrity of the protein drug . • Prevents contamination or loss of efficacy during storage and transportation. 24
BIOASSAY : • Bioassays are essential for evaluating the biological activity and efficacy of protein drug delivery systems. • These assays assess the functional activity of the protein drug within the delivery system and its ability to obtain the desired therapeutic response. • Bioassay are of two types: in vitro and in vivo. • In case of in vitro bioassays response of cells to hormones and growth factors is monitored. • In case of in vivo bioassay pharmacological response of animals to proteins is monitored. 25
UV spectroscopy : UV spectroscopy is a valuable technique used to analyse protein drug delivery systems, providing insights into their composition, stability, and interactions. 1. Protein concentration determination: • UV spectroscopy is used to quantify the concentration of proteins in the delivery system. • Based on their absorbance at specific wavelength • This information is crucial for accurate dosing and formulation consistency. 26
2. Detection of Aggregation: • Aggregation of proteins within the delivery system can be detected by changes in UV absorbance. • Typically manifested as an increase in absorbance at higher wavelengths due to light scattering by aggregated particles. 3.Interaction Studies: • UV spectroscopy can assess interactions between proteins and delivery vehicles or excipients. • Shifts in absorbance peaks can indicate binding or complex formation. 27
ELECTROPHORESIS : Electrophoresis is a widely used technique for analysing protein drug delivery systems, providing valuable information about the composition, purity, and integrity of proteins within the system. SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis): • This technique is used to separate proteins based on their molecular weight. • SDS denatures proteins and imparts a negative charge proportional to the protein’s mass, allowing for size-based separation during electrophoresis. • SDS-PAGE is commonly used to assess the purity of protein drug formulations and detect protein degradation . 28
FTIR(Fourier-transform infrared spectroscopy) : Fourier-transform infrared spectroscopy (FTIR) is a powerful technique used to analyze protein drug delivery systems, providing insights into their composition, structure, and interactions. Interaction studies: • FTIR spectroscopy enables the study of interactions between proteins and drug carriers or excipients. • Changes in peak positions, intensities, or shifts indicate molecular interactions. • Such as hydrogen bonding or electrostatic interactions, between proteins and formulation components. 29
BRADFORD ASSAY : • The Bradford assay is a common method used to quantify protein concentration in protein drug delivery systems. • In the Bradford assay, a protein sample is mixed with Bradford reagent, typically containing Coomassie Brilliant Blue dye and a strong acidic solution such as phosphoric acid or sulphuric acid. • The mixture is then incubated to allow dye-protein binding to occur. • After incubation, the absorbance of the sample is measured at 595 nm using a spectrophotometer. • The intensity of the blue colour is directly proportional to the protein concentration in the sample. 30