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PharmDr. Veronika Mikušová, PhD. ... Virus removal: Filtration Precipitation Chromatography And/or viral inactivation (problem: narrow window between succesful viral inactivation

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  • Protein delivery PharmDr. Veronika Mikušová, PhD.

  • Protein structure

    • primary structure: amino acid chaines in well- defined 3D structure

    • secondary structure: α helices, β sheets

    • tertiary structure: proper positioning of the different subunits relativer to each other

    • quarternary structure: individual protein molecules interact to build a larger well-defined structure ex: haemoglobin

  • • Formation and stability of sec., tert. and quarternary are based on relatively weak physical interactions:

    ▫ Electrostatic interactions

    ▫ Hydrogen bounding

    ▫ Van der Waals forces

    ▫ Hydrophobic interactions

    ▫ Not on covalent binding

    which are relatively weak = protein structure can be rather easily changed (loss of pharmacological properties)

  • • Amino acid chaines can be modified by covalently attaching non-amino acid sections:

    ▫ sugars = glycoproteins

    ▫ phosphate groups

    ▫ sulphate groups

    • These groups may be essential for the pharmacological effect of the protein

  • Proteins and GIT

    • Pharmaceutical protein molecules are large • The GIT muconasal surface is a large interface that is

    protected by a monolayer of epithelial cells, connected by tight junctions - it acts as a physical and chemical barrier towards the absorption of proteins and peptides

    • Diffusional transport through epithelial barriers (GIT) is slow unless specific transporter molecules are available

    • Conditions in GIT are extremely hostile to this proteins = enzymatic degradation is fast

    • => large majority of pharmaceutical protein is therefore delivered via parenteral route

  • Possible mechanisms of proteins

    absorption in GIT:

    • Passive transport: simple or facilitated diffusion,

    along concentration gradient, independent of chemical energy (but low lipophilicity and large Mr limit this process, beneficial for di/tripeptides)

    • Active transport: against concentration gradient

    • Endocytosis: energy consuming process – engulfment of the molecules for absorption

  • • Transcellular pathway: across the cells 1. by passive diffusion: depends on physico-chemical

    properties of the molecule: size, charge and lipophilicity

    2. by a specific carrier: peptide and amino acid transporters transport molecule from lumen into the cell – substrate specificity

    • But all compounds absorbed through transcellular pathway are substrate for p-glycoprotein mediated efflux which transports the molecules from inside the cell back into the intestinal lumen

    • The lipophilicity of a molecule is one of the main factors that governs transport via the transcellular route as molecules need to pass through the lipid bilayer of the intestinal membrane - a significant challenge

    3. Transcytosis, Receptor-mediated endocytosis

  • • The transepithelial transport of macromolecules by intestinal epithelial cells occurs to a limited extent through absorption into lymphatic circulation via M-cells of Peyer's patches – minimal degradation of proteins - promising mechanism

    • Paracellular pathway: transport of peptides across the aqueous channels in the cell junctions

    • Not ideal for macromolecules (restricted to relatively small hydrophilic molecules)

    • The addition of a novel functionality to the protein or the use of novel delivery systems – may enable optimal absorption

  • • Proteolytic enzymes: present at various regions along the GIT (trypsin, chymotrypsin, exopeptidases, endopeptidases etc.)

    • Acidic nature of gastric medium – denaturation and degradation of protein

    • Hydrolytic, irreversible cleavage of proteins into AA and small absorbable oligopeptides

  • • conserving the integrity of these large molecules is essential to:

    ▫ ensure an optimal therapeutic effect

    ▫ and to minimize effects such as the induction of unwanted immune responses (immune response may neutralize therapeutic activity in chronic dosing schedules and cause serious side-effects)

  • • Many functional groups are available for chemical degradation (and preferred 3D structure is irrevesibly disturbed) by for example:

    ▫ Heat

    ▫ pH changes

    ▫ Changes in ionic strenght

  • • Preferred shelf life for pharmaceutical produc is at minimum 2 years

    • But most protein degrade too fast when formulated as aqueous solutions even when kept in the refrigerator

    • Therefore they have to be stored in in a dry form and be reconstituted before administration

    • They are usually dried by freeze-drying and the choice of proper excipients during this step (lyoprotectants) is extremely important

  • Sources of pharmaceutical proteins

    • Most proteins used in therapy are produced by: ▫ recombinant DNA technology ▫ or hybridoma technology

    • They are known as biotechnology products or bioitech products

    • Ex: ▫ Human isulin ▫ Erythropoietin ▫ Monoclonal antibodies ▫ Cytokines ▫ Interferons

  • Production

    • They are all produced in cell cultures by procaryotic or eucaryotic cells (E.coli, mamalian cells as Chinese hamster ovary cells or transgenic animals and genetically modified plants)

    • Not all are exactly identical with endogenous product • Clinical trials has proved their efficacy and safety • Isolation of the expressend protein from the culture

    medium is a multistep process consisting of several different steps (chromatography and filtration)

    • For every protein a tailor made purification protocol has to be developed to remove impurities while ensuring integrity

  • • Majority of protein drug are biotech-derived molecules

    • But there are still proteins of major therapeutic importance isolated from blood from humans or animals ▫ Albumin ▫ Blood clotting factors (factor VIII from the blood of

    human volunteers) ▫ Antisera from patients or animals (horses, sheeps)

    • Special purification protocols have to be developed, with particular emphasis on reduction of viral contamination

  • Formulation of pharmaceutical

    proteins for parenteral administration

    Physical instability • Depends on environmental conditions • Examples:

    ▫ Elevated temperature – denaturation of proteins in aqueous solutions

    ▫ Low temperature – may induce destabilization ▫ Adsorption of the protein monomer on the walls of the

    container – protein aggregation ▫ Shaking, exposure to shear forces – protein

    aggregation (hydrophobic parts of the molecule are exposed to hydrophobic interface air/water –> unfolding of the protein -> aggregation)

  • Chemical instability

    • Full prevention of all chemical degradation reactions is difficult (because of many AA involved)

    • Formulator should consider which chemical degradation pathways are relevant

    • At neutral pH peptide bonds between AA are stable, only Asn-Gly and Asn-Pro are relatively labile

  • • Pathways of degradation of protein: ▫ Fragmentation ▫ Isomerisation ▫ Deamidation ▫ Oxidation ▫ Disulphide scrambling ▫ Oligomerization ▫ Aggregation ▫ Cross-linking ▫ Denaturation

  • Deamidation

    • Rather common degradation in water

    • Asn and Gln can be deamidated

    • Kinetics depends on pH and neighbouring AA

    Glu Gln

  • Oxidation • Met, Cys, His, Try, and Tyr are sensible • Oxidative milieu may also cause free Cys units to form disulphide

    bridges or disulphide bind scrambling ▫ oxidation of Met to sulfoxide can be associated with the loss of

    biological activity for many peptide hormones (i.e. corticotropine)

    ▫ oxidation can be iniciated by metal ions, visible light – photoxidation

    ▫ pH independent, may increase at low temperature (better solubility of O2 in water)

    ▫ the most important controling parameter: the degree of solvent accessibility to the side chains of proteins (burried side chains oxidize very slowly)

  • Isomerization, racemization

    • Naturally occuring L-forms turns to D-forms, it changes activity of the protein

    • Slow process occuring in vivo

  • Proteolysis • acidic hydrolysis of

    peptide bonds of Asp

    • hydrolysis can take place at either N- terminal and/or C- terminal peptide bond

  • Incorrect disulfide formation

    • sulfhydryl groups and disulfide bonds are an important factor affecting the properties of proteins

    • the interchange of disulfide bonds can result in incorrect pairings, leading to an altered 3D structure

  • Denaturation (the loss of the globular or 3D structure)

    • thermal denaturation (often depends on pH; for lysozyme 42°C/pH=2, 65°C/pH=6.5)

    • cold denaturation (proceeds often below -20°C) • chemical denaturation (urea, guanidinium

    hydrochlorid) • pres