Pharmaceutics Exam 2 Study Guide (OCR) PDF

Study guide (Exam 2) § Exam date: Wednesday 10/30/2023 § Number of questions: 49 questions § Total points: 100 points § Please bring your calculator for the math problems. § There will be some questions from lectures (1...

Study guide (Exam 2) § Exam date: Wednesday 10/30/2023 § Number of questions: 49 questions § Total points: 100 points § Please bring your calculator for the math problems. § There will be some questions from lectures (1-14). § Some questions from the biopharmaceutics topic will be taken on paper. § Students are supposed to understand and be able to explain the following terms and concepts (classes 15- 28): 42 questions exam soft, 7 questions on paper (Active learning), 5-6 questions exam 1 material, formulas will be provided, 1-2 FITB Drug Stability 1. Understand the Q10 method for shelf life determination. - Q10: measure of the rate of change of the chemical reaction as a consequence of the temperature being increased by 10ºC - Q10 = 3 - Estimates the shelf life for products that is going to be stored in different conditions than specified in the product label § Room temp vs refrigeration 2. Know the drug stability testing process. - FDA requires a demonstration of drug stability - Stability testing includes chemical, physical, and microbiological attributes of drugs that are susceptible to change during storage and are likely to influence the stability of the pharmaceutical product - Factors that are considered: § Temperature § Humidity § pH § Light intensity § Drug concentration 3. Estimation of shelf life using Q10 method. - Q10= R2/R1 4. Discuss the stability testing protocols and frequency of testing. - Accelerated stability testing is used to determine the type of degradation products which may be found after long term storage § Speeds up drugs degradation process to allow faster submission process of data to FDA § minimum of tree time points - Long term stability testing is also called real-time stability testing § Normally performed for longer duration of the test period to allow significant product degradation under recommended storage conditions § every 3 months over the first year, every 6 months over the second year, annually after that. - If no significant change occurs during the six-months accelerated and long term stability testing, the product will be placed in the market with a shelf life up to 2 years - Intermediate: if significant change occurs at any time during 6 months testing at the accelerated storage condition Pharmaceutical packaging 5. Know the different types of containers according to the material of construction - Primary (container-closure system): closure + container - Secondary: contains primary package, associated components (like information leaflets, spoon, etc.), contained in a box or something similar - Tertiary: contains multiple secondary packs, facilitates handling and transport, and prevents damage associated with handling, transport, storage - Container: receptacle that holds the article (API + excipients) and it in direct contact with the article (ampules, vials, bottles, syringes, and pen injectors) - Closure: a material that seals an open space of a container and provides protection for the contents, prevents contamination (caps, lids) - The purpose of packaging: i. To protect the product from the environment ii. To protect the product from damage and maintain the product integrity during shipping and storage iii. To provide an appropriate presentation of the product to the consumer - USP classifies primary packaging according to: § Material of construction (glass, plastic, metal) § Their ability to protect the contents from external conditions § Dose: single unit package vs. multiple unit package 6. Know the difference between single and multiple dose containers - Single dose containers: holds a quantity of drug intended as a single dose and, when opened, cannot be resealed with assurance that sterility has been maintained i. Advantages: protection of dose from the environment, offer greater product stability and assurance of sterility, can be used to package preservative-free preparations, and they can reduce the incidence of errors such as the withdrawal of an incorrect dose and hence improve patient safety. ii. Disadvantage: cost more b/c more material goes into packaging them - Multiple dose containers: permits withdrawal of successive portions of the contents without changing the strength or endangering the quality or purity of the remaining portion iii. Advantages: more economical (one bottle containing a one month’s supply) iv. Disadvantages: if you change the needle but not the syringe then an infection outbreak could happen because the syringe is exposed to the needed an outside world, also in a multi-unit container the remaining doses are exposed to the environment every time the container is opened which can lead to contamination of the product - USE MULTI-UNIT AND SINGLE-UNIT WHEN TALKING ABOUT TABLETS, USE MULTI-DOSE OR SINGLE-DOSE WHEN TALKING ABOUT INJECTABLES Understand different packaging materials *** - Liquids are in contact with the primary pack, as opposed to solids (tablets and capsules) - Liquids require greater quality from a pack, so they ‘take nothing out of the product and add nothing to it” - Injectables require even greater quality from the pack to maintain sterility and freedom from other possible contaminates - Packaging materials can be: glass, plastic, metals - Glass: o 4 categories according to their degree of hydrolytic resistance to water attack ­ o Glass can be colored (i.e. amber) to package drugs that are susceptible to degradation by sunlight o Glass used in pharmaceuticals falls into four categories according to their degree of hydrolytic resistance to water attack o Glass holds acidic formulations o Contains a sodium ion (soda lime glass) o Disadvantages of using glass for containers § More expensive compared to plastic § Not resistant on impact § Leaching of alkali – what does this mean? - Plastic: o Advantages: § Light weight, resistant to impact § Versatility of design (shape) § Plastic squeeze bottles § Inexpensive o Disadvantages: § Permeability of the containers to atmospheric oxygen and moisture § Leaching of the constituents of the container to the product § Adsorption of drugs to the container § Transmission of light through the container (light sensitive drugs) o Types of plastic: § Polyvinyl chloride (PVC): advantage; this material is rigid and has good clarity, making it useful in blister packaging of tablets and capsules § Newer plastics: amorphous polyethylene (APET) and polyethylene terephthalate glycol (PETG), advantages; improved moisture and gas barrier compared with PVC - Metals: o Tin: § Advantages: light weight, can protect from light and has low permeation of oxygen and water § Disadvantage: more expensive than aluminum o Aluminum: § Advantages: light weight, protect from light and has low permeation of oxygen and water o Lead: § Used with radioactive pharmaceuticals 7. Understand the meaning of well-closed and tight-closed containers - Well closed containers: i. Protect the contents from contamination by extraneous solids and from loss of the article under normal conditions of handling, shipment, and storage ii. Used for products that do not require complete protection from air or moisture iii. Provides a basic level of protection against dust and other contaminants but its not completely airtight - Hermetic (tight) containers: iv. Impervious to air or any other gas under the ordinary or customary conditions of handling, shipment, and storage v. Commonly used for products that are highly sensitive to air and moisture such as injectables and highly sensitive drugs vi. Offers highest level of protection, ensuring that the contents remain sterile and free from contamination  - Well-Closed Container: Prevents solid contents from spilling and restricts accidental opening but is not necessarily airtight. Suitable for short-term storage or items less sensitive to environmental exposure. § Think screw top bottle - Hermetic Container: Completely airtight and prevents any gas, liquid, or microorganisms from entering or leaving. Used for long-term preservation and high-sensitivity items. § Think canned food 8. Define sorption and leaching of contents and how they can affect the API **** - Interactions btw packaging and the drug could impact safety and efficacy of drug products - Leaching: when chemicals move out of the packaging material into the drug i. Compounds leached from plastic containers are generally polymer additives, such as the plasticizers (additives that are added plastic polymers to make plastic flexible), stabilizers, and antioxidants ii. More leaching happens to a liquid or semisolid packaged in plastic iii. Little leaching occurs with tablets or capsules iv. Increased temp and excessive agitation will increase the likelihood of leaching v. *** special concern with IV bags where leaching of contents to fluids may pose health hazards to patients - Sorption: when the chemicals move out of the drug into the packaging material vi. Binding of molecules to polymer materials, divided into both adsorption and absorption vii. Unionized (uncharged) species of molecules has a greater tendency to undergo sorption viii. Can occur with API and/or excipients ix. Sorption results in a decrease in the concentration of the molecules and so lower dose of API administered to patients 9. Understand the closure and child resistance packaging - Designed to prevent children under 5 y/o from opening the package within a reasonable period but it’s not difficult for adults to use properly Biotech products 10. Describe the importance of biotechnology in pharmaceutical sciences. - Biotechnology is the use of living organisms for the production of useful products - Pharmaceutical biotechnology is the biotechnical manufacturing of pharmaceutical products - 28% of the market - One of the nation’s fastest growing areas - Relies on cutting edge discovery 11. Interpret the difference between small molecule drugs and biotech drugs. **** - Small molecule (aspirin) i. Molecular weight < 500 Da ii. Conventional medicine iii. High toxicity and side effects iv. Saturated market - Biotech drug (Herceptin) v. Large molecules vi. High efficiency vii. Poor stability 12. Know examples of biotech products that were developed to replace conventional products. - Type 1 diabetes  i.Body can’t produce enough insulin which is used to convert glucose to energy ii.Use of therapeutic protein to replace endogenous protein has been established for treatment iii.The first successful treatment type of type 1 diabetic patient with animal-derived insulin iv. Insulin used to be extracted from the pancreas of pigs and cows v. You could have an immune reaction to animal insulin, so we no longer rely on it to be a source for human insulin - Human growth hormone vi. Protein essential for growth and development vii. Children born with a deficiency suffer from dwarfism viii. hGH was isolated from the pituitary glands of human cadavers, 50 of them were required to treat one hGH deficiency for just one year (pit-HGH) ix. Isolation process is difficult and there is contamination risk x. Creutzfeldt-Jakob disease is a fatal degenerative neurological disorder in patients receiving pit- HGH (b/c of contamination during isolation) xi. To isolate 5 mg GH, 500,000 sheep brains are needed: one gallon of the engineered bacterial cells can produce 5 mg HGH, and bacteria can produce a billion copies in 15 hours 13. Describe the structures of DNA and RNA 14. Describe the “Central Dogma” - The Central Dogma means that genetic information goes from DNA to RNA to proteins. - DNA à RNA à Protein; cannot go in reverse order, the central dogma states that genetic information only flows in one direction - The sequence of a gene decides the order in which 20 amino acids will join to make a protein. - A codon is a set of three nucleotides, and each codon codes for one amino acid. - The order of codons in DNA, after being copied into mRNA, decides the amino acid sequence of the protein ---------------------------------------- 15. List the procedures and enzymes that are involved in the recombinant DNA technology **** - Find a target gene and a plasmid. - Cut both with special (restriction) enzymes. - Join (ligate) the gene to the plasmid. - Put plasmids into bacteria (bacterial transformation) - Purify the protein made by the bacteria. o Restriction Enzyme: Cuts DNA at specific spots. o Ligase: Glues two pieces of DNA together. o Bacterial Transformation: Putting plasmids into bacteria.  - A wide variety of expression host: bacteria, yeast, plants, and transgenic animals 16. Explain the difference between antigen and epitope - Antigen: The whole molecule that causes an immune response. - Epitope: A small, specific region on the antigen that the immune system directly interacts with. - One antigen can have multiple epitopes. - An antibody has a binding site called the paratope (antigen-binding site), the antigen has many epitopes on it and the epitopes bind to the paratope on the ANTIBODY à antigen 17. Describe the structure and components of an antibody - Antibodies are a group of immunoglobulins produced by B-cells to specifically recognize foreign antigens and trigger appropriate actions from the host immune system - 150 kDA protein - >30% of biopharmaceuticals in clinical trials - Applications: a. Diagnosis b. Therapeutics c. Targeting agents d. Purifications and other applications in scientific research 18. Tell the antibody production process - Antigens are macromolecules that are recognized as foreign objects in the immune system - Antigens: proteins, polysaccharides, parts of bacteria, viruses, and other microorganisms - Antibodies are not generated against the entire antigen molecule but to particular groups on the antigen called the EPITOPE - Many different antibodies can be made against a single antigen 19. Explain the difference between polyclonal antibody and monoclonal antibody - Polyclonal antibodies: are a mixture of antibodies produced by different B cells in response to an antigen. Each antibody recognizes a different epitope on the same antigen. a. Target multiple epitopes on the same antigen. - Monoclonal antibodies: Identical antibodies produced by a single B cell clone. They recognize and bind to only one specific epitope on an antigen. b. Target one specific epitope on an antigen. Understand the role and structure of plasmids. **** - Autonomously replicating DNA molecule - Circular and double stranded - Found in bacteria - Many identical plasmids can exist within a single bacterial cell - Plasmid used in genetic engineering is called a ‘vector’ - ALLOW PRODUCTION OF A GENE PRODUCT (PROTEIN) - Gene insertion: o Scientists can insert a desired gene into a plasmid, creating a "recombinant plasmid." - Transformation: o This plasmid is then introduced into the host cell, where it is replicated along with the host cell's DNA. - Protein synthesis: o Once the gene is expressed, the host cell's ribosomes translate the mRNA into the corresponding protein. - Example: o Insulin production: By inserting the human insulin gene into a plasmid and transforming bacteria with it, researchers can produce large quantities of insulin protein for medical use. Discuss the structural differences between heavy and light chains in antibodies. - Antibody structure: Y shaped protein that has 2 identical heavy chains and 2 identical light chains, connected by disulfide bonds - Heavy Chain: has two regions. CONTAINS MORE AMINO ACIDS (HEAVY) 1. Constant: the same for antibodies of the same type (IgA, IgD, IgE, IgG, IgM), but different for different types. These types have different roles in the immune system. 2. Variable: changes between antibodies from different B cells but stays the same for antibodies made by the same B cell. - Light Chains: Has successive domains: one constant domain and one variable domain Explain the roles of the Fab and Fc regions in antibodies. - Fab: composed of one constant and one variable domain from each heavy and light chain of the antibody. WHERE THE ANTIGEN BINDS - Fc: modulating the activity of immune cells. Its composed of two heavy chains that contribute to two constant heavy chains. o Insures that each antibody generates an appropriate immune response for a given antigen o Binds to various cell receptors or other immune molecules to destroy foreign objects Explain the roles of Mab (monoclonal antibody) and Pab (polyclonal antibody) - PolyAb: derived from different B cells; a mix of immunoglobulin molecules produced against a specific antigen; each recognizes a different epitope o Can be generated more rapidly; less expensive; less technical skills required - Monoclonal Ab: derived from a single B cell; identical immunoglobulin molecules produced against a single epitope o Homogeneity and consistency are principal advantages Describe the process of hybridoma technology for monoclonal antibody production. **** 1. Inject antigen into a mouse. Some of the B cells will produce antibodies against the antigen 2. Isolate B cells from the mouse spleen 3. Normal B cells will die within a few days 4. Myeloma: tumor cells that grow indefinitely 5. Hybridoma: fusion of a B cell and a myeloma cell; maintains the ability of B cell to produce antibody and the ability of myeloma to grow indefinitely 6. Hybridoma cells will be screened, the best cell will be cultured, and the Mab will be purified Analyze the differences in immunogenicity between chimeric, humanized, and fully human antibodies. - Chimeric Ab: 75% of human sequence. Mouse heavy and light chain variable regions linked to human constant region - Humanized Ab: >90% human sequence - Human Ab: 100% human sequence. Human antibody is generated in mice (transgenic mice) - Repeating use of mouse antibodies leads to severe immune reaction (human anti-mouse antibodies, HAMA response) Describe the structure and function of single-chain variable fragments (scFv). - The large size of an antibody molecule limits its application in drug delivery - Fv is the variable domain of Fab - ScFv: single chain Fv o Obtained by connecting the VH and VL domains by a linked in a single polypeptide o Retains the same binding affinity of the parent antibody and has decreased immunogenicity (smaller size) o Used as a targeting agent in targeted drug delivery systems - *** an antibody is larger in weight but smaller in size, an antigen is smaller in weight but larger in size *** Formulation of Biotech products 20. Describe the challenges in protein drug development. - Large molecules have difficulty in cellular uptake - The biologically active structure is maintained by weak covalent forces that can be easily disturbed by mild manufacturing/ storage conditions and result in poor stability - Factors affecting stability a. Temperature b. pH c. Humidity d. Ionic strength e. Enzyme f. Mechanical forces (pressure, shearing and shaking) 21. Know the different types of instabilities of protein drugs. - Physical instability: Changes in the secondary, tertiary, or quaternary structure a. Aggregation b. Surface absorption c. Precipitation - Chemical instability: formation of new chemical entities by bond formation or cleavage d. Hydrolysis e. Oxidation f. Deamidation g. Disulfide exchange 22. Interpret why protein drugs are not stable. - Each protein drug exist as an unfolded polypeptide that lacks any stable (long lasting) three dimensional structures - When the protein is folded into its three-dimensional structure its known as its native shape - The correct 3D structure is essential to its function - How to stabilize these? a. Avoid any physical or chemical factors that might cause protein degradation (factors affecting stability above) b. Proteins are most stable in conditions that mimic their natural environments c. Add additives that can stabilize their native proteins d. PEGylation: modification with PEG e. Formulation using nanotechnology - Storage: f. Solution: unstable, a freezer is needed, difficult to handle and transport, costly  g. Dried form: stable even at room temp, stable in the fridge, convenient to handle and transport, cost effective 23. Describe the spray drying process. - Protein drugs are often formulated as liquid dosage forms which are unstable and have limited shelf life that requires storage in the freezer so proteins can be formulated as dry powder - There are 2 drying methods: spray drying, and freeze drying (lyophilization) - Spray drying: 1. The solution is sprayed as a fine mist into the drying chamber using a special nozzle called an atomizer. 2. The liquid in the droplets evaporates quickly after being sprayed. 3. Hot gas dries the droplets, turning them into powder that falls to the bottom of the chamber. 4. A cyclone at the bottom separates the powder from the air, and the powder is collected in a container. 24. Describe the freeze-drying process. (lyophilization) - Sublimation vs Evaporation a. Sublimation: where a solid directly transforms into a gas without passing through a liquid phase (solid à gas) b. Evaporation: where a liquid turns into a gas (liquid à gas) - Freeze drying is a sublimation process - Removing water from a frozen sample using a vacuum at low pressure 25. Explain the importance of lyophilization in protein drug formulation. - Procedure: a. Freeze the material at -50ºC to -80ºC b. Primary drying i. Reduce surrounding pressure ii. Enough heat is applied iii. 95% of the water is sublimated iv. Takes several days c. Secondary drying i. Low pressure ii. Temp is higher than primary drying. Maybe above 0ºC iii. Sublimate tightly bound in water d. Vacuum is broken with inert gas (N2) e. Final humidity ~ 2% - Why its important: f. Convenient for storage g. Convenient for shipping h. Degradation of proteins in solid state is much less than that occurs in a solution i. Cost effective j. Best for long-term storage k. Allows protein formulations to be stored at room temp during shelf life l. It’s the most important technique in protein formulation m. 46% of FDA approved biologic products are freeze dried products - Considerations: n. Protein should be remained in the native state  o. Protein unfolding should be minimized p. Residual moisture must be very low (