Biomanufacturing Notes PDF

1. Introduction to Biomanufacturing Biotechnology = the use of living systems & organism to develop/make products/"any technological application that uses biological systems/living organisms/derivatives thereof to make/modify products/processes for specific use Biomanufacturing = the manufacturing...

1. Introduction to Biomanufacturing Biotechnology = the use of living systems & organism to develop/make products/"any technological application that uses biological systems/living organisms/derivatives thereof to make/modify products/processes for specific use Biomanufacturing = the manufacturing engineering & science that enables the production of biotechnology products Clinically pulled high value manufacturing Significant development potential ○ Dated & stagnant technology platform ○ Limited application of operations search & management => more engineers needed ○ Poor control systems implementation ○ Processes have prohibited cost of goods (COGS) Cooksey Report = primarily pharmaceutically focused 2 key gaps in the translation of UK health research 1. Translating ideas from basic clinical research into the development of new products & approaches to treatment of disease & illness 2. Implementing those new products & approaches into clinical practice *Translational research = the process of taking the findings from basic or clinical research and using them to produce innovation in healthcare settings Product Development Timeline Key metrics: ○ Average time to market = 10-15 years ○ Average cost = $1B ○ Failure Rate = 90% ○ Gram's stain Lectures Page 2  Gram +ve => crystal violet stain ○ Outermost layer of peptidoglycan = cellular substance that gives bacteria cell rigidity ○ Mostly Cacci Gram -ve => crystal violet stain washes away ○ Outermost membrane of lipopolysaccharide = blocks the strain from adhering to the peptidoglycan ○ Mostly Bacilli Stem Cells DNA (gene) → mRNA (transcription) → tRNA + Amino Acid → PROTEIN Adenine Adenine Thymine Uracil Guanine Guanine Cytosine Cytosine Lectures Page 3 Common Biotechnology Product Types 1. Vaccines = biological preparation that provide active acquired immunity to particular diseases ○ Usually contain agents that resemble disease-causing microorganisms and are made of weakened or killed forms of the microorganism, or its components ○ Large volumes => easily scalable ○ Low production costs ○ Short development & delivery times => critical for pandemic response ○ Manufacturing process types ▪ Egg-based Vaccine Manufacturing - 6 months □ Most flu vaccines currently ▪ Cell-based Vaccine Manufacturing - 6 months ▪ Recombinant Protein Vaccine Manufacturing - 2 months ▪ Plant-based Vaccine Manufacturing - 1 month ▪ mRNA Vaccine Manufacturing 2. Monoclonal Antibodies (mAbs) ○ 2 main mechanisms of immunity 1. Cellular 2. Humoral => antibodies = specific to a given antigen (=disease causing agent) ○ 5 classes of antibodies: IgG, IgA, IgD, IgE & IgM ▪ All 5 classes secreted by activated B cells as glycoproteins Lectures Page 4  ○ mAbs are identical immunoglobulins, generated from clones of a single B-cell ○ mAbs recognize unique epitopes (binding sites) on single antigen ○ Derivation from a single B-cell clones and targeting a single epitope differentiates monoclonal Abs from polyclonal Abs ○ mAbs generation requires collection of B-cells (lymphocytes) that are fused with myelomas (cancerous B-cells) to create an immortalized hybridoma that can undergo many passages ○ Single cells are required to assure clonality that is achieved through limiting dilutions ○ Limiting dilution is a technique that serially dilutes plating concentrations of the heterogeneous population, such that each well eventually contains a single cell ○ The single cell is then used to create an expansion, which is screened for appropriate expression ○ mAb production techniques were first developed in 1975 Typical Manufacturing Process Clinical Process - Cell as Product Lectures Page 5 3. Introduction to Biosafety Biological Hazards = A potential hazard to humans, animals and/or the environment, caused by a biological organism or material produced by such an organism Biosafety Principle = CONTAINMENT = series of safe methods for managing infectious agents/biological hazards in the laboratory reduce or eliminate human and environmental exposure to (potentially) hazardous agents Biosafety Levels = set of precautions defined by the agents or organisms that are being used in a lab setting 4 levels Each level builds on the previous one BSLs dictate the type of work allowed to take place in a lab setting BSLs are defined by ○ Risks related to containment ○ Severity of infection ○ Transmissibility ○ Nature of the work conducted ○ Origin of the microbe ○ Agent in question ○ Route of exposure Elements of Containment 1. Laboratory Practice & Technique ○ Most important containment element is strictly adhering to standard microbial practices and techniques = basic hygiene practices that are common to and apply to all laboratories ○ Levels of Containment 1. Primary Containment = Protection of personnel and immediate laboratory environment from exposure to biologically hazardous/infectious materials/agents Lectures Page 6  □ Aims to minimise and protect the operator and immediate environment from exposure to biologically hazardous/infectious materials/agents □ Good microbiological technique □ Use of appropriate safety equipment including: Use of biological safety cabinets (BSCs) Enclosed containers Personal protective equipment (PPE)* Vaccines for personal protection 2. Secondary Containment = Protection of environment external to the laboratory from exposure to biologically hazardous/infectious materials/agents □ Aims to minimise and protect the environment external to the laboratory from exposure to biologically hazardous/infectious materials/agents Separate working areas from public spaces Decontamination facilities Separate hand washing facilities Ventilation/specialist air handling Air locks Change rooms/shower facilities 2. Safety Equipment ○ Biological safety cabinets (BSC) = principle device used to contain infectious splashes or aerosols generated by microbiological procedures ▪ THREE categories/classes based on how the BSC works and what it protects □ Class I: Personal and environmental protection □ Class II & III: Personal, environmental and product protection Lectures Page 7 3. Facility Design ○ 4 levels of Biocontainment lab design, management and construction ○ Every level is designed with specific criteria dependent upon the nature of work performed in them ○ The faculty is designed to reduce/prevent the escape of microorganisms in order to: ▪ Improve security ▪ Protect public/environmental health ▪ Protect employees ▪ Protect research A risk assessment of any specific agents will determine the level and appropriate combination of these elements required for any experimental work Lectures Page 8 4. Introduction to Good Manufacturing Practice (GMP) & Ethics Good Manufacturing Practice (GMP) = High level assurance for safety, efficacy & quality (Quality is built in testing is part of GMP, but alone does not provide a good level of quality assurance) Applies to both Active Pharmaceutical Ingredients (APIs) & Finished Pharmaceutical Products (FPPs) Good Manufacturing Practices for FPPS 1. Quality assurance 2. Good manufacturing practices for pharmaceutical products 3. Sanitation and hygiene 4. Qualification and validation 5. Complaints 6. Product recalls 7. Contract production and analysis ○ the contract giver and accepter, and the contract 8. Self-inspection and quality audits ○ items for self-inspection and self-inspection team ○ frequency of self-inspection ○ self-inspection report and follow-up action ○ quality audit and suppliers’ audits, and approval 9. Personnel 10. Training 11. Personal hygiene 12. Premises 13. Equipment 14. Materials 15. Documentation 16. Good practices in production 17. Good practices in quality control (QC) Basic Requirements for GMP Clearly defined and systematically reviewed processes Qualification and validation is performed Appropriate resources are provided: ○ qualified and trained personnel ○ premises, space, equipment and services ○ materials, containers, labels ○ Procedures (Standard Operating Procedures), storage, transport ○ laboratories and in-process control Clear, written instructions and procedures Lectures Page 9  Trained operators Records of actions, deviations and investigations ▪ Records for manufacture and distribution ▪ Proper storage and distribution ▪ Systems for complaints and recalls GMP = continuous urge for improvemet Involvement of the Management Annual Product Quality Review Quality Risk Management Complaints Handling Self – Inspection GMP Summary & Conclusions GMP compliance is not an option Quality should be built into the product GMPs are very similar and are really Good Common Sense Good practices cover all aspects of manufacturing prior to supply The role and involvement of senior management is crucial Ethics = moral principles that govern a person's behaviour or the conducting of an activity Elements of Ethical Procedures to Conduct Clinical Study *COSHH = control of substances hazardous to health Lectures Page 10 5. Measurement & Characterisation of Biological Manufacturing Characterisation Types Species of origin Correlation with the tissue of origin Differentiation status Transformation status Stability (e.g. susceptibility to transformation Finite or continuous life span Cross-contamination with other cells (Hela cells) Means of Characterisation Morphology ○ Easy and fast ○ Optical microscopy ▪ Variable depending on culturing conditions & personnel ○ Scanning electron microscopy (SEM) can be used to assess the cell morphology and size ○ transmission electron microscopy (TEM) to visualise cellular ultrastructure and intracellular organelles Species Identification ○ Chromosomal analysis to evaluate number & structure of chromosomes ○ Cytogenetic techniques: karyotyping, fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH) Specific Markers Cell/Tissue Assays Cell surface markers Flow cytometry e.g. CD11c (dendritic), CD31 (endothelial) Gene expression Polymerase chain reaction (PCR) = e.g. T2Runx2 (osteogenic marker), qualitative & real-time SOX9 (chondrogenic marker) Loop mediated isothermal amplification (LAMP) Flow cytometry Secreted cytokines/proteins Enzyme-linked immunosorbent assay e.g. VEGF (angiogenesis), NGF (ELISA) = direct, indirect, sandwitch (neurotrophic factor) Lectures Page 11 Unique Markers DNA sequencing e.g. human leucocyte antigen (HLA) = highly ELISA polymorphic & unique to an individual Flow cytometry Intermediate filament proteins: Immunostaining e.g. glial fibrillary acidic protein (GFAP; astrocytes), desmin (muscle cells), cytokeratin (epithelial cells) Differentiated products: Immunostaining e.g. melanin (melanocytes), haemoglobin (erythroid Spectroscopy cells), serum albumin (hepatocytes) ELISA High-performance liquid chromatography (HPLC) Flow Cytometry Powerful technique that ○ allows for detection of surface markers of cells ○ allows for detection of intracellular factors ○ allows detection of secreted factors by cells ○ allows for detection of DNA content Principle of operation ○ Forward scatter (FSC) correlates with cell size ○ Side scatter (SSC) correlates with internal complexity Limitations ○ Some information can be obtained ○ To distinguish between 2 cell types ▪ Size has to be different ▪ Internal complexity (i.e. amount of granules) has to be different ○ If the previous 2 parameters are the same => no distinction can be made Fluorescence Flow Cytometry Lectures Page 12  Fluorophores ○ Fluorophores (or fluorochromes) are molecules that emit fluorescence upon excitation with light. ○ Fluorophores generally include proteins (such as antibodies) and peptides, and small organic compounds ○ Fluorophores commonly used in flow cytometry include: ▪ PI: propidium iodide ▪ Alexa Fluor® dyes (Thermo Fisher Scientific) ▪ FITC: fluorescein isothiocyanate ▪ DAPI: 4′,6-diamidino-2-phenylindole ▪ PerCP: peridinin chlorophyll protein ▪ PE: phycoerythrin (derived from red algae) ▪ APC: allophycocyanin (derived from red algae) ▪ GFP: green fluorescent protein (derived from Box jellyfish) ▪ CFP: cerulean fluorescent protein (derived from Aequoera Victoria jellyfish) ▪ mRFPs: monomeric red fluorescent proteins (e.g. mCherry; isolated from Discosoma sea anemones) Principle of Fluorescence (Excitation = emission spectra of fluorophores) Fluorescence – Activated Cell Sorting (FACS) FACS is a specialized type of flow cytometry Provides method for sorting heterogeneous cell mixtures ○ one cell at a time ○ using specific light scattering and fluorescent characteristics of each cell Provides fast, objective and quantitative recording of fluorescent signals from individual cells Provides physical separation of cells of particular interest Lectures Page 13 6. Upstream Technology: Fermentation, Automation & Bioreactor Design Upstream processing = the stage(s) of bioprocessing where cells are grown to the desired quantity in bioreactors, and all stages related to this. => Everything related to the mass expansion/production of cells and product formulation. Downstream = the stage(s) of bioprocessing after the cells have been harvested, and all stages related to this. => Everything related to cell processing after the cells have been harvested to ensure product consistency, purity and safety. Cell Culture Types of cell cultures ADHERENT/ANCHORAGE DEPENDANT SUSPENSION CELL CULTURE CELL CULTURE Appropriate for most cell types, Appropriate for cells adapted to including primary cultures suspension /non-adhesive cell lines (e.g., hematopoietic [blood]) Requires periodic passaging, but Easier to passage, but requires daily cell permits easy visual inspection under counts and viability determination to inverted microscope monitor growth patterns Cells are dissociated enzymatically or Does not require enzymatic or mechanically mechanical dissociation Growth is limited by surface area, Growth is limited by concentration of which may limit product yields and cells in the medium, which allows easy scale-up scale-up Requires tissue-culture treated vessel Can be maintained in culture vessels that are not tissue-culture treated, but requires agitation (shaking/stirring) for adequate gas exchange Used for cytology, harvesting Used for bulk protein production, batch products continuously, and many harvesting, and many research research applications applications Basic requirements for cell growth ○ Nutrient supply = food ○ Removal of waste ○ Moisture/humidity Lectures Page 14  ○ Controlled temperature -> Usually 37 °C ○ Correct pH and osmolarity ~ 7.2-7.45 ▪ This requires regulation with CO2(usually 5% CO2 in air) ○ Dissolved oxygen ○ Substrate for attachment (for adherent cultures only) ○ Agitation/mixing (for suspension cultures only) Medium Supplementation = Important to sustaining proliferation and maintaining cell metabolism SUPPLEMENT ROLE/FUNCTION BASAL A liquid/gel designed to support cell growth. MEDIUM An energy source containing compounds to: (NATURAL/ ✓ Regulates cell cycle ARTIFICIAL) ✓ Maintains pH and osmolarity ANTIBIOTICS Used to control the growth of bacteria/fungal contamination. Routine use not recommended since: - May mask mycoplasma - Resistant bacteria - Metabolic disruption SERUM ✓ A source of hormones, growth factors and attachment factors. ✓ One of the most important media components. PHENOL RED ✓ Acts as a pH indicator ✓ Colour changes as result of cell metabolism - Mimics some steroid hormones inc. oestrogen, so non- compatible with oestrogen-sensitive cells - May interfere with sodium-potassium homeostasis - May interfere with colorimetric assays AMINO ACIDS ✓ Source of amino acids (building blocks of proteins) *Cells unable to synthesise their own ✓ Required for proliferation  L-Glutamine = essential amino acid CARBOHYDRA ✓ Energy source TES Most media contain glucose and galactose May contain maltose and fructose VITAMINS ✓ Growth and proliferation PROTEINS & ✓ Very important in serum-free media PEPTIDES  Albumin: Binds water, salts, free fatty acids, hormones and vitamins & toxin removal  Apoprotein: protective agent, stable at neutral and high pH and high temperatures. Inhibits serine proteases including trypsin  Fibronectin: Cell attachment  Transferrin: Iron transport Lectures Page 15  SUPPLEMENT ROLE/FUNCTION INORGANIC SALT ✓ Regulates osmotic balance ✓ Regulates membrane potential by provision of: Sodium, Potassium & Calcium ions BUFFERS Regulate pH Gaseous CO2 balances the carbonate/bicarbonate ions ▫ Cell cultures with natural buffers need to be maintained at 5-10% CO2 (usually via an incubator) ✓ Low cost and non-toxic HORMONES & ✓ Can be used to replace serum GROWTH FACTORS ✓ Improved/controlled proliferation ✓ Controlled differentiation and specialist functions - Can be very expensive - Optimal concentrations/use highly variable Serum = the most important media components ○ Usually isolated from calves Complex mixture and source of: ▪ Carbohydrates ▪ Vitamins ▪ Lipids (fats) ▪ Hormones ▪ Albumins ▪ Minerals ▪ Growth factors ▪ Trace elements ▪ Growth inhibitors ▪ Binding proteins ▪ Amino acids ▪ Protease inhibitors ▪ Proteins ▪ pH buffer ○ Function = Provision of basic nutrients including: ▪ Growth factors and hormones □ Involved in growth and specialised cell function(s) ▪ Binding proteins □ To promote attachment and spreading factors ▪ Protease inhibitors □ Protects cells from proteolysis (protein/peptide breakdown) ▪ Increases media viscosity □ Protects cells from mechanical damage ▪ Acts as buffer □ Stabilizes pH ○ Advantages & Disadvantages of serum in media ✓ Contains various growth factors and hormones which stimulates cell growth and (healthy) functions ✓ Helps in the attachment of cells ✓ Acts as a spreading factor ✓ Acts as a buffering agent which helps in maintaining the pH of the culture media ✓ Functions as a binding protein ✓ Minimises mechanical damage or damaged caused by viscosity Lectures Page 16  - Testing required to maintain the quality of each batch before using - May contain sone growth inhibiting factors - Increased risk of contamination - Presence of serum in media may interfere with the purification and isolation of vell culture products - Lack of uniformity in the composition of serum - Batch-to-batch variation Bioreactor = a device that uses mechanical means to influence biological processes The role of a bioreactor is to provide a controlled environment to achieve optimal growth/or product formation in the particular cell system employed Bioreactor requirements ○ Maintain asepsis => sealed/sterilised vessel to avoid contamination ○ Aeration/agitation => homogeneous flow (cells need O2, nutrients, pH, buffer etc.) ○ Controlled/optimised growth conditions => temperature, pH, flow rate to endure predictable proliferation/cell growth ○ Minimal evaporation => to avoid loss of cells/nutrients ○ Cell parameter monitoring/sampling => To understand Critical Material Attributes (CMAs) & Critical Process Parameters (CPPs) which influence product Critical Quality Attributes (CQAs) ○ Reduced labour/operators/staff => ↑productivity & ↓human error ○ Permit scale up => Consideration of reactor dimensions ○ Flexibility => To enable multiple cell cultures/types/ changes to be made as required Critical Material Attribute (CMA) = Physical, chemical, biological or microbiological property or characteristic of an input material to the process that should be within an appropriate limit, range, or distribution to ensure the desired quality of output material. Critical Process Parameter (CPP) = A process parameter whose variability has an impact on a CQA and therefore should be monitored or controlled to ensure the process produces the desired quality. Critical Quality Attribute (CQA) = Physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. Lectures Page 17 Physiological Parameter MOTITORED PARAMTER SENSOR TECHNOLOGY CONTROL STRATEGY pH Electrochemical probe Acid/base addition Optical probe CO2 overlay addition/sparging Dissolved O2 Electrochemical probe CO2 overlay addition/sparging Temperature Thermocouple Heating jacket Resistance based probe Cooling water Nutrients & metabolites Spectroscopy Heating jacket Liquid chromatography Cooling water Chemical reaction-based Media perfusion/flow rate analyser adjustment Viable cell density & Electrical probe Volume adjustment distribution Spectroscopy Temperature reduction Microscopic imaging Agitation probe Cell-Therapy Specific Parameters MOTITORED PARAMTER SENSOR TECHNOLOGY CONTROL STRATEGY Endogenous factors Surface marker & Gene Media addition (cell signalling molecules, expression Bolus addition (i.e. i.e., cytokines, GFs) addition of GF etc.) Media perfusion/flowrate adjustment Surface marker & Gene Flow cytometry Off-line feed-forward expression Transcriptomics/ control targeted transcript profiling Computational (i.e. RNA profiling) modelling Pass or fail release criteria Lectures Page 18 Culturing Platforms for Cell-Based Products Culture Systems: Common Configurations 1. Static Culture = dishes, flask, hyper flasks ○ Suitable for anchorage dependant cultures ○ Hydrophilic surface promotes cell attachment ✓ Simple & well established technique ✓ Maintains sterility ✓ Relatively inexpensive => economical ✓ Easy observation of cells -> inverted microscope ✓ Effective for small scale culture - Often monoculture - Requires manual handling - Time consuming => laborious - Limited applicability to in vivo microenvironment - Limited cell density & up-scalability 2. Suspension Culture = roller bottles, spinner flasks, microcarriers, continuous stirred reactors ○ Suitable for anchorage dependant and suspension cultures ○ Cells can be seeded in suspension or onto scaffolds (i.e. microcarriers) ✓ Maintains sterility ✓ Relatively inexpensive => economical ✓ Interval media exchange => continuous exposure to fresh nutrients => ↑ nutrient & O2 diffusion ✓ Greater scalability compared to flasks/dishes - Difficult individual handling - Requires racks in heated cabinet/incubator - Limited applicability to in vivo microenvironment => poor aeration and turbulences (i.e. high shear stress) - Limited scalability Lectures Page 19 3. Perfusion Culture = columns/hollow fibres, microcarriers, continuous stirred reactors Suitable for anchorage dependant & suspension dependant cultures A continuous culture method whereby there is a constant media flow in and out but the cells are retained inside the bioreactor ✓ Well established culture system ✓ Constant medium perfusion: ✓ Mitigates nutrient limitations since continuous exposure to fresh nutrients & continued removal of waste products ✓ ↑↑↑ cell concentrations & ↑↑↑ product yield => ↓↓ media working volumes ✓ Medium/High scalability - Can produce high shearing stress on cells - Product/cell retention ○ Some product may be retained within the bioreactor ○ Very high cell densities may be limited by vacuum capacity of the bioreactor - Mixing capacity, O2 demand, &/or fluid viscosity may limit final cell density - Filter fouling = operational failure 4. Rotating Walls Vessel Cells & cell assemblies exposed to low-shear, low turbulence environment Can be used to support cell proliferation & differentiation on 3D scaffolds ✓ Medium can be changed, sampled or modified without stopping the rotation ✓ Efficient gas transfer ✓ Inline monitoring of pH, dO2 & glucose/lactate ✓ Mimics in vivo environment - ↑ sedimentation velocity and collision may induce cell damage - Requires an incubators - Effective only at small volumes (< 10 L) - Difficult to scale up 5. Direct Mechanical Stimulation Mechanical action(s) applied to cells within substrates to simulate in vivo biomechanical properties including: ○ Hydrostatic pressure ○ Compression ○ Shear ○ Tensile strain ✓ More representative of the in vivo biomechanical environment ✓ Cells can be grown within 3D substrates/scaffolds to replicate native ECM ✓ Promotes cell growth and/or differentiation to desired phenotype ✓ May permit continuous evaluation/ monitoring of tissue whilst maintaining cell/tissue viability/integrity ✓ Stimulates synthesis of ECM macromolecules`= in vitro tissue maturation - Can become expensive => Bespoke/ customised parts - Bioreactors may lack the full in vivo complexity/intricacy - 3D constructs are inherently more difficult to monitor - Mechanical behaviour(s) of bioreactor systems highly variable - Clamping/loading issues with some systems Lectures Page 20 6. Tissue Specific Culture Systems Tissue engineering aims to generate biological substitutes that recapitulate morphological, biochemical and mechanical properties of native tissues Cells + scaffold = functional tissue/model In vivo cells and tissues are in a dynamic environment ○ Biomechanical cues ○ Mechanical cues Biomimetic bioreactors apply mechanical loading at physiologically low levels to stimulate: ○ Cell growth and/or differentiation to desired phenotype ○ Biosynthesis of extracellular matrix (ECM) macromolecules => Huge potential to provide biologically relevant in vitro biomimetic tissues Lecture Summary Upstream processing refers to the stage(s) of bioprocessing where cells are grown to the desired quantity in bioreactors, and all stages related to this ○ i.e., cell isolation, cell cultivation, media preparation and supplementation etc. A “bioreactor” refers to a device that uses mechanical means to influence biological processes The role of a bioreactor is to provide a controlled environment to achieve optimal growth/or product formation in the particular cell system employed ○ Bioreactor platforms vary in terms of their design, operation and control parameters Cell parameter monitoring and sampling is essential for understanding Critical Material Attributes (CMAs) & Critical Process Parameters (CPPs) which influence product Critical Quality Attributes (CQAs) Lectures Page 21 7. Downstream Technology: Filtration, Centrifugation & Purification Steps in Downstream Processing Isolation of Protein from Cells Many different proteins exists within one cell => a number of steps needed to extract protein of interest & separate them from many contaminants In the case that a protein of interest is not secreted by the cell into the culture medium, the first step of any purification process is the disruption of the cells in order to for the protein to be released ○ This can be achieved by a process called homogenisation ▪ Homogenisation is any of several processes used to make a mixture of two mutually non-soluble liquids the same throughout ▪ During homogenisation, proteases are released during cell lysis, which will start digesting the proteins in the solution. ▪ If the protein of interest is sensitive to proteolysis, it is recommended to proceed quickly, and to keep the extract cooled, to slow down the digestion. ▪ Alternatively, one or more protease inhibitors can be added to the lysis buffer immediately before cell disruption Types of Filtration Retentate = components that do not pass through the membrane Permeate = components that pass through the membrane Lectures Page 22  1. Cross Filtration ▪ Flow parallel to membrane surface ▪ Does not cause build up, therefore does not suffer from reduced flow overtime 2. Dead End Flow ▪ Flow perpendicular to membrane surface ▪ Causes build-up of filter cake on membrane, causing reduced flow ▪ overtime ▪ Filter cake is the solid mass of residues remaining on membrane Microfiltration = Separates soluble contaminants remaining within the supernatant Supernatant may include: ○ other proteins ○ bio-molecules ○ un-used culture media Pressure driven process => Separates components in a solution or suspension based on molecular size & particles size range: 10μm (starches) to 0.04μm (DNA, viruses, and globular proteins) Ultrafiltration Used to further separate any contaminants able to pass through the microfiltration membrane Uses a pressure gradient => solution moves by induced pressure gradient & separates particles size range: 0.1μm to 0.001μm & usually based on molecular weight: typical range 200 – 300,000 g/mole Microfiltration vs. Ultrafiltration Microfiltration ○ proteins act as the permeate ○ separates larger particles ▪ e.g. colloids, fat globules, cells ○ located upstream to reduce load and fouling capacity on ultrafiltration membrane Ultrafiltration ○ proteins act as the retentate ○ separates smaller particles ▪ e.g. macromolecules However, the two processes are basically identical Lectures Page 23 Protein Extraction from Liquids - Salting Out Following solubilisation, the protein of interest can be purified based on its solubility, which is usually dependent on overall charge, ionic strength, polarity Ammonium sulphate (NH4SO4) commonly used to “salt out” Salting out takes away water by interacting with it, making the protein less soluble, since the hydrophobic interactions among proteins increases The concentration of the salt is increased until the protein becomes completely insoluble and precipitates Different proteins consist of different variations of amino acids, thus the salt concentration at which individual proteins salt out differs from protein to protein Differential Centrifugation Following homogenisation the sample is spun to separate unbroken cells, nuclei, other organelles and particles that are not soluble in the buffer used Different spinning speeds allow for particle separation Lectures Page 24 Column Chromatography Used to separate a mixture of chemical substances into its individual compounds Widely used method for the purification or separation of chemical compound mixtures Based on the principle that different compounds distribute themselves to a varying extent between different phases Two phases 1. stationary (absorbent): solid; sample interacts with this phase 2. mobile (solvent): liquid; sample flows over the stationary phase and carries along with it the constituent to be separated The compound mixture moves along with the mobile phase through the stationary phase and separates depending on the different degree of adhesion of each component in the sample to the stationary phase Size-Exclusion Chromatography = molecular sieve chromatography When an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography Separates molecules based on their size Stationary phase composed of cross-linked gel particles. Extent of cross-linking can be controlled to determine pore size Smaller molecules enter the pores and are delayed in elution time Larger molecules do not enter and elute from column before the smaller ○ elution = the process of extracting one material from another by washing with a solvent Affinity Chromatography Uses specific binding properties of molecules/proteins Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to the protein of interest Lectures Page 25 Electrophoresis Charged particles migrate in electric field toward opposite charge Proteins have different mobility that is defined by charge, size and shape Agarose is used as gel substrate for nucleic acids Polyacrylamide is used as gel substrate mostly for proteins ○ more resistance for larger molecules than smaller Smaller proteins move through faster Determination of Protein Primary Structure Determine which amino acids are present (amino acid analysis) Determine the N- (anime group) and C- termini (unbound carboxyl group) of the sequence (amino acid sequencing) Determine sequence of smaller peptide fragments (most proteins >100 amino acids) Some type of cleavage into smaller units necessary Protein Cleavage Protein cleaved at specific sites by: ○ enzymes: trypsin (cleaves @ C-terminal of (+) charged side chains), chymotrypsin (cleaves @ C-terminal of aromatic amino acids) ○ chemical reagents: cyanogen bromide Different cleavage reagents help to determining primary protein structure After cleavage, a mixture of the peptide fragments is produced ○ Can be separated by chromatography Lectures Page 26 8. Shipment & Storage of Biologically Derived Products Why store cells? Regrowth Reuse Transportation Important Bioprocess Considerations Safety ○ Maintaining sterility ○ Leaching Efficacy ○ Protecting the dose adequately ○ Delivering the dose Technology options ○ Options vary massively with scale ▪ Large scale = lots of options ▪ Small scale = limited choice New Technology = Closed Vial Technology Allow new industry to maintain sterility Minimize investment costs in facility Offer key advantages for patient quality and ease of use to pharmaceutical companies Cryopreservation The use of very low temperatures to structurally preserve intact living cells and tissues Unprotected freezing is normally lethal to cells while controlled cooling can be used to produce stable conditions that preserve life Benefits ○ generation of safety stocks ○ saves time and money ○ preservation of cells ○ insurance against phenotypic drift ○ standard for experiments Principles of cryopreservation ○ High levels of ice formation and increased solute concentration have a negative impact on cell viability ○ Optimal cooling rate for cell viability is 1°C/min – 3°C/min ○ Cryoprotectants ▪ dimethyl sulfoxide (DMSO) and glycerol are the two most widely used cryoprotectants ▪ encourage dehydration and minimize solution effects Lectures Page 27  Cryopreservation procedure 1. Check for contamination ▪ Sources □ contaminated cell lines □ improper aseptic technique ▪ Types □ microbial: bacteria, mycoplasma, fungi, viruses □ cellular: cross contamination ▪ Signs □ turbid media □ rapid decline in pH – colour change □ morphological/structure changes □ filamentous structures 2. Media preparation ▪ Classical Cell Culture Media: □ 5 – 10% (v/v) DMSO □ 20% (v/v) foetal bovine serum (FBS) or bovine serum albumin (BSA) ▪ ATCC® serum – free freezing media □ all in one media □ 10% (v/v) DMSO with proteins and additives for cell survival ▪ Cell Suspension □ 3×106 to 5×106 cells/mL □ 1 mL total volume 3. Freezing cells in a controlled-rate chamber ▪ Controlled rate freezer □ programmable electronic freezing unit □ reliable, consistent rate of cooling ▪ Vial selection □ Several types of vials exist for storage at ultra-low and cryogenic temperatures plastic vials ◊ internal thread ◊ external thread straws glass ampoules (heat sealed) Lectures Page 28  □ Considerations for vial type selection storage temperature liquid submersion head space effect on warming material stresses 4. Recovering cryopreserved cells 5. Post thawing considerations ○ Thaw as quickly as possible ▪ add cold medium in centrifuge tube ▪ thaw the cells in 37°C water bath ~ 2 minutes (small ice remaining) ▪ add frozen cells in centrifuge tube dropwise to avoid osmotic shock ▪ centrifuge & re-suspend the pellet in growth medium ○ Cell recovery – measuring viability of cells ▪ microbial cells □ serial dilutions ▪ animal/human cells □ stain □ animal embryos ▪ morphology Lectures Page 29 Workshop 1 Summary Currently we don’t have the ability to mimic/replicate the structural complexity of the native tissue. In order to realise the potential of any engineered solution and to get it from “bench to bedside” we must consider: ○ What are we trying to produce and for what purpose? (to define critical criteria and requirements) ○ Standardisation of protocols (to directly compare studies/techniques) ○ Need to consider if it is reproducible/up-scalable ○ Need non-destructive testing platforms/measures of quality ○ Who is the customer? ○ How will you get your product to them? Workshop 2 Summary Three principal tissue engineering approaches have been researched: ○ direct implantation of freshly isolated or cultured cells; ○ in vivo tissue regeneration; ○ implantation of tissues assembled in vitro from cells and scaffolds MSC’s/EPCs Implantation Direct cell implantation involves isolating individual cells or small cellular aggregates from the recipient or a donor, which are expanded in culture and injected into the damaged tissue directly. Cells sense their physical 3D environment by translating extracellular signals into biochemical signals, which trigger expression/repression of particular genes that, subsequently, regulate cell function These extracellular signals can promote or restrain cell proliferation, migration and differentiation, trigger ECM remodelling, or promote enhanced tissue organization Understanding how to manipulate signalling to promote the desired end effects is a critical key to successful tissue repair and reconstruction The function of many cell types and, subsequently, tissue growth patterning and architecture, is regulated by four major sources of external signalling The type of physical stimulation that would promote appropriate cell function and subsequent tissue-engineered construct regeneration is relatively easy to be determined However, the level (magnitude) of the physical stimulation to be applied still remains largely speculative: ○ physiological? ○ foetal? ○ pathological? The determination of the appropriate level of physical stimulation is even more problematic for tissues that it is not possible to directly measure their physical environment. Workshops Page 30 Workshop 3 Summary A reproducible and consistent cell line is essential for any bioprocess Due to the large number of cell population doublings needed to make cell-based products, there exists a concern for genetic drift and cell line stability In order to limit the risk of genetic drift, cells will need to be initially expanded, validated via rigorous quality control, and cryopreserved as a cell bank Cell bank = a collection of appropriate containers, whose contents are of uniform composition, stored under defined conditions. Each container represents an aliquot of a single pool of cells Master Cell Bank (MCB) = an aliquot of a single pool of cells, prepared from the selected clone under defined conditions individual vials from the MCB can then be serially subcultured to produce working cell banks Working Cell Bank (WCB) = prepared from aliquots of a homogenous suspension Manufacturers may prepare their own cell banks or may obtain cellular supply from external sources Manufacturers should describe the type of banking system used along with size of cell bank, container and closure system, cryopreservation and storage methods Manufacturers should describe the procedures used to avoid microbial contamination and cross‐contamination by other cell types present in the laboratory A labelling system which can withstand the process of preservation, storage, and recovery from storage without loss of labelling information must be used The cell bank should be stored in either: ○ liquid nitrogen (ultra‐low temperature freezer) for long-term storage, or ○ vapour phase of liquid nitrogen Cell stability under the freezing and storage conditions should be validated using cell recovery or viability data Storage of MCB and WCB should be in two or more locations Access of cell bank should be restricted Exponential cell growth described by: Growth rate described by: Workshops Page 31