Therapeutic Proteins

Give examples of the conditions therapeutic proteins are indicated for

Cancer, haemophilia, hepatitis C, hereditary deficiencies, arthritis, immunodeficiency, cosmetic, influenza and other viruses, hereditary emphysema

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Describe features of therapeutic proteins (1)

Unable in solution, high mwt, complex structure/conformation, sensitive to environment (T, pH, polar solvents, stress), prone to enzymatic degradation, susceptible to adsorption, unfolding, aggregate formation, loss of activity

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Describe features of therapeutic proteins (2)

Prone to microbial contamination. Increases viscosity and low solubility at high concentration. Require care during storage and shipping. Very expensive, cell culture, complex purification steps, low production yields. Sterile suites for cell culture

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Describe features of therapeutic proteins (3)

Sterile suites for purification. Cold chain. Strength (mcg to 100 mg). Route of delivery (IV, SC, IM, ID). Dosing frequency (weekly, monthly, quarterly). Healthcare (product in vial IV administration), expensive for acute conditions

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Describe features of therapeutic proteins (4)

Home use (pre-filled syringe). Storage temperature (2-8 degrees Celsius, RT for plasma derived proteins). Shelf life (12-36 months)

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Describe the effect of oxidation on therapeutic proteins (1)

Metals and peroxide contaminants in excipients and delivery devices catalyse oxidation. Amino acids prone to oxidation - Cys, Met, Trp, Tyr. Oxidised proteins are functionally inactive

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Describe the effect of oxidation on therapeutic proteins (2)

Unfolding associated with enhanced susceptibility to proteolytic degradation. Oxidation affects structure, activity, rate of degradation of proteins, contributes to pathological conditions. Oxidation affects stability, purity, safety

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Describe the effect of oxidation on therapeutic proteins (3)

Aggregation occurs due to exposure of protein to air - water interphase

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Describe features of protein aggregation (1)

Common issue during manufacture, storage and transportation. Aggregation may arise from mechanisms and stress conditions. High temperatures, pH, mechanical stress. Types of aggregates - soluble/insoluble, covalent/non-covalent

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Describe features of protein aggregation (2)

Reversible/irreversible and native/denatured. Presence of aggregates of any type is undesirable. Aggregation may cause adverse reactions on administration

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Describe features of immunogenicity

Common immunological phenomenon. Protein aggregates in biological drugs have potential to trigger an immune response in the patient. Decrease effectiveness of drugs. Could potentially cause harmful side effects

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What is required to take a protein from a 'bug' to the clinic? (1)

Purity (critical for safety - proteins are parenteral medicines). Sterility, viral contamination, pyrogen removal. Final dosage form - can be solid (freeze dried) or solution

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What is required to take a protein from a 'bug' to the clinic? (2)

Excipients used for several functions - solubility enhancers, anti-aggregation, adsorption agents, buffer components, preservatives, anti-oxidants, osmotic agents

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How do therapeutic proteins work in the body?

Elimination by B and T cells. Proteolysis by endopeptidases and exopeptidases. Small proteins <30 KDa filtered out by the kidney quickly. Unwanted allergic reaction may develop (even toxicity). Loss due to insolubility/adsorption

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Describe features of the storage and use of therapeutic proteins

Large unstable molecules. Biological forms held together by weak non-covalent forces. Easily destroyed by relatively mild storage conditions

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What are the four main interactions in a protein structure?

Electrostatic forces, H bonding, van der Waals, hydrophobic forces

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Describe features of the bonding and levels of structure in a peptide (1)

Combination of secondary structures result in 3D structure (defines function of protein). Fold into compact globular structures. Non-covalent interactions occur in secondary/tertiary structures

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Describe features of the bonding and levels of structure in a peptide (2)

Hydrophobic interactions/H bonds/electrostatic interactions/van der Waals, hydration. Water molecules bound to protein internally/externally. Water molecules trapped inside protein are bound tightly to H bond donors/acceptors

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Describe features of the bonding and levels of structure in a peptide (3)

Both loosely and strongly bound water have important impacts on protein stability and function. Dried enzymes become more active after they absorb 0.2 g water/g of protein

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Describe features of the bonding and levels of structure in a peptide (4)

When a protein solution is cooled below -40 degrees Celsius, a fraction of water doesn't freeze. Non-freezable water is tightly bound water

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What is the effect of non-covalent processes on activity? (1)

Non-covalent processes lead to loss of activity (protein pharmaceutical inactivation) due to hydrophobic interaction (between protein and bottle or between protein molecules). Leads to aggregation/precipitation

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What is the effect of non-covalent processes on activity? (2)

Denaturation, adsorption, aggregation, precipitation

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Describe features of aggregates (1)

Concerns (potency, bioavailability, injection site reactions, obstruction of capillaries and needles, immunogenicity). Challenge for pharmaceutical development

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Describe features of aggregates (2)

Loss of active, filtration resistance, high viscosity, heterogenous, complex mechanism of formation, potential to go undetected

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Which factors have an impact on aggregation?

Extrinsic, intrinsic. Agitation, temperature, light, interference, extremes of pH, ionic strength, mini impurities, excipients, structural. High concentration. Manufacturing operations, storage, distribution and handling

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What is the effect of degradation processes on a protein structure? (1)

Degradation processes lead to changes in primary structure e.g. racemisation, oxidation (Met, Cys), disulphide exchange, deamidation, proteolysis

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What is the effect of degradation processes on a protein structure? (2)

Trypsin (cleaves peptide bonds on carboxy side of basic side chains, Lys, Arg). Chymotrypsin (cleaves peptide bonds on carboxy side aromatic side chains). Carboxypeptidases cleaves from C-terminus. Amino peptidases cleaves for amino terminus

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What are the methods to promote protein stabilisation during storage? (1)

Smooth glass walls (reduce adsorption/precipitation), avoid polystyrene or containers with silanyl or plaster coatings, dark opaque walls reduce oxidation caused by light. Air tight containers or Ar atmosphere reduced air oxidation

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What are the methods to promote protein stabilisation during storage? (2)

Refrigeration. Low temperature (reduce microbial growth/metabolism/thermal/spontaneous denaturation, reduce adsorption). Freezing (best for long term storage), freeze-thaw can denature proteins

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Describe features of freeze-drying proteins (1)

Therapeutic proteins in solution can often undergo degradation as water can promote both chemical/physical degradation processes. Can occur even when dosage form is kept in a refrigerator (cold chain) throughout storage lifetime

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Describe features of freeze-drying proteins (2)

Freeze-drying can improve stability. Water is removed by sublimation and not by evaporation to give the protein formulation as a solid cake which is then reconstituted by addition of water prior to administration

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What are the three stages of freeze drying?

Freezing step of an aqueous protein and excipient solution. Primary drying. Secondary drying (each step requires optimisation in terms of formulation used and exact processes followed)

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Describe features of freeze drying

Presence of water in protein solutions promotes chemical and physical degradation, reduces shelf-life. Freeze drying removes water through sublimation and not evaporation (phase diagram)

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What is spray drying?

Atomisation of a liquid feed into very small droplets within a hot drying gas leading to flash drying of the droplets into solid particles (diagram)

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Describe features of excipients (1)

Cryoprotectants used during freeze drying (e.g. reducing sugars sucrose and trehalose). Addition of polyols (glycerol or PEG to aid solubilisation). Use surfactants to reduce adsorption and aggregation

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Describe features of excipients (2)

Solubility enhancers - non-glycosylated proteins, can use amino acids, surfactants and polyols. Anti-aggregation/adsorption agents, reduce hydrophobic interaction, especially at interfaces, albumin 1%, phospholipids, surfactants

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Describe features of excipients (3)

Buffer components - pH is key to protein solubility and physicochemical stability, use phosphate citrate of acetate. Preservatives/anti-oxidants - Met, Cys, Tyr, Trp and His, all susceptible to oxidation, removal of oxygen, use ascorbic acid

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Describe features of excipients (4)

Preservatives needed in containers designed for multiple injections (phenol, paraben)

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Give examples of recombinant insulins

Degludec, insulin glargine U300, insulin glargine U100, insulin determir, human NPH insulin, insulin lispro aspart and glulisine, fast acting insulin aspart (diagram)

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What is the role of carbohydrate? (1)

Extracellular protein. Hepatocytes have receptors on the outside of cells which can recognise damaged oligosaccharides. Removed from blood and broken down. Controls lifetime of proteins as protein ages more sialic acid residues are cleaved of protein

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What is the role of carbohydrate? (2)

Glycosylation analogues, recombinant EPO, long acting ESA, CERA, pegylated EPO

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What is isoelectric focusing?

A technique for separating different molecules by differences in their isoelectric point

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State features of analytical needs (1)

Single protein purified in large amounts from all other proteins in production cell. Large molecule, very complex matrix. Characterisation affected by change in production (media, temp, oxygen), changes in purification/formulation

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State features of analytical needs (2)

Purification from cell line to product, homogenous and pure enough to be injected into humans

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Outline recombinant protein production

Cell bank vial, T flask, roller bottles, seed bioreactor, production bioreactor, centrifuge, chromatography columns, viral removal, UF tank, ultrafiltration, 0.2 micron filter, package bulk dissolution and freeze at -80 degrees Celsius

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Describe features of ion-exchange chromatography

Protein mixture added to column containing cation exchangers. Proteins move through column at rates determined by net charge at pH being used. Cation exchangers, proteins with more negative net charge move faster/elute earlier

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Describe features of affinity chromatography

Solution of ligand added to column. Protein mixture is added to column containing a polymer bound ligand specific for protein of interest. Unwanted proteins washed through column. Protein of interest is eluted by ligand solution

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Describe features of electrophoresis

Protein sample placed on gel. Separate proteins in first dimension on gel ***** with isoelectric focusing. Separate proteins in second dimension on SDS-polyacrylamide gel

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State features of circular dichroism

Asymmetric atoms, secondary structures, asymmetric environment (of aromatic amino acids) - diagrams

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What are the other techniques involved in peptide production?

MALDI-TOF MS, electrospray ionisation (droplet formation, droplet fission, desolvation, collisional cooling further desolvation, focusing m/z analysis), LCMS (determine antibody mwt), accurate mwt determination, characterisation (fingerprint map)

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State the analytical tools to evaluate proteins (1)

Amino acid sequence/modifications (MS, chromatography). Folding (S-S bonding, MS, NMR, circular dischoism). Subunit interactions (chromatography, MS). Heterogeneity (size, charge, hydrophobicity, chromatography, MS)

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State the analytical tools to evaluate proteins (2)

Glycosylation (anion exchange, enzymatic digestion MS). Pegylation (chromatography, MS). Bioactivity (bioassay, ELISA, signal transduction). Aggregation (size exclusion, light scattering, microscopy). Proteolysis (electrophoresis, chromatography, MS)

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Therapeutic Proteins

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