Pharmaceutical formulation and processing Flashcards

Question

You are a virtual QP, and you received a call from your multiproduct CMO that they found a yellow powder during granulation of your product which is white powder. What do you do? What are your concerns? I was shown a picture of a granulator with the yellow contaminant. Then I was shown another picture showing the root cause of this issue – which was a valve connector that accumulated product residues previous batch.

Answer 1

The talk led to nitrosamine contamination and recall.

Answer 2

The key steps are: 1. Blending of the API with excipients (e.g. diluents, disintegrants). 2. Addition of a binder solution (e.g. purified water with povidone or starch paste) to form a wet mass. 3. Wet massing or kneading to ensure uniform distribution of moisture. 4. Granulation through a sieve or screen to form granules of desired size. 5. Drying using a fluid bed dryer or tray dryer to remove excess moisture. 6 .Sizing or milling to break up oversized granules and achieve a consistent particle size distribution. 7. Final blending with external phase (e.g. lubricants like magnesium stearate, glidants). 8. Compression into tablets using a tablet press. 9. Optional: Film coating or enteric coating, if needed.

Answer 3

For tablets, CQAs typically include: Uniformity Tablet weight variation Disintegration time Dissolution profile Hardness Friability Content uniformity Appearance Moisture content CPPs are process parameters that, when varied beyond a certain limit, may impact one or more CQAs. CPPs during wet granulation include: Blending time and speed (affect content uniformity) Binder addition rate and volume Granulation endpoint (e.g. torque, wet mass consistency) Drying temperature and time (affects moisture content, stability) Milling screen size and speed (affects granule size distribution) Compression force and dwell time (affects hardness, disintegration) Tablet press speed (impacts weight uniformity and friability)

Answer 4

To manufacture an ATMP in the UK, the site must hold a: • Manufacturer’s Authorisation (MIA or MIA(IMP)), depending on whether the product is for commercial or clinical trial use. • HTA License under The Human Tissue (Quality and Safety for Human Application) Regulations 2007 (SI 2007/1523), if handling human tissues/cells such as leukapheresis material. • The site must also be GMP-compliant and inspected by the MHRA, with an up-to-date GMP certificate. Additionally, clinical sites (hospitals administering the product) must be HTA-approved to receive, store, and handle human-derived materials.

Answer 5

Model Answer: Key GMP controls include: • Grade A isolators operated under positive pressure with a Grade D background. • Use of validated closed systems (e.g., CliniMACS Prodigy) to minimise contamination risks. • Validated disinfection and decontamination cycles (e.g., vaporised hydrogen peroxide for isolator gassing). • Single-use systems to avoid cross-contamination between patients (as these are autologous products). • In-process critical process parameters (CPPs) such as controlled time from leukapheresis to processing (e.g., max 96 hours), and controlled-rate freezing (e.g., 1°C/min) for cryopreservation.

Answer 6

Model Answer: Under the 2007 HTA Regulations (SI 2007/1523), donor material must be screened for: • HIV 1/2 • Hepatitis B and C • Syphilis • HTLV I/II • Other risk-based markers depending on donor risk assessment. Testing must occur within 30 days before leukapheresis. If the patient has an active infection, leukapheresis must be postponed until resolved, to avoid impacting cell viability and manufacturing outcomes.

Answer 7

Model Answer: For clinical trial ATMPs, if a batch is OOS for non-safety-related parameters (e.g., potency), a clinical justification can be made. The QP can certify that the batch was manufactured in accordance with the approved CTA and GMP, but final use is a clinical decision. The investigator must: • Conduct a risk-benefit assessment, • Obtain informed consent from the patient, • Seek regulatory approval (e.g., via the MHRA clinical trials unit), if required. However, OOS for sterility, endotoxin, mycoplasma, or identity cannot be released due to patient safety concerns

Answer 8

Model Answer: Key QC tests include: • Sterility • Endotoxin • Mycoplasma • Viability (e.g., flow cytometry) • Identity (e.g., CD3+, CD4/CD8 phenotype) • Vector copy number (must be <5 to avoid oncogenic risks) • Potency: e.g., impedance-based cytotoxicity assay to measure cancer cell killing capability. Due to the short shelf-life (e.g., 6 months), release may be done on partial results (e.g., interim sterility) with full results available post-release, based on risk assessment and control strategy.

Answer 9

Model Answer: No. CAR-T products are ATMPs, and unlike vaccines and plasma-derived products, they are not subject to official batch release testing by NIBSC. Under Directive 2001/83/EC Article 114 and UK regulations, official batch release applies to: • Immunological medicinal products (e.g., vaccines), • Blood/plasma-derived products. ATMPs are excluded from NIBSC batch release but still require QP certification before use.

Answer 10

Model Answer: A key CPP is the holding time from leukapheresis to start of manufacturing, as prolonged storage reduces cell viability. • Initial limit: 72 hours. • Updated based on data: reduced to 26 hours. • Controlled by: • Time-stamped transport logs. • Acceptance criteria at goods-in (viability testing, temperature logs). • Deviations raised and investigated if exceeded. Another example is controlled-rate freezing: 1°C/min to preserve cell integrity.

Answer 11

Model Answer: • Autologous: Cells are collected from the patient and returned to the same patient. Each batch is unique. • Risk: patient-specific variability, limited material, identity mix-ups. • Allogeneic: Cells are from a healthy donor and used to treat other patients. • Risk: immune rejection, need for HLA matching. CAR-T is usually autologous, which poses traceability and batch segregation challenges that the QP must oversee.

Answer 12

Model Answer: The QP must ensure: • Unique identifiers (e.g., barcodes, patient ID codes) are applied at collection, maintained through manufacture, testing, and release. • Chain of custody and chain of identity documentation is robust. • Cross-checking during goods-in, batch processing, and final labelling. • Segregation of material at all stages. • Staff training on patient-specific handling procedures. Failure in traceability could result in administering the wrong product — a critical patient safety risk.

Answer 13

API, suspending agent, vehicle (water or alcohol-based), flavouring agent, colouring agent, buffering agent (for pH), flocculating agent, preservative, and viscosity enhancers.

Answer 14

A: A cream is typically an emulsion made up of two phases—aqueous phase (water-based) and oil phase (lipid-based). Common ingredients include: • Aqueous phase: Purified water, humectants (e.g. glycerol, propylene glycol), buffering agents • Oil phase: White soft paraffin, liquid paraffin, cetostearyl alcohol, isopropyl myristate • Emulsifiers: To stabilise the emulsion (e.g. polysorbates, cetomacrogol, sodium lauryl sulfate) • Preservatives: To prevent microbial growth (e.g. methylparaben, propylparaben, phenoxyethanol) • Antioxidants: To prevent oxidation of oils (e.g. vitamin E [tocopherol], butylated hydroxytoluene [BHT]) • Thickening agents/viscosity enhancers: e.g. carbomers, xanthan gum • Active pharmaceutical ingredient (API): Incorporated depending on the intended therapeutic effect

Answer 15

Tablets contain various excipients that serve specific functions in the formulation. A helpful mnemonic is A–I: • A = Active Pharmaceutical Ingredient (API): The medicinal substance. • B = Binder: Helps hold ingredients together (e.g. starch paste, povidone, hydroxypropyl cellulose). • C = Coating agent: Improves appearance, taste masking, and stability (e.g. hydroxypropyl methylcellulose, PEG, Eudragit). • D = Disintegrant: Aids tablet breakup in GI tract (e.g. crospovidone, sodium starch glycolate, croscarmellose sodium). • E = — (Not typically assigned). • F = Filler (Diluent): Adds bulk to tablets (e.g. lactose, microcrystalline cellulose, dibasic calcium phosphate). • G = Glidant: Improves powder flow (e.g. colloidal silicon dioxide, talc). • H = Anti-adherent: Prevents sticking to equipment (e.g. talc, magnesium stearate). • I = Lubricant: Reduces friction during compression (e.g. magnesium stearate, stearic acid).

Answer 16

A: No, infectious disease marker testing is not part of the final QC testing for the finished ATMP or gene therapy product. These tests are performed early in the process to assess the suitability of the starting material, particularly when the starting material (e.g., peripheral blood for leukapheresis) is of human origin. In autologous ATMPs like CAR-T, these markers — HIV 1 and 2, Hepatitis B and C, HTLV-I, and Syphilis — are part of donor (or patient) eligibility screening. This testing falls under the Human Tissue Authority (HTA) in the UK, in line with the Human Tissue (Quality and Safety for Human Application) Regulations 2007, which implement EU Directive 2004/23/EC and Commission Directive 2006/17/EC. The primary purpose is to ensure biological safety, traceability, and compliance with donor suitability criteria. In contrast, final GMP QC tests for ATMPs include sterility, endotoxin, mycoplasma, vector copy number, potency, identity, and viability. These are performed under GMP and are essential for QP certification and batch release.

Answer 17

In contrast, Final QC testing for ATMPs includes: • Sterility • Endotoxin • Mycoplasma • Vector copy number • Potency • Cell identity and viability

Answer 18

Because the starting material is patient-specific and cannot be replaced with a banked cell line due to the autologous nature of the therapy.

Answer 19

1. Active pharmaceutical ingredient (API) – the drug, usually insoluble in water. 2. Suspending agent – helps keep solid particles dispersed (e.g. xanthan gum, methylcellulose). 3. Wetting agent / Surfactant – reduces surface tension to allow proper wetting of solid particles (e.g. polysorbates like Tween 80, sodium lauryl sulfate). 4. Vehicle – usually purified water or a mix of water and other solvents. 5. Buffering agents – to maintain pH. 6. Preservatives – to prevent microbial growth (e.g. parabens, benzyl alcohol). 7. Flavouring and sweetening agents – for oral suspensions, to improve palatability. 8. Colorants – optional, for appearance. Note: Oil is only included in emulsion-based suspensions or oil-based vehicles, but most suspensions are aqueous.

Answer 20

Steps: 1. Blending – uniform mix of API and excipients. 2. Binder addition – e.g., purified water (CPP: quality must meet Ph. Eur). 3. Wet Massing – forming a granulated mass. 4. Drying – controls on temperature and time (CPPs). 5. Milling/Sieving – controls on screen size (CPP). 6. Blending with lubricant – e.g., magnesium stearate to reduce sticking. 7. Compression – tablet formation (CPP: compression force, speed). 8. Coating (if required) – for stability/appearance. CQAs Identified: • Granule particle size • Powder flow • Tablet weight uniformity • Appearance (shape, color) • Density • Disintegration time CPPs Identified: • Mixing time and speed • Binder solution quality and quantity • Drying temperature/time • Milling screen size • Compression force

Answer 21

Potency is assessed using an impedance-based cytotoxicity assay where the CAR-T product is incubated with CD19⁺ target cells derived from a qualified cell bank. The system measures real-time killing of target cells by monitoring electrical impedance. This method provides a functional readout of the CAR-T cells’ ability to recognize and eliminate antigen-positive targets. The assay uses reference cancer cell lines to ensure consistency across batches. In an impedance-based assay (e.g. xCELLigence system): • Cancer target cells are seeded onto a special plate with gold microelectrodes at the bottom. • These adherent cells attach and spread, generating a baseline electrical impedance. • When CAR-T cells kill the cancer cells, the dead cells detach from the plate. • As cells detach, the impedance drops — and this change is detected in real-time.

Answer 22

1. Formulation • Selection of excipients (e.g. bulking agents, cryoprotectants). • Solution preparation and sterile filtration (if aseptic). 2. Filling • Aseptically fill drug solution into vials/trays. • Partial stoppering of vials in Grade A environment. 3. Loading into Freeze Dryer • Load into pre-cooled shelf in lyophiliser under aseptic conditions. 4. Freezing • Controlled nucleation or uncontrolled freezing to form solid ice matrix. • Typical temp: –40 to –50°C. 5. Primary Drying (Sublimation) • Chamber pressure reduced (vacuum), shelf temp raised to allow sublimation. • Ice → Vapour; most of water removed here. 6. Secondary Drying (Desorption) • Shelf temp increased further (e.g. 20–40°C) to remove bound water. 7. Stoppering under Vacuum or Inert Gas • Stopper vials while under vacuum or inert gas atmosphere. 8. Unloading • Unload from lyophiliser into Grade A or B area. 9. Capping and Final Packaging • Fully stopper and cap, then label and store under appropriate conditions.

Answer 23

CQA Justification Residual Moisture - Affects product stability and shelf life Appearance (cake structure)- Collapsed/particulate indicates poor drying or contamination Reconstitution Time- Impacts usability and delivery Potency/Assay- Ensures therapeutic efficacy pH after reconstitution- Critical for drug stability Sterility- Essential for parenteral products Endotoxin level- Particularly for injectables Container Closure Integrity- Prevents microbial ingress, ensures sterility

Answer 24

CPP Impact Freezing rate and shelf temperature-Affects ice crystal size, cake porosity, and sublimation efficiency Chamber pressure during primary drying P-Affects sublimation rate and product temperature Shelf temperature during primary and secondary drying- Impacts residual moisture, cake collapse Duration of drying phases-Ensures complete water removal without degradation Stoppering pressure and timing- Impacts container closure integrity Vacuum leak rate- Affects process efficiency and sterility assurance

Answer 25

Buzz words: 1. Impeller Speed: • What it is: The rotation speed (RPM) of the impeller, the main mixing blade in the granulator. • Purpose: Controls the mixing intensity and powder movement. Helps distribute the granulating liquid throughout the powder bed to start forming granules. • Impact: • Too high → over-granulation (large, wet lumps). • Too low → poor mixing, uneven granules. ⸻ 2. Chopper Speed: • What it is: The rotation speed (RPM) of the chopper blade in the granulator. • Purpose: Breaks down larger wet lumps of granules into smaller, uniform particles. It prevents agglomeration and controls granule size distribution. • Impact: • Too high → overly fine granules (poor flow). • Too low → large, uneven granules. ⸻ 3. Granulation Time: • What it is: The total duration (minutes) of the granulation process — how long the impeller and chopper run with the granulating liquid. • Purpose: Controls how long the powders are mixed and granulated to form granules of the desired size and consistency. • Impact: • Too long → over-wet or overworked granules (dense, possibly sticky). • Too short → incomplete granulation (powders not binding properly). • Flowability → about how well powder/granules move. • Compressibility → about how well they compress into a solid form. Inlet Air Temperature: • This is the temperature of the air entering the fluid bed dryer (or any drying system). • It’s the hot air that dries the wet granules by evaporating moisture. Outlet Air Temperature: • This is the temperature of the air leaving the drying chamber after it has picked up moisture from the granules. • It reflects how much moisture has been removed—higher outlet temp = less moisture remaining. Fluidisation (or fluidization): • It refers to the process of suspending solid particles (like granules or powders) in an upward flow of air or gas, making them behave like a fluid. • This happens in a fluid bed dryer, where hot air is blown from the bottom to lift and mix the granules, ensuring even drying. Think of it like popping popcorn—the hot air moves the kernels around so they heat evenly. In drying, the air lifts the granules, allowing uniform heat and moisture removal. Turret Speed (in a tablet press): • The turret is the rotating part of the tablet compression machine that holds the dies and punches. • Turret speed refers to how fast the turret rotates—usually measured in revolutions per minute (RPM). As the turret rotates: • Powder is filled into each die. • The punches compress the powder into tablets. • The tablet is ejected, and the process repeats. ⸻ Why is turret speed important? It’s a CPP (Critical Process Parameter) in tablet compression. • Too fast turret speed: • Less time for powder filling → can cause weight variation. • Less dwell time (time under compression) → may lead to weak tablets (low hardness, friability issues). • Too slow turret speed: • Reduces productivity. • Might not affect quality, but inefficient. 1. Weighing and Dispensing • Purpose: Ensure correct quantities of raw materials. • CPPs: Accuracy of weighing. • CQAs: Material identity, potency, and purity. ⸻ 2. Wet Granulation • Purpose: Mix powders with a granulating liquid to form granules, improving flowability and compressibility. • CPPs: • Impeller speed. • Chopper speed. • Granulation time. • Temperature (if heated). • CQAs: • Granule size distribution. • Bulk/tapped density. ⸻ 3. Drying (e.g., Fluid Bed Dryer) • Purpose: Remove moisture from granules. • CPPs: • Inlet/outlet air temperature. • Fluidisation air velocity. • Drying time. • CQAs: • Loss on drying (LOD). • Granule moisture content. ⸻ 4. Milling/Sieving • Purpose: Reduce granule size to uniform distribution. • CPPs: • Mill speed. • Sieve size. • CQAs: • Granule size distribution. ⸻ 5. Blending (Including Lubrication) • Purpose: Mix dried granules with additional excipients (e.g., lubricants like magnesium stearate). • CPPs: • Blender speed. • Mixing time. • CQAs: • Blend uniformity (assessed by sampling top, middle, bottom). ⸻ 6. Compression (Tablet Press) • Purpose: Compress the blended powder into tablets. • CPPs: • Turret speed. • Fill depth. • Pre-compression and main compression force. • Dwell time. • CQAs: • Tablet weight. • Hardness. • Friability. • Disintegration time. • Uniformity of dosage units (content uniformity). ⸻ 7. Coating (Optional) • Purpose: Apply a coating for taste masking, protection, or controlled release. • CPPs: • Pan speed. • Spray rate. • Atomization pressure. • Bed temperature. • Distance between spray gun and tablet bed. • CQAs: • Coating uniformity. • Weight gain. • Appearance (e.g., color, gloss). ⸻ 8. Packaging • Purpose: Protect the tablets from environmental factors (moisture, light). • CPPs: • Packaging line speed. • Seal integrity (e.g., blister packs). • CQAs: • Pack integrity. • Labeling accuracy.

Answer 26

Simple Process Flow: 1. Powder blending → 2. Roller compaction → 3. Ribbon formation → 4. Milling (to granules) → 5. Lubrication blending → 6. Tablet compression. Words: Dwell Time (in tablet compression): • Definition: Dwell time is the amount of time the tablet punches remain in contact with the powder under compression force (i.e., how long the powder is compressed before the punches move apart). • It’s the time duration during which the upper and lower punches maintain pressure on the powder within the die cavity. • Measured in milliseconds (ms). ⸻ Why is dwell time important? • Longer dwell time allows better consolidation of the powder, leading to: • Stronger tablets (higher hardness). • Reduced risk of capping/lamination (tablet defects). • Better binding of particles. • Short dwell time (due to high turret speed or small punch size) can cause: • Weak tablets. • Poor mechanical strength. ⸻ How is it controlled? • CPPs affecting dwell time: • Turret speed: Faster turret = shorter dwell time. • Punch head flat length: Longer head flat = longer dwell time. (Some presses use extended dwell time punches for this reason.) Fill Depth (in tablet compression): • Definition: Fill depth is the depth of powder filled into the die cavity during tablet compression. • It determines how much powder gets into each die cavity before compression happens, which directly affects: • Tablet weight. • Tablet thickness. ⸻ Why is fill depth important? • It’s a CPP (Critical Process Parameter) because: • Too much fill depth → tablets could be overweight or too thick. • Too little fill depth → tablets could be underweight or too thin. • It ensures uniform dosing (critical for content uniformity) and consistent tablet dimensions. ⸻ How is fill depth controlled? • By adjusting the lower punch position during the filling stage: • The lower punch controls how deep the powder fills. • Changing the fill cam setting alters the fill depth. • CPPs influencing fill depth: • Filling speed. • Powder flowability. • Turret speed (indirectly, faster speed = less time to fill). What is Ribbon Density? • Ribbon density refers to the density of the compacted sheet or “ribbon” formed during roller compaction in dry granulation. • It’s a CQA (Critical Quality Attribute) because it indicates how well the powder has been compacted between the rollers. ⸻ Why is Ribbon Density Important? • It directly affects: • Granule properties after milling (size, strength, flowability). • Compressibility of the granules (how well they form tablets later). • Content uniformity and tablet weight variation downstream. ⸻ How is it calculated? • Usually measured as: \text{Ribbon Density} = \frac{\text{Mass of ribbon sample}}{\text{Volume of the ribbon sample}} (can be expressed in g/cm³) • Some companies use in-line sensors or off-line tests on ribbon segments to assess density. ⸻ Controlled By Which CPPs? • Ribbon density is influenced by key roller compaction CPPs: • Roller pressure / compaction force • Roller gap width • Roller speed • Feed screw speed Dry Granulation Process (Roller Compaction) 1. Weighing and Dispensing • Purpose: Accurate measurement of raw materials. • CPPs: Weighing accuracy. • CQAs: Material identity, potency, purity. ⸻ 2. Blending (Pre-Mixing) • Purpose: Homogeneously mix powders (API + excipients). • CPPs: • Blender speed. • Mixing time. • CQAs: • Blend uniformity. ⸻ 3. Roller Compaction (Granulation Step) • Purpose: Compress powder mixture between two rollers to form ribbons or flakes (no liquid used). • CPPs: • Roller pressure/compaction force. • Roller speed. • Gap width between rollers. • CQAs: • Ribbon density (affects downstream flowability and compressibility). • Granule size distribution (after milling). • Bulk/tapped density. ⸻ 4. Milling/Sieving (Post-Compaction) • Purpose: Mill the compacted ribbons into granules of desired size. • CPPs: • Mill speed. • Sieve size. • CQAs: • Granule size distribution. • Flowability. ⸻ 5. Lubrication (Final Blending) • Purpose: Add lubricants (e.g., magnesium stearate) to granules. • CPPs: • Blender speed. • Mixing time. • CQAs: • Blend uniformity (including lubricant distribution). ⸻ 6. Compression (Tablet Press) • Purpose: Compress granules into tablets. • CPPs: • Turret speed. • Fill depth. • Compression force. • Dwell time. • CQAs: • Tablet weight. • Hardness. • Friability. • Disintegration time. • Content uniformity. ⸻ 7. Coating (if applicable) • Same as wet granulation (optional).

Answer 27

1. Manufacturing Steps Solutions (e.g., Paracetamol oral solution) • Step 1: Dispense and weigh raw materials (API, excipients, purified water). • Step 2: Prepare purified water according to EP/BP specifications. • Step 3: Dissolve excipients (e.g., preservatives, sweeteners) in water under stirring. • Step 4: Add API gradually while maintaining agitation to ensure complete dissolution. • Step 5: Adjust final volume with purified water. • Step 6: Filter (e.g., 0.45 µm) to remove particulates. • Step 7: Fill into containers under non-sterile, GMP-controlled conditions. Suspensions (e.g., Amoxicillin suspension) • Step 1: Dispense and weigh raw materials. • Step 2: Prepare dispersion medium (water + suspending agents + preservatives). • Step 3: Wet the API (to prevent clumping) and add to dispersion medium. • Step 4: Homogenize to achieve uniform distribution of suspended particles. • Step 5: Adjust final volume, mix thoroughly. • Step 6: Fill into containers with gentle agitation to maintain uniformity. Emulsions (e.g., oil-in-water) • Step 1: Prepare aqueous phase (water + hydrophilic excipients). • Step 2: Prepare oil phase (API + lipophilic excipients). • Step 3: Heat both phases (often ~60-70°C) to match temperatures. • Step 4: Slowly add the oil phase into the aqueous phase under high-shear mixing. • Step 5: Homogenize to achieve fine droplet size and stable emulsion. • Step 6: Cool to ambient temperature and fill. Syrups (e.g., simple syrup or medicated syrup) • Step 1: Prepare concentrated sugar solution (heat water, dissolve sugar). • Step 2: Prepare a separate solution of excipients/API in water. • Step 3: Combine the sugar solution and excipient/API solution while mixing. • Step 4: Adjust final volume, mix thoroughly. • Step 5: Filter and fill. ⸻ 2. Critical Process Parameters (CPPs) • Mixer speed: Ensures uniform mixing; too high may introduce air, too low may cause non-uniformity. • Homogenizer speed/height: Controls particle/droplet size distribution, especially for suspensions/emulsions. • Addition rate of APIs/excipients: Too fast may cause clumping or incomplete dissolution. • Temperature: Crucial during dissolution or emulsification (e.g., heating phases in emulsions). • Mixing time: Sufficient to ensure uniformity but avoid degradation or over-shearing. ⸻ 3. Critical Quality Attributes (CQAs) • Appearance: Clear for solutions, uniformity for suspensions/emulsions, absence of phase separation. • pH: Ensures stability, solubility, and preservative efficacy (typically specified range). • Preservative content: To prevent microbial contamination in multi-dose containers. • Related substances: Impurity profile must be within limits. • Viscosity: Especially critical for suspensions and emulsions for dose uniformity and pourability. • Microbial limits: Typically ≤100 CFU/mL (BP/EP) for non-sterile liquids. • Assay: API content within specified limits.

Answer 28

1. Cell Banking * Steps: o Create Master Cell Bank (MCB) and Working Cell Bank (WCB). o Store in liquid nitrogen. * CPPs: o Thawing temperature/time. o Cryopreservation conditions (liquid nitrogen, ≤ -150°C). * CQAs: o Cell identity (genetic stability). o Cell viability. o Sterility, mycoplasma, virus-free status. ________________________________________ 2. Upstream Processing (Cell Culture) * Steps: o Thaw WCB → expand cells in shake flasks → transfer to seed bioreactors → large-scale production bioreactor. * CPPs: o Temperature (e.g., 37°C for mammalian cells). o pH, dissolved oxygen (DO), CO₂ levels. o Agitation speed (ensures mixing without damaging cells). o Nutrient feed rates (glucose, amino acids). * CQAs: o Cell viability, growth rate. o Product titre (concentration). o Metabolite levels (e.g., lactate, ammonia). ________________________________________ 3. Harvest / Clarification * Steps: o Remove cells/debris from culture medium (centrifugation or depth filtration). * CPPs: o Centrifuge speed, flow rate, filter pore size. * CQAs: o Product yield. o Host Cell Protein (HCP) and DNA content (impurities). ________________________________________ 4. Downstream Processing (Purification) * Steps: o Capture step (e.g., Protein A chromatography for mAbs). o Polishing steps (e.g., ion exchange, viral filtration). * CPPs: o Flow rate of chromatography columns. o pH, buffer composition, conductivity. o Filtration parameters (pressure, pore size). * CQAs: o Purity (removal of HCP, DNA). o Potency (bioactivity). o Viral clearance (critical for safety). ________________________________________ 5. Formulation * Steps: o Buffer exchange (e.g., diafiltration), addition of stabilisers/excipients. * CPPs: o Mixing speed, temperature. o Sterile filtration (0.22 µm). * CQAs: o Concentration, stability. o pH, osmolality. ________________________________________ 6. Fill & Finish (Sterile Filling) * Steps: o Aseptic filling into vials/syringes under Grade A (isolator) with Grade B background. * CPPs: o Filling volume, stopper placement, capping torque. o Sterile filtration (final 0.22 µm filter). * CQAs: o Sterility, endotoxin levels. o Particulate matter. o Container Closure Integrity (CCI). ________________________________________ 7. Storage and Distribution * Steps: o Store under controlled conditions (e.g., 2-8°C). * CPPs: o Temperature control (validated cold chain). o Humidity (if applicable). * CQAs: o Product stability, integrity (no degradation). ________________________________________ Regulatory References: * EU GMP Annex 2 (Biological medicinal substances and products). * Part II (ICH Q7 for APIs). * EU GMP Annex 1 (for sterile processes, Fill/Finish). * ICH Q5C (Stability of Biotech products). * ICH Q5D (Cell substrate characterization).