formulation issues for proteins and peptides Flashcards
(21 cards)
why oral route not good for protein delivery
-enzymatic degradation in GI tract
-stomach acidic environment denatures proteins
-large and hydrophilic, difficult to cross membrane of intestinal lining
-first pass metabolism
-sensitive to heat, pH, doesnt survive GI tract
routes of administration for proteins
-parenteral delivery preferred
-intravenous, intramuscular, subcutaneous
-minor routes: intrathecal, intra arterial, intracardiac
requirements for parenteral formulations
-must be sterile
entry of viruses/bacteria into bloodstream via injections evade defences, rapid dissemination throughout circulatory system, result in severe infection, fatal if not treated fast
how to ensure sterility of produce
-sterilisation regimes
packs, syringes heat treated/irradiated before use, ensure no contamination after packing and before injection
-use all sterile ingredients
difficult for materials with biological origin, cant heat sterilise or irradiate proteins bc they’ll denature, filtration sterilisation
problems with filtration sterilisation and why good
problems: proteins may clog filter, cant remove viruses so needs additional purification steps, loss of product if protein aggregates
good bc it can remove bacteria and doesnt expose protein to heat and harsh chemical
how does the circulation place constraint on formulations and affect clearance time
formulating into vein immediately goes to bloodstream (passes into heart, pulmonary circulation then from heart to tissue), drug returns to heart though liver and metabolism begins, round trip takes 10-30 seconds, blood flow in tissues is slow so absorption is efficient
what does efficient circulation require
stable formulations, avoid particulates if possible as they can block small vessels or trigger immune response
describe pulmonary and systemic circulation journey
pulmonary circulation=through lung from right ventricle to left atrium
systemic circulation=through body from left ventricle to right atrium
proteins must be stable to aggregation (no aggregation) when formulated for systemic injection, protein aggregation is the first step to what and what risks does it have, how to prevent aggregation
-protein aggregation is first step to particle formation, if they get bigger than 3-5um it can block capillaries and risk pulmonary embolism
prevent aggregation:
control temperature (lower=slows aggregation)
add stabilising excipients (additives like sugar, amino acids, surfactants, PEG polymers to reduce interactions)
adjust pH and ionic strength
use proper containers, minimise agitation, maintain low protein conc
what happens to protein formulation in storage, filtered, injected, bloodstream
storage=denaturation/aggregation due to temperature, light or pH changes, oxidation, precipitation, hydrolysis
filtered=adsorbs to filter, loss of protein, shear induced unfolding
injected=degradation by enzymes, immune recognition
in bloodstream=dilution instability, aggregation, rapid clearance if recognised as foreign, adsorption
why do proteins adsorb to a surface
-proteins have both hydrophilic and hydrophobic regions
-proteins mostly hydrophilic on the outside and hydrophobic regions are buried inside to avoid water
-on very hydrophobic surfaces, the proteins can adsorb and form hydrogen bonds/polar interactions like w water so the hydrophobic parts of protein get attracted to the hydrophobic surface (hydrophobic effect)
-on adsorption the proteins denature and other regions are exposed and more adsorption occurs
how does protein avoid contact wiht highly charged/hydrophobic surfaces
use hydrophilic glass vials, avoid hydrophobic vial caps, care needed with syringe and filter choice
consequences of protein adsorption at surfaces
precipitation, loss of API, inaccurate dose
what happens when protein encounters air-water interface
-aggregation and precipitation
-exposure of hydrophobic regions at air interface
-proteins partly unfold to minimise interfacial energy
how to minimise exposure to air water interface
-avoid vigorous shaking of vials
-ensure vials, syringes are filled as much as possible to minimise air bubbles
-avoid multiple doses from same vial
what happens when co-solvents are added to proteins
(similar risks to air water interface exposure)
-hydrophobic excipients can bring hydrophobic regions in protein together
-on dilution in bloodstream, risk of self association and aggregation
what happens when surfactans are added to proteins
-similar to solvents
-solvating hydrophobic regions can cause aggregation when these regions coalesce on dilution in bloodstream
-high conc of surfactant can denature protein eg. sds in protein gel electrophoresis
two major mechanisms surfactants stabilise proteins
-preferentially locating at interface so it precludes protein adsorption
-and/or associating with proteins in solution, stabilising them against close approach and inhibiting aggregation
effect of pH and adding salts to proteins
effect of pH
-protein stability and activity vary dependent on pI
-aim to formulate close to pH 7.4 for injectiin
effect of adding salts
-can stabilise highly charged proteins
-can salt out some proteins
effects of concentration and viscosity
smaller needle=better compliance larger needle=easier formulation
-protein conc formulated as highas possible to reduce injection volume
-but highly concentrated solutions are more viscous and harder to inject through small needles and more prone to aggregation
–How do proteins drugs differ from small molecule drugs in structure and stability?
–How do the structures of proteins at the primary, secondary and tertiary level affect the ways in which they can be used as therapeutic agents?
–What are the major differences in formulation requirements for proteins compared to small molecule drugs?
–What are the factors which might affect protein therapeutics during storage, formulation and use?
