PBB Midterm Review PDF

PBB Midterm Review Lecture 1 what is biotech and what is biopharmaceuticals biotech → use of living organisms or part of living organisms to improve quality of life insulin, viral vaccine...

PBB Midterm Review Lecture 1 what is biotech and what is biopharmaceuticals biotech → use of living organisms or part of living organisms to improve quality of life insulin, viral vaccine biopharmaceutical → drugs made from bio tech process ex. disinfectant drug → used to sterilize and clean food → ex. IPA (isopropyl alcohol (disinfectant that can evaporate and leave the surface → products need to be regulated as a drug bc you don’t want traces of disinfectant to end up in the finished product ( need to biodegradable and not toxic) how are proteins/gene expressed in prokaryotes and eukaryotes; similarities and differences for cell growth: Use Eukaryotic Cells when you need to study complex cellular mechanisms, perform functional assays, or require proteins with specific modifications relevant to human biology. Use Prokaryotic Cells for rapid growth, ease of manipulation, and when producing large quantities of proteins that do not require complex modifications. Eukaryotes Similar Prokaryotes Both eukaryotes and prokaryotes use the central dogma of molecular biology: whole animals bacteria DNA is transcribed into mRNA, which is then translated into proteins. PBB Midterm Review 1  Eukaryotes Similar Prokaryotes Ribosomes: Both types of cells complex cells with unicellular, without a nucleus; use ribosomes for protein mebrane-bound transcription and translation synthesis, where mRNA is organelles, including a occur simultaneously in the translated into amino acid nucleus where cytoplasm. ; same host cell sequences. transcription occurs. replicated mRNA undergoes extensive processing, including capping, polyadenylation, and splicing to remove introns before translation - 5' Capping: Addition of a modified guanine nucleotide to the 5' end, which protects the mRNA mRNA is usually not Genetic Code: Both organisms and aids in ribosome processed; it is often utilize the same genetic code, binding. translated as soon as it is with codons corresponding to Polyadenylation: Addition synthesized. - no introns to specific amino acids. of a poly-A tail to the 3' splice out end, which also stabilizes the mRNA and facilitates its export from the nucleus. Splicing: Removal of introns (non-coding regions) and joining of exons (coding regions) to produce a mature mRNA. Gene expression is Regulation is often simpler, regulated at multiple primarily at the transcriptional levels (not co-linear) level, often through operons →Transcriptional (clustes of genees Regulation: Involves regulatated together, various transcription responding to environmental factors and changes enhancers/silencers that → protein- coding is colinear control the initiation of with translated mRNA PBB Midterm Review 2  Eukaryotes Similar Prokaryotes transcription. →Post-Transcriptional Regulation: Includes mRNA stability, alternative splicing, and RNA interference (RNAi). →Translational Regulation: Involves factors that influence the initiation and efficiency of translation. →Post-Translational Regulation: Includes modifications such as phosphorylation, glycosylation, and ubiquitination that can alter protein function or stability. Translation initiation Initiation occurs at the Shine- involves the recognition Dalgarno sequence, which of the 5' cap structure helps align the ribosome with and scanning for the start the mRNA. codon (AUG). Proteins often contain signal sequences that Prokaryotic proteins generally direct them to specific remain in the cytoplasm, as organelles (e.g., nucleus, prokaryotes lack membrane- mitochondria, ER). The bound organelles. Some transport mechanisms proteins may be secreted into can be quite complex and the external environment, but involve various proteins this process is simpler. and pathways. applications of biotech reliable disease diagnosis, new drug therapies, genetic screening, vaccines, PBB Midterm Review 3  higher yield crops, pest control foods with health benefits basic structure of gene regualtory sequences ( promoters, enhancers, silencers ) and coding sequences (exons) and non-coding sequences (introns and UTR’s) promoter Location: Located at the beginning of the gene, upstream of the coding sequence. Function: Serves as the binding site for RNA polymerase and transcription factors, initiating the transcription process. The promoter determines when and how much of a gene is expressed. Transcription start site Location: Just downstream of the promoter. Function: The specific nucleotide where transcription begins. The first nucleotide of the coding sequence is often designated as +1. exons and introns Exons: Location: Interspersed along the gene. Function: These are the coding regions that are transcribed into mRNA and translated into protein. Introns: Location: Found between exons. Function: Non-coding regions that are transcribed into pre-mRNA but are removed during RNA processing (splicing) before translation. 5’ untranslated region (UTR) Location: Located between the promoter and the start codon of the first exon. PBB Midterm Review 4  Function: This region is transcribed into mRNA but is not translated into protein. It plays roles in the regulation of translation and stability of the mRNA. coding sequence Location: The sequence of exons that follows the 5' UTR. Function: This is the portion of the gene that is translated into a protein. It is made up of codons, each corresponding to a specific amino acid. 3’ untranslated region (UTR) Location: Located after the stop codon of the last exon. Function: This region is also transcribed into mRNA but not translated into protein. It contains regulatory elements that influence mRNA stability, localization, and translation efficiency. terminator Location: Located downstream of the 3' UTR. Function: Signals the end of transcription. This region is recognized by RNA polymerase to stop transcription and detach from the DNA. regualtory elements Enhancers: Location: Can be found upstream or downstream of the promoter, sometimes far away from the gene. Function: Enhance the transcription of a gene by providing binding sites for transcription factors. Silencers: Location: Similar to enhancers, they can be located at varying distances from the promoter. Function: Repress gene expression by inhibiting transcription factor binding. diff between dna and rna PBB Midterm Review 5  DNA RNA stable due to its double stranded unstable bc single stranded, contains ribose structure - less susceptible to which is more prone to degeneration hydrolysis double stranded sugar phospate single stranded sugar phosphate ribose deoxyribose base pair (A,T,C,G) single nucleobase (A,U,C,G) role in synthesis of proteing(mRNA), role in stores and transmits genetic info, regulation and catalysis (ribosomal RNA or rRNA, serves as blueprint trnasfer RNA or tRNA found in nucleus of eukaryotic cells found in nucleus and cytoplasm and cytoplasm of prokaryotic cells Lecture 2 drug discovery process drug discovery→initial feasibility → pre-clinical studies→ clinical studies→ submission→license→sale of product→post market surveillance what is accomplished at each stage of drug discovery process Drug Discovery product stumbled upon, research done on diseases and how the human body responds to it gene identification →how gene plays role in disease progression protein interaction → how protein interact to influence disease progression ex. covid 19→ needed to wear N95 masks → after research, cloth masks were allowed how does disease react when introduced to the new found drug and if the body responds well to the drug Initial Feasibility q’s asked: can we make the necessary drug? is the tech avail? is it cost effective (initial risk benefit analysis - want more benefits compared to PBB Midterm Review 6  risks in order to progress further in clinical trial q’s for initial risk benefit analysis does it give required response in preliminary biological experiments? any major regulatory obstacles? (ex. showstopper- obstacle in development due to current regulatory climate hindering sale of product; ex. unable to make final dosage form - patents required) any social obstables (ex. ethics? ex. india → diabetes→ ppl are hindu, dont tale e-coli meds from bovine broth, company needs to find alternatives; ex. mRna based tech→ vaccines→ #of ppl around world don’t believe in vaccines (covid 19) any show stoppers cost effective drug pharmacodynamics Pre-Clinical Studies (animal studies)/ what are pre-clinical studies used for usually rodent based, why? less expensive/ less complicated respond similar to humans (mammals, warm blood, similar organs and sizing ratio) drug pharmacokinetics how drug moves through body over time drug pharmacodynamics effect of drug on body - tissues/receptors bioavailability → min amount of drug needed in blood stream to produce desired effects of drug in human body metabolic studies → study metabolites of the breakdown of your API - measure concentration of metabolites to detect level of drug in body to figure out initial starting dose in body→evaluating potency, purity PBB Midterm Review 7  toxicity and safety acute (immediate symptoms of discomfort/uneasiness) and chronic (repeated use/exposure of products show symptoms) toxicity reproductive toxicity (evaluate product will not lead to sterile or infertility) and teratogen (testing to see how well the fetus will develop) mutagenicity →how drug can change genetic code of DNA (silent mutations, key protein changes can affect future generations as well) carcinogenicity → don’t want product to cause cancer all tests need to be pharmacologically relevant(animal model needs to metabolize drug product similar to how a human would. exception → if you have don’t have model that is pharmacologically relevant → ex. psychiatric disorders→ schizophrenia → cannot replicate behaviour in animal models. Clinical Studies (phase 1,2,3) - human studies/ what happens in each of the phases of the clinical studies follow established gcp guidelines, developed by ICH(protocols that need to be adhered to are authorized by research ethics board, outlined by ICH guidelines) ex. infringing rights of participants by collection additional samples beyond scope of clinical test most expensive part of drug develeopment process, each phase needs to be successfully completed before next can start phase 1: safety studies (establish if product is safe in human system) - human toxicity (identify limit of tolerance) - typically healthy subjects (cannot expose product to a “sick” body) less than 100 subjects phase 2: dosing studies - optimal dosing regimen, drug-drug interactions, food-drug interactions (some drugs cannot be taken w PBB Midterm Review 8  dairy - not optimal for absorption), contraindications, focused on treatment pop, rescue treatments less than 1000 subjects phase 3 (final phase - drug should be made with exact manufacturing process): efficacy studies- focused on treatment pop, multi centered trials, random and double blind (vials are coded, no one knows who gets drug or placebo), comparators included, rescue treatments greater than 1000 subjects Submission/Licences applications to authorities (CTA, labelling, NDS) submissions are reiterative process, lots of back and forth once NDS approved→ notice of compliance and DIN received→ need DEL to conduct business After Licensing sale of product - establishment licence (DEL) needed to start manufacturing drugs and sell them phase 4/ post market surveillance secondary indications, off-label use (ex. ozempic → clinically proven to treat diabetes, also known to treat weight loss→clinicians prescribe ozempic off label to obese ppl→not known for the conditions its been given for), long term side effects Lecture 3 what are the advantages and disadvantages of using … for biopharmaceutical production: bacteria, animal cells, yeast, plants bacteria (commonly used - e-coli) → not using solid growth media for the purpose of using bacteria for drugs advantages disadvantages model system for years inability to do post - (understanding of transcriptional modifications PBB Midterm Review 9  advantages disadvantages prokaryotic growth, →prokaryotes lack machinery metablism, food industry to recognize introns from (fermentation tech well extrons and cannot splice established→yogurt)) introns→ need instructions through processed mRna, and revers transcription lipopolysaccharides (LPS) on the surface (+ = thick lps layer on bacterium cell ; - = thin lps layer) → if protein does not have transport system, will be trapped, bacteria cell needs to split open, release components well characterized of lps layer ; bacterial infection→ immune response activated after lps layer components exposed to bacteria *need to ensure components of lps layer are eliminated in final dose* high levels of heterologous( give bacterium instructions on how to transcribe and intracellular accumulation of build portein → allows us o protein extract protein for drugs) proteins can be achieved endotoxins ( toxins produced inside and remain inside → only released at cell death) and grow rapidly in exotoxins (toxins produced simple/inexpensive media inside bacteria but exported out (ex. e-coli → 20 mins in of cell through bacterial pump) ideal conditions) *make sure to neutralize any toxins (eliminate or activate) in purification process* eukaryotic - animal cells → most common cell systems are CHO (Chinese hamster ovary) cell line BHK 1 (baby hamster kidney) cell line → cultivated PBB Midterm Review 10  in bioreactor that grow and multiply to extract proteins advantages disadvantages correct post-transcriptional modifications (have proper nutritional requirements are machinery to remove introns complex and stick extrons together cell lines well characterized like HELA and MRC 5 (both addition of animal products human cell lines) fermentation tech studied → grows slowly→ bacterial growth media→ bovine contaminants may use the serum→ protein fraction of ingredients faster and starve bovine blood the eukaryotic cells susceptible to contamination and physical damage bc of the thin phospholipid bilayer yeast (usually sacchromyces cerevisiae) → used for baking and brewing advantages disadvantages inclusion bodies → when yeasts produces protein not part of its regular genome, they package protein in inclusion body within its cell - compartment that they stuff molecular biology well unwanted protein and hide it studied from everyone else - complication of hiding protein → when to get protein? → slice open yeast cell, but also solubilize inclusion body to get to the protein safe organisms expression is low → approx 5% grows quickly in inexpensive downstream purification is media challenging PBB Midterm Review 11  advantages disadvantages large scale fermentation tech is developed due to baking and growing in food industry can do most post - transcriptional modifications → doesnt need MRNA to convert→ can use genomic dna to get yeast to produce protein plants (more similar to human protein production)→ cell lines can be used to express recombinant proteins → in comparison to bacteria, insects and yeast, plant cell lines show greater similarity in post transcriptional modifications in human proteins advantages disadvantages lower production costs → contamination → weeds, fungal effort/ resources required contamination, insects/animals for plant to take root are that can eat the plants or relatively cost effective ex. contaminate them canola crops; tomato plants immunogenic response in from agriculture biology humans → sensitivity to fruits, POV→ many crop species nuts, oils → make sure plants are well understood can eliminate immunogenic proteins risks to pollinating species → versatile production for potentially lose out on many types of products engineered plant characteristics two products in one → more than one product produced long growing time in one plant allow for long term storage at good temperature PBB Midterm Review 12  know what good practices govern the the manufacturing of pharmaceuticals. cell culture facilities → controlled environment good practices = GLP, GMP, cGMP, GCP, GDP ICH (international conference on harmonization) Europe, USA, Japan quality, RA, pre-clinical, clinical safety result= harmonized way of evaluation quality of pharmaceuticals → global set of rules that makes it easier for drugs to enter the market globally Lecture 4 what is the overall process flow of upstream process for biopharmaceutical production/ what happens at each step cell bank → small scale propagation → large scale propagation → induction of product cell bank → production of recombinant cell → propagation of homogenous cell line (every cell line in the line carries the modification) → production of master cell bank (100-200 vials) → propagation of 1 vial of master cell bank → creation of working cell bank w 300-500 vials (2 tier cell bank used when you have a big manufacturing site/batch) production of recombinant cell→ insertion of gene of interest that codes for desired protein of interest (must be mRNA (copy of DNA (cDNA), do not use genomic data (bacteria cannot distinguish introns from extrons) should have an inducible element for control ) small scale propagation → 1 vial form wcb or 1 vial from mcb taken to increase cell volume and number of cells (each vial is 1.5 to 2ml in volume → slowly expand vial to 2L) grown in artifical media ( animal based → bovine milk/ serum) and in define controlled practices raw material → bovine serum(part of bovine blood) routinely used → no evidence that the serum can transmit disease (needs to be BSE free PBB Midterm Review 13  (proteins that cause disease)) → can only use serum from New Zealand, Australia, US (NZ and AUS have flawless record of being BSE free whereas US has had incidents of meat supply having mad cow disease) bench top has access ports for sensors, nutrient flow, waste outflow and sample collection fermentor, chemostat, bioreactor are enclosed systems that cultivate cells of interest large scale propagation and protein induction→anywhere from 100L to 1000L → one step process common bioreactors/ culturing methods avail for cell growth batch → add growth media w cells to encourage healthy growth and ferment for entire duration of time → don’t add or remove anything continuous stirred tank →motor that runs and stirs and new media is added to bioreactor→ semi-permeable membrane allows media to pass in but cells cant pass out, keeping the cells safe→ most common airlift→ movement of cells bathed in fresh media → air movement currents→ uses air to enhance mixing and circulation of culture media→ improves mass transfer ( essential for cell growth) bubble column → uses gas bubbles to promote mixing and mass transfer within a liquid medium→ useful for cultivating microorganisms or cells in fermentation processes fluidized bed → employs fluidization to enhance mass transfer and improve the growth of microorganisms or cells→ solid particles suspended in fluid or gas flow packed bed→ packed bed of solid materials, such as support particles or immobilized cells, through which a liquid medium flows photo→ algae type host system → emit wavelengths of UV light to encourage photosynthesis PBB Midterm Review 14  protein induction→ cultivate parameters → temp, nutrient flow, agitation, aeration, foaming, pH how is gene inserted into a vector construct/ how are cells genetically modified pUC19 plasmid vector → gene of interest is inserted into the multiple cloning site (MCS). The MCS has multiple restriction enzymes that are used as tools to open plasmids and insert gene of interests. These restriction enzymes have specific recognition sequences that allow foreign DNA to enter the cell. They break down the sequence of both the MCS and gene of interest and use DNA ligase to join the sticky ends of both the multiple cloning site and gene of interest together to insert it into the plasmid The lacZ gene produces β-galactosidase which breaks down lactose for the use of energy. Since the rhGH gene has been inserted with the help of the restriction enzymes on the MCS, it has disrupted the lac Z gene, causing it no longer produce β-galactosidase. To start transcription of the gene of interest, the Plac promotor needs to bind to the rhGH gene. However, because the LacR binding site is in between the MCS with gene of interest and the Plac promotor, it is unable to bind. Cells get energy through glucose first and then through lactose, so if the host cell is receiving more glucose than lactose, the transcription of the gene of interest cannot happen. To trick the system, IPTG is introduced into the plasmid, to mimic the effect of having too much lactose in the cell system. Since the lac Z gene, that would normally be used to break down the IPTG is unavailable, the LacR binding site pulls off from the pUC19 plasmid and binds to the IPTG to break down the lactose for energy. Now that the LacR binding site is no longer in between the gene of interest and the Plac promotor, the promotor can bind to the GOI and start transcription Amp Gene →The Amp gene in the pUC19 plasmid is an antibiotic resistance gene. This gene allows the host bacteria that carries the plasmid to survive when introduced to an antibiotic. Since the bacteria E- coli carries the plasmid with the gene of interest, the Amp gene allows the E-coli to survive in the presence of antibiotic PBB Midterm Review 15  what is an inducer? its purpose? examples? induction → process where signal is sent to cells/culture to start producing protein of interest (antibiotics, temp, nutrient addition/omission, growth phase) purpose → activate transcription for genes, control metabolic pathways, used in fermentation processes clapping signal that is used to start transcription ex. IPTG induced production of goi @ each cycle of growth, diff genes get turned on/off→ if goi gets turned on during late stage, use promoter and put in front of goi and use growth phase as protein inducer ex: lac operon of e-coli acts as an inducer by binding to the repressor protein, allowing the transcription of genes necessary for lactose metabolism; iptg synthetic analog of lactose, IPTG is commonly used in molecular biology to induce the expression of genes under the control of the lac promoter. purpose of MCB and WCB and storage requirements master cell bank → usually 100 - 200 vials, homogenous, gold standard (for producing batch of product → form same culture → from same colony in petri dish free from extraneous agents(bacterial/viral contaminants), sterile, genetically stable (make sure genetic code is intact and not mutated monitor vials for genetic stability as representation of the entire cell banks genetic stability) Working cell bank → usually 300-500 vials (1 vial = 1 lot), homogenous, free from extraneous agents, sterile, genetically stable stored in large freezers flash frozen with liquid nitrogen (-80 degrees centigrade for storage, flash frozen at -70 degrees centigrade); back up power sources and stored in at leaste two seperate locations water in cytoplasmic host cell’s explain when they start to freeze so flash freezing prevents damage on living tissue PBB Midterm Review 16  Genetic stability (stability testing of cell line is continually monitored through stability program and must be documented in NDS) gene sequence → confirm genetic sequence of goi to see if its correct protein sequence → correct protein structure protein activity → develop assay to confirm if protein is activated peptide chain modifications → test or changes in polypeptide chain cytogenetics and chromosome structure → confirm identity of host cells karyotyping → human cell line→ 23 pairs of chromosome (if you identify cell w 8 chromosomes, you’re not working w human cell line) signature sequences → molecular fingerprints at DNA level → unique to cell species to confirm Lecture 5 what is downstream process flow/ what happens at each step; what techniques/methods are used at each step (how is api extracted from host cell, how is it concentrated(filtration, centrifugation, purification) and purified ) harvest raw material → api extraction→ removal of cell debris→ filtration→ centrifugation→ low level chromatography→ high level chromatography→ sterile filtration→ final product goal = final product needs to be >95% pure; all impurities identified; process is predictable and consistent; sterile; defined dosage harvest raw material (crude harvest→ two types → extracellular & intracellular extracellular ( protein expressed outside of cell and pumped to internal environment ) recovery of cells→ concentration of broth intracellular ( protein expressed in cell (common) ) recovery of cells → cellular disruption → removal of cell debris → concentration of broth PBB Midterm Review 17  cell disruption (api extraction)(physical>chemical → don’t have to remove chemical afterward and can prevent chemical affection protein composition detergent (chemical) enzymatic action (chemical) sonication (physical) → sound waves → range of frequency→ resonance weakens protein → bacterial cell wall collapses allowing poi to release homogenization (physical) → counter oscillating blades → disrupts protein-protein interaction but not intramolecules and thus poi still intact nitrogen explosion (physical) → high pressure condition→ similar to hyperbaric chamber→ gas expansion = bubbles → cytoplasm pop holes → poi release osmotic pressure (physical ) → dunking cell in hypotonic solution (no salt) protease inhibitors need to be added - why? → When cells are disrupted, their internal proteolytic enzymes (proteases) can be released. These enzymes may begin to degrade proteins of interest, which can compromise experimental results or yield. → to prevent degradation of poi removal of cell debris and concentration(filtration & centrifugation) filtration separates by particles → size, viscosity (high = longer to go through pore size), shape (some particles can’t fit through shape of pore size), compressibility dead end and cross flow filtration dead end → static filter→ medium has pores that are smaller than the particles to be filtered→decrease of filter w time, filter cake will clog the filter→ eventually need to stop to clean → can lead to contamination, slow process= takes more time PBB Midterm Review 18  cross flow filtration → dynamic → feed is pressurized and fed tangentially to the filter → velocity up to 6m/s→ no dead end, shearing action reduces thickness of filter cake and fluid keeps passing, increasing filtration rate → allow small particle to pass through → optimal for poi collection (disadvantage → hihgh energy consumption, more expensive but worth it if product is in high demand centrifugation separates by particle density ( high density = more it will separate at bottom) and centrifugal force ( high force = heavier protein will be pulled) and time and duration (amount fo time for centrifugation → alllows for sediment particle pusher centrifuge → combination of centrifugal force and a mechanical pusher to separate solids from liquids→ allows for a continuous feed of material, making it suitable for large-scale operations→ Solids are expelled from the centrifuge in a continuous manner, which minimizes downtime. (Best for low solid content, continuous feeding, and pushing solids along the drum) decanter centrifuge → separates solids from liquids by using high centrifugal force generated by the rotation of a cylindrical bowl →Solids are deposited against the drum wall and form a sediment layer, while the lighter liquid phase is forced out through a separate outlet→Effective for high solid content slurries, continuous operation, and versatile in handling different materials. purification (low level chromatography → separate large property of protein from poi → gradually migrate to high level chromatography) typically column chromatography→ usually 2-4 chromatographic steps ( each column works w a diff physical property of poi to separate it out → goal = to provide high resolution of product and to remove any contaminating agents * you lose product during purification * PBB Midterm Review 19  types of chromatographic columns → disposable are avail and can be custom made ion exchange → based on pH (cation or anion exchange soulubility in diff pH conditions gel filtration → based on size ( higher size, heavier= higher polypeptide chain, more complex ; smaller protein will travel slower bc trapped) hydrophobic → based on hydrophobic patches on proteins (bind to proteins hydrophobic in nature) affinity → binding to specific antibody/complex (fishes out poi) metal chelate → binding to certain metals(zinc/copper) hydroxyapatite→ binding to calcium/phosphate ions how is column chosen? what is product like → use columns that can bind to protein based on physical chemistry ( physical>chemical) what are the contaminants → how many contaminant agents you want to eliminate; make sure another column or purification technique is used to eliminate this is it safe for humans to use → won’t react with poi or affect human consumption interchangeable resin →regenerate resin → take existing, neutralize and regenerate for new product (avoid using used resin → general practice = new product to prevent contamination dependent on properties of API and can be chilled (try and maintain cool temp → prevent protein degradation) feed forced → gravity, vacuum, positive pressure can be disassembled, easily modified and easy to re-validate RA issues with columns → column material may leach into product, may co -purify a contaminant ( may need to add additional purification step to eliminate contaminant), provide high purity but low yield bc PBB Midterm Review 20  product is lost, analytical test development (phase 1 and 2 of clinical trial → test may not be validated, may have wide pass/fail criteria, need to have optimal manufacturing process. by phase 3 → all tests must be validated and pass/fail criteria needs to be established) sterile filtration / what types of sterilization are available? which are used in biopharmaceuticals? why? filter, radiation, chemical thermal (cannot use for biopharmaceuticals specifically in biologics becasue biologic product is a protein which has a structure that is sensitive to environmental changes like extreme heat) for biopharmaceuticals →use 0.2um filter → removal of microorganisms from heat sensitive solutions, not heat sterilized, sterile filtration, contaminations, virus can come out of this, monitored flow rate ( final product → batch profile (confirm what you have produced is what you intended)→ aseptic filling, automated, limited human interactions (variables → injection rate, dispensing volume, molding change) → must show at least 3 equivalent lots for phase 3 clinical trial, equivalent in terms of process, yield, testing protein sequence → can identify protein of interest secondary modifications i.e glycosylation → has proteins been modified by host cell beyond transcription chromatograms → determine identity and purity of structure yield consistency → measurement of yield excipients → amount measured table showing all results certificate of analysis validation→ cleaning, manufacturing, testing notifications of changes before and after establishment license granted and examples PBB Midterm Review 21  process changed before establishment license → usually in phase 1 and 2 of production (phase 3 material must be made with process that will be licensed) process changed after establishment license minor changes →ex. diff filter, new supplier of media → Regulatory authorities must be notified and approve change → to avoid: validate many diff supplier for process major changes→ ex. diff manufacturing method, new media, new purification method→ Regulatory authorities must be notified immediately, bridging clinical trials may be necessary Lecture 6 what is a QOS and how to complete QOS/elements covered Quality Overall Summary (QOS) → summary of quality data → drug substance and drug product drug substance → general info ( nomenclature, structure, physiochemical properties) → manufacture (process, critical steps, controls, batch production details) →characterization ( secondary structures, biological activity, impurities) → control of drug substance (pass/fail specifications, validated methods) → reference standards or materials (established substance standards) → container closure system (construction, material composition, suppliers) → stability( existing stability data and stability monitoring protocols Drug product → description and composition of drug (dosage form, strength, diluents, container type) → pharmaceutical development (reference to R&D and proof of efficacy, compatibility of components, sterility, process development)→ manufacture (manufacturer info, batch formulation, manufacturing process and controls, critical steps, process validation)→ control of excipients (identification, analytical tests, method validation, source) → control of drug product ( pass/fail specifications, validated methods, batch analysis, and impurity profile)→ reference standards or materials (established substance standards) → container PBB Midterm Review 22  closure system (construction, material composition, suppliers) → stability( existing stability data and stability monitoring protocols QOS Appendices → facilities and equipment (flow of material, equipment specifications, process and cleaning protocols) → adventitious agents safety evaluation (contaminant assessment, containment protocols, viral clearance testing)→ excipients (identification and suitability) what types of QOS templates are avail for biopharmaceuticals biotech/biologics conventional biotherapeutics blood products vaccines PFSI covered in Quality module Product specific facilities informations (PSFI) → floor plans, equipment, control Lecture 7 how are biologics regulated in Canada biologic → drug prepared using biological material through conventional manufacturing methods or recombinant dna → ex. bacterial/ viral vaccines, blood and its derivatives, hormones and enzymes, gene therapies BRDD → regulations→ policy docs → guidelines → templates→ global collaborative initiatives evaluate and monitor biologics for their safety, efficacy, quality and public awareness and education regulations → food and drug act ( what the law is)→ food and drug regualtions (how to reinforce that law and how that law will be met) responsible for review and approval of all types of drug submissions for: Schedule C → radiopharmaceutical in nature PBB Midterm Review 23  Schedule D → Biologics (drugs used for purpose of improving human life) food and drug regulations includes NDS, CTA and regulatory requirements: division1: old drugs division 1a: DEL division 2: GMP division 3: regulations to support radio-pharmaceuticals division 4: regulations to support biologics division 5: human critical trials division 8: new drug submission how are subsequent entry biologics regulated why BRDD offices are organized the way thy are BRDD → biologic and radiopharmaceutical drugs directorate oversees regulation of biologics, radio-pharmaceuticals, blood & blood products, viral and bacterial vaccines, gene therapeutic products, transplant tissues/organs organized into 3 centres → reviews safety and efficacy of products, test and analyze product samples, conducts inspections and OSEs, and monitors lot release programs centre for biologics evaluation → regulates and reviews vaccines, allergenic extracts, blood proteins, cell and cell based medicines, gene therapies, tissues and organ transplantation centre for evaluation of radio pharmaceuticals and biotherapeutics → regulates and reviews radiopharmaceutical, monoclonal antibodies, cytokines, hormones, enzymes, biotech derived products centre for regulatory excellence statistics and trials→ supports submission management, sponsor interactions, collaborates with international regulatory partners, oversees clinical trials, BRDD performance data, risk management PBB Midterm Review 24  organized into 3 supporting offices office of policy and international collaboration→ develops policies, guidances, directives, standards, in partnership with international partners and sponsors office of quality and information management→supports BRDD QMS, generally certified ISO 9001, specific lab work certified ISO 17025 (lot release testing), medical devices under ISO 13485 office of business integration → supports meeting with stakeholders and sponsors, general operation support to BRDD unique sections of biologics NDS module 1 → regional info (specific to region and not “common” in common technical doc (CTD) GMP and establishment license→ ensuring products are fabricated to meet standard that give assurance and confidence→ biologics → raw material, stability and sterile products raw material tested against specifications → BSE free pharmacopoeial standards characterize mcb and wcb → cell line identification validated test methods supplier certificate of analysis stability expiration dates → shelf life storage conditions performed on cell banks and lyophilized and reconstituted product start date of biologic PBB Midterm Review 25  moment poi expressed in large scale propagation→ degrading the moment its expressed, thus, start date begins @ that moment when poi is combined w excipients→ start date begins then sterile products manufactured in separate and enclosed areas, supervised by microbiologist use validated sterility procedures use hermetically ( not exchange of product) sealed containers, uses of robots, form - fill seal process changes environmental grade levels ( 5 grade level rooms E→ A ; become more sterile from E → A labeling requirements for → radiopharmaceutical and biologics, components, kits, info requirements on package → applies to inner and outer labels, reference product monograph CPID-B (certified product information document-biologic)→ outline how drug is being fabricated/formulated → supplement to QOS (analogy → movie poster: idea of if you’ll like movie, CPID (trailer) → more detail, QOS (movie) → module 3,4,5 (details get repeated for easy review) module 2 → A. Overall TOC, B. Introduction, C. QOS, D. Nonclinical Overview, E. Clinical Overview, F. Nonclinical Summary, G. Clinical Summary QOS summary(findings, outlines outcome of data/results vs overview (how trials are conducted) QOS = 4 parts part 1: G. General info part 2: S. Drug Substance part 3: P. drug product part 4. A. Appendices PBB Midterm Review 26  module 3 → quality batch analysis (P.5.4) → evidence showing ability to produce consisted product facilities and equipment (A.1)→describe manufacturing facility, equipment, work flow master production docs (R.1.2) (PERs (positron emitting radio- pharmaceuticals) only)→ formulation and method of manufacture (radioactive component integrated in negative space → clean room environment (pressure cascade → positive pressure→ gush of air against of you to prevent mix of components) Lot release doc (R.3)/how does lot release work → unique for biologics bc of the risk they carry → proposed format of a protocol for lot release based on risk of product characteristic ex. live ebola vaccine (viral pathogen so higher risk biologic compared to humatrope. Assigned in 4 groups Group 1 - Pre-Approval Stage → products that are still under testing and research → NOC not issued yet but lot release letter issued Group 1A - clinical trial material → products still under research in clinical trials Group 1B - consistency testing → products still under research for consistency in manufacturing process Group 2 - Sample Testing and protocol review → high level of assessment → BRDD tests each lot and reviews sponsor protocols/results; letter of lot release issued by BRDD to sponsor Group 3 - Protocol review and Periodic testing → moderate level of assessment→ BRDD reviews sponsor protocols/ results, letter of lot issue, periodic testing at discretion of BRDD (no quarantining of lots required but periodic testing occurs. Ex. batch 1,2,3,4 get released but batch 5 is quarantined for release → interval of testing increases as batch’s pass the testing process PBB Midterm Review 27  Group 4 - notification and periodic testing → low level of assessment → notify HC when you release product but don’t send any batch’s to BRDD for testing → all lot test results may be audited at future date group assignment (at beg. start at 2 or 3 and with passed tests move to group 4) determined by: product indication → target pop, disease state nature of product→ complexity of analysis and API production history→ no reworked lots inspection history→ compliant w GMPs testing history→analytical testing for purity, potency post-market experience→recalls, ADRs Module 4→ non clinical study reports Module 5→ clinical study reports canada’s view on biosimilars similar to innovators product but not identical → enters market after innovator product similar vs identical need to develop own manufacturing process ex. make own cell line diff to match exact chemical structure similar action as novel drug → safety quality, efficacy proof of equivalence or better in terms of safety, quality and efficacy treated as NDS and NOT ANDS (not generic drug) bc the products are a biologic sponsor must include comparability studies with innovator must address choice of innovator biologic and rational for not including an innovator biologic as the comparator PBB Midterm Review 28  proof of equivalence for chemical, bio activity, bioavailability, safety, toxicology, pharmakinetic, pharmadynamic studies, effectiveness Lecture 8 what divisions of the regulations pertain to biologics - what is covered under schedule c and d no person can sell any drug describe in c and d unless premises in which drug were manufactured and the conditions and process were safe to use schedule/part c → drugs (not radionuclides) sold or represented for use in preparation of radiopharmaceutical schedule d → biologics allergenic substances used for treatment or diagnosis of allergic or immune disease→ peanuts/gluten through allergen patch test (purified samples of potential allergens) pituitary extracts → hormones released by pituitary glands → recombinant dna tech ex. humatrope aprotinin→ protein used to prevent blood from clotting → similar to heparin blood and blood derivatives → serum albumin fraction that have antibodies cholecystokinin→ enzymes assisting digestion → especially in infants drugs obtained by recomb dna procedures → human insulin drugs other than antibiotics prepared from micro-organisms → toxoid prep → bacterial toxins being inactivated and training immune system how the toxins look like without getting sick glucagon → control blood sugar levels gonadotropins → treatment for fertility, pregnancy, ivf treatments human plasma through plasmapheresis → methods of collecting plasma immunizing agents PBB Midterm Review 29  insulin interferon monoclonal antibodies, their conjugates and derivates secretin snake venom urokinase what do divisions 3,4,8 describe Part C, Division 3 → regulation on radiopharmaceutical defining terms ex drug license, master lot prohibition of sale authorities can collect sample for verifying quality, efficacy and safety (raw material in process or fdf packaging component samples) animal components obtained from healthy animals → infection free no labeling to indicate that it is a prescription “Pr” ex. short hald life → if radiologists order drug and miss appt, need to dispose radiopharmaceutical and order new one covers info on: define radiopharmaceutical, “component”, “kit” labeling requirements→ safety measures for use, radiation levels, contraindications, radioactive symbol, handling instructions, disposing instructions, “components” and “kits” technetium-99m sold with impurity sodium pertechnetate within safe limits for imaging purposes info requirements on the package insert Part C, Division 4 → regulation on biologics definitions → date of manufacturer etc prohibition of sale → must have NDS, DIN #, DEL PBB Midterm Review 30  exclusion of probiotic drugs date of issue→ date poi expressed, date API mixed w excipients, date when product gets put on cold truck compartmentalization/segregation of manufacturing facilities QA/QC physically separate from manufacturing covers info on: authority collect samples for verifying quality, efficacy and safety animal quarters supervised and animal components obtained from healthy animals infection free → necropsy records of all animals → segregate / report infectious animals labeling requirements → indicating it is a prescription “Pr” specific regulations for → bacterial vaccines, products analogous to bacterial vaccines, viral vaccines, bacteriophages, insulin products, anterior pituitary extracts Part c, Division 8→ regulations for new drugs define new drug, canadian reference product requirement for submission to sell and advertise need for approved compliance with regulations requirement for clinical testing covers info on: need for demonstrated quality, safety, efficacy requirement for maintaining records for future use → demonstrating accurate record keeping requirements for impartiality in cases of dissatisfaction sale of medicated feeds sale for animal experimental studies labeling PBB Midterm Review 31  conditions of experimental study suspension or cancellation of experimental studies certificate biopharmaceutical to market → clinical (pre-cta meetings and cta approval) and quality (chemistry and manufacturing, quality information, QOS) → when phase 3 is successful, apply for NDS Include → modules 1 - 5 → all info confirmed w on-site eval (OSE) Lecture 9 what is QA and QC Quality Assurance (QA) → planned, systematic activities implemented in quality system so quality requirements for product or service will be fulfilled - ASQ (American society for quality) → totality of features and characteristics of product or service that bear on its ability to satisfy given needs Quality Control (QC) → observation techniques and activities used to fulfill requirements for quality - ASQ definition→ part of quality management focused on fulfilling quality requirements What is quality standard → set of defined criteria that characteristics of quality can be measured against → GMP, ISO, ICH what do GMPS regulate regulations for drug manufacture, testing, packaging, storage, handling, tracking and distribution why have GMPs safety, efficacy, potency, identity, truthful to its claims what GMPs criteria are inspected during audit and why premises→ well designed, cleanable surface, air filtration present, compartmentalization, animal facilities equipment → well designed, cleanable surface, manipulate function as needed, maintenance, records, design qualification PBB Midterm Review 32  personnel→ trained in hygiene and microbiology, responsible, accountable, performance reviews, records sanitation→ established sanitized procedures, procedures on decontamination, undesirable product, pest control, microbial monitoring and endotoxins, cleaning validation, personnel health/ hygiene raw material testing → tested against specifications, BSE free, use of pharmacopeial standards, mcb and wcb characterized, validated test methods, cell line identification manufacturing control→ show that you have a controlled method to manufacture the product, material handling/storage, critical procedures validated, reconciliation of whats left over, accuracy and precision checks, tamper free raw materials quality control department→ taking raw material samples and checking them against specification standards, documentation, material release, incident reports and corrective and preventative actions packaging material testing→ type of material product packaged in (suppled by external supplier → provides proof of safety of packaging material), material leaching, labels verified, non reactive composition finished product testing→ tested against specifications, validated test methods, use or pharmacopeial standards, sterile and hermetically sealed (refrigerated in frozen conditions to ensure product remains active before taken to the market) records→ record everything, master production docs, master batch records, regulatory compliance and inspections, raw material lots, finished product lots samples → finished product sample from each lot , raw material samples from each lot why → investigation → can trace back which lot was used and confirm and prove raw material is safe and meeting specifications how much of sample→ enough for you, HC and another third party to verify test results PBB Midterm Review 33  stability→ support expiration date and shelf life for product, establish storage conditions, performed on cell banks ( genetic stability testing) sterile products→ manufactured in separate and enclosed area→supervised by microbiologist that uses validated sterility procedures, use of robots, form-fill seal process, environmental grade levels medical gases → all GMP regulations apply with exception of: sample retention and stability (compressed gas→ hard to get samples → material bleeds and cannot hold onto samples over time. ex. sprays → overtime, compressed gas no longer that as its eventually leaked out) Lecture 10 how eukaryotic and prokaryotic biopharmaceutical production are diff and how they are similar for prokaryotes → upstream and downstream process for eukaryotes→ major diff = growth of diff organisms and subsequent downstream treatment production of vector containing gene → bacterial plasmid not used, instead viruses used insertion of vector dna into cell→ goi incorporated directly into gene of host cell → part of host cell genome, through viruses→ tool to move genetic material in host cell → no need to make reverse transcription happen production of mcb and wcb → same as prokaryote (want cell line that has capability of producing goi, diff in testing→ ensure host cell can acc carry goi, goi has been intubated into a particular site of chromosomal structure that doesn’t disrupt host growth type of testing to confirm no contamination → screen out bacterial/viral/fungal contaminants (only want eukaryotic cell)→ ICH guideline → vial safety eval (outlines # of tests that can be done on cell banks to ensure no viral contaminants are introduced PBB Midterm Review 34  viral testing → cell lines tested to make sure only virus that has successfully integrated into host genome gets protection and ensures no other viral contaminants get introduced lysogenic life cycle → integrated within host genome and remains dormant within host genome→ every time host cell replicates, virus also replicates, never leaves host → host remains alive lytic life cycle → virus replicates machinery of host cell, causes cell death/lysis→ no longer capable of producing poi bc host cell destroyed ideal to remain in lysogenic life cycle so cells are able to grow/multiply and able to produce poi - ex. shingles (same virus) → revers of lysogenic to lytic cycle → lysogenic life cycle → replicates along w cells in body, remains dormant until body reaches early/ late 40s → changes to lytic cycle → expression of shingles propagation→ bioreactors slightly diff but process same → animal products used in media can be source of contamination so additives added into growth media to prevent growth contaminants induction of product → inducers specific to eukaryotic systems harvesting → less harsh as no bacterial cell wall to destroy purification → column chromatography still preferred→ no lps layer (if lps layer→ pyrogen trigger fever→ likely contaminant introduced in manufacture process)→ sterile filtered and filled (still use 0.22nm filter) common source of contamination biological contaminants → bacteria, viral and fungal (taken in process controls , sample throughout process to ensure no contaminants are present) other contaminants → chemical (elements of cleaning solution → cleaning equipment→ ensure safe chemicals are able to degrade safely; chemical components of growth media that might be co-purified) and physical ( PBB Midterm Review 35  microscopic pieces of metal thorough wear and tear of equipment, dust, sand and gravel) common bacterial and viral contaminants bacterial → staphyloccocus aureus (associated w skin infection, can result in skin infection if product infected w this virus), e-coli, pseudomonas aeruginosa, salmonella sp sources → raw material, personnel, equipment, packaging, environment for biopharmaceutical → ensure contaminant free cell line viral → hepaptitis, HIV 1 and 2, HTLV, CMV sources → personnel, airborne sometimes viral validation → proactive approach bc cannot remove virus if contaminated→ need to throw away lot → tests done by through ICH Q5A viral testing dependant on → origin of tissue cell line, viral mediated transformation, method of manufacture PBB Midterm Review 36