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Protein and Insulin
Protein formulation and insulin
| Question | Answer |
|---|---|
| What is protein denaturation? | Disruption of the higher order structure, such as the secondary and tertiary protein structure Can be reverisble or irreverisble Can be caused by thermal stress, ph extremes or denaturing chemicals. Protein typically unfolds |
| How and where is dennaturation likely to occur? | Great at air water interface because the air water interface is more mobile. Shaking allows a continuous creation of a new interface, thus providing a massive surface area that lead to massive scale denaturation |
| How does protein aggregation happen? | Can result because a protein adsorbs and then unfolds at the air water interface generated by shaking or shear, exposing the hydrophobic amino acids normally located in the interior. The amino acids side chains interact with each other and form aggregates |
| What needs to be considered for protein formulations? | For dry powder: croprotection and lyo protection For solution: solubility Both: preservation Some excipients can be added to the reconstituting solution rather than the formulation directly. |
| List the formulation excipients | Bulking agents, tonicity modifieers, cryo/lyo protectants, buffer system, preservative, albumin, amino acids, carbs, polyhydric alcohol, propylene glycol, cyclodextrins, chelating agents, surfactants |
| Explain bulking agents | Protein drugs are potent and products have small quantities. Bulking agents are needed to allow for pharmceutical processing and producing a lyophilised product. Examples: mannitol, glycine, hydroxyethyl starch. |
| Explain tonicity modifiers | If a solution is non-isotonic can lead to haemolysis, crenation of red blood cells. Examples: mannitol, dextrose or sodium chloride |
| Explain cryo and lyo protectants | Cryo = protects against freezing Lyo = protects against drying Examples cryo: polyethylene glycol Cryo + lyo = sucrose and trehalose |
| explain buffer systems | Control pH and can also impact on protein solubility. Generally inorganic buffers are used |
| Explain preservatives used in protein formulation | Biological products, susceptible to MO growth, especially multi dose vials. Reconstituted lyophilised powders need to be stable for two weeks. Preservative can be in powder or in the solution used for reconst. Careful about turbidity/precipit. |
| Name common preservatives used in protein formulation | Benzyl alcohol, methylparaben, phenols |
| explain why albumin is used in protein formulation | Human serum albumin (HSA) at 0.1-1% is used to stabilise and prevent adsorption of peptides and proteins onto surfaces. To stop the proteins/peptides adsorbing, albumin preferentially adsorbs to the surface instead. Indirectly stabilises and inc recovery |
| explain why amino acids are used in protein formulation | Reduce surface adsorption, inhibit aggregate formation, stabilise proteins against heat denaturation. Can also increase protein solubility: glycine, asp, glu, arg, lys his most effective. Most commonly used is glycine. |
| explain why carbohydrates are in protein formulation | Stabilise proteins against a variety of stress situations: heat, lyophilisation, and improve stability. Can also increase solubility of proteins. Examples: sucrose and trehalose (non reducing sugars) |
| explain why polyhydric alcohols are in protein formulation | Stabilise the native protein structure. Examples: glycerol, glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol. All trihydric or higher Glycerol also reported to suppress aggregation. |
| explain why cyclodextrins are included in protein formation | Minimise protein aggregation. Encapsulate the hydrophobic amino acid side chains, stop them from interacting with each other and aggregating. |
| Explain why surfactants are in protein formulation | Preferential adsorption of surfactant onto interface. Protein cannot adsorb and unfold, aggregate at surface. Can also bind to the protein and reduce the available hydrophobic surface area, reducing its ability to self associate and aggregate. |
| Explain why chelating and reducing agents are in protein formulation | Anions and cations will bind directly to the protein and reduce the proteins solubility (divalent cations). Remove the ions using EDTA maintains protein solubility. EDTA also inhibits metal-catalysed oxidation of sulfhydryl groups. |
| How should proteins be handled in a hospital setting | Need knowledge of the properties of proteins/peptides to avoid problems Prtoein may aggregate if reconstituted with vigorous shaking, or may adsorb to the surface area of an IV admin set during delivery, incompatibilities and causes of precipitation |
| What is the diluent used for reconstitution and how long can it be stored for after? | Water For Injections USP and about 14 days after reconstitution |
| Explain the self assembly behaviour of insulin | -Quarternary strcture, exists as monomer at very low concentration. Higher concentration, exists as dimer. In presence of zinc ions and ph of 4-8, 3 dimers come together and form a hexamer (most stable form). At conc'n of 2mM +, hex's form at ph7, no zinc |
| What are the formulation factors that can affect self assembling insulin | Electrostatic repulsion between insulin monomers at high pH. pH reduced, charge repulsions are reduced. Increased ionic strength screen the charge repulsions Chelation of zinc ions causes hex's to deaggregate to dimers Sodium glycocholate hex's to mono |
| Outline regular insulin (humalin r) formulation | Human insulin, short acting, short duration of activity. made up of zinc-insulin crystals dissolved in clear fluid |
| Explain hexameric insulins | Unmodified human/porcine insulins form hexamers in contact with zinc in bloodstream. Hexamer form is too big to bind to the receptor, slowly has to dissociate back to a monomer. Not ready for the body when insulin required in large dose after a meal |
| How does the mechanism of release work for NPH insulin? | Formulation is a crystalline suspension of human insulin, zinc and protamine. Insulin crystal disso with the protamine zinc and insulin precipitating out, crystal becomes a hexamer. The hexamer then dissociates to dimer, which can then diffuse |
| What are the two types of insulin recombinant analogues | One that is faster acting and more bioavailable than natural insulin, for supply after a meal The other is one that needs to be less bioavailable and releases more slowly over 24 hours to supply basal insulin levels for the day |
| What is lispro insulin? | Rapid acting, human analog. Lys B28, Pro B29 at c terminal of b chain are reversed. Blocks formation of insulin dimers and hexamers. Allows for more monomers to be available postprandial. |
| What is insulin aspart? | Proline B28 replaced with aspartic acid, removes contatc between Prob28 and Blyb23 at monomer interface, creates a lower tendency to self associate and form hexamers. Fast acting insulin. |
| What is apidra insulin? | Asparagine b3 replaced by lysine, lysing b29 replaced by glutamic acid. Affects self association and lowers isoelectric point to ph 5.1 If its least soluble point is lower than physiological ph, then its solubility is enhanced. |
| What is glargine insulin lantus? | Added 2 arginines to c terminal of b chain, moves isoelectric point from 5.4-6.7, glargine more soluble in acidic pH, takes longer to dissolve at physiological ph = long acting insulin. AsparA21 replaced by glycine, reduces deamination and dimerisation |
| How does lantus act as a long acting insulin? | Lantus has a ph of 4, so most of the material preciptates after injection. A small amount can be used immediately, but the rest in the bloodstream. As it is used, small amounts of the prepitate move into solution in the blood, providing a basal level of |
| What is levemir? | long acting analog, threonine b30 removed and replaced with c14 fatty acid attached to b29. Long action achieved by slow absorption at injection site from self association. Distribution to target tissues slowed by albumin binding. |
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LDM
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