Q.C. of parentrals 1

2 1. Sterility test 2. Pyrogen test 3. Particulate evaluation 4. Container closure integrity test 5. Leakage test 6. Safety test 7. pH test 8. Osmolarity/isotonicity 9. Clarity test 10. Uniformity of dosage unit

3 11. antimicrobial preservative efficacy 12. Antioxidant preservative efficacy 13. Redispersibility 14. Reconstitution time 15. Water content for non aq. Products 16. Functionality testing of devices for prefilled syringes – includes syringability, seal integrity

Protein and peptide drug delivery preformulation 4

5  Preformulation studies of therapeutic peptides serves basis for the formulation of dosage form  Preformulation data should be generated for 1. Isoelectric point 2. pH solubility profile 3. Physical stability 4. Changes in solubility 5. Chemical stability

6 Native Functional Protein RefoldingUnfolding Partly or Fully Unfold Protein Non covalent ChangesCovalent Changes I.Aggregation II.Surface Adsorption III.Precipitation I.Aggregation II.Surface Adsorption III.Precipitation a)Deamidation b)Racemization c)Oxidation d)Hydrolysis e)Disulfide exchange a)Deamidation b)Racemization c)Oxidation d)Hydrolysis e)Disulfide exchange Mechanism involved in degradation of protein

 pH :  Solution pH is important for stability purpose. For simple peptides pH of minimum degradation should be identified.  Peptides are usually formulated at slightly acidic pH (3-5). For proteins pH is set away from isoelectric pH to avoid aggregation.  Neutral pH 6-7 – concerned about oxidation of cystine, methionine in addition to deamidation and hydrolysis.  pH above 8 these reactions occur more readily. 7

Preformulation studies  Physical stability  Proteins are polymeric in nature  Their charged groups are preferentially oriented at the protein surface  Proteins are prone to physical changes in addition to chemical changes  Loss of native, three dimensional structure is referred as denaturation  After denaturation proteins undergo further inactivation through several physical changes.  It can adsorptively lost to surfaces in which it is in contact.  It can form aggregates with other protein molecules resulting in precipitate formation 8

9  Factors influencing denaturation of proteins are : temperature, pH, solvent media, agitation and shaking  Proteins are very surface active, spontaneously adsorb to foreign surface  The loss due to surface adsorption are particularly significant with peptides at low concentration  Adsorptive loss can be minimized by making the surface neutral by the way of a polymer coating.

PHYSICAL CONSIDERATIONS: Physical instability can lead to loss of protein function. This can be influenced by Temperature, Pressure, PH, Concentration of denaturing agents (e.G., Guanidine hydrochloride) Exposure of the macromolecule to mechanical disruption Denaturation or unfolding of the protein macromolecule resuts in loss of tertiary structure of the protein, formation of a disordered protein. 10

Reversible Versus Irreversible Protein Denaturation 11

Changes in solubility  Most physical properties are due to charges on the molecules and the ability to bind water  Charge groups are due to the β and γ carboxyl group of side chains of aspartyl and glutamyl residues and α carboxyl group of c-terminal amino acid  When pH is below 2 proteins have net positive charge  As pH increase the carboxyl group become more ionised until at unique pH for each protein, the isoelectric point (pI), the net charge is zero 12

13  As pH increased beyond pI, the protein has more negative charge due to further ionization and neutralization of positively charged amino groups.  pH of the vehicle has major effect on the secondary structure of the protein as change in optical rotation of proteins.

Chemical and Physical Considerations in Protein and Peptide Stability Proteins undergo chemical changes through several pathway Hydrolysis, oxidation, beta elimination, disulphide exchange, racemisation. primary reaction mechanism for the deamidation A.Formation of a deprotonated amide nitrogen B.Rate-determining nucleophilic attack on the side chain carbonyl C.formation of intermediate D.Hydrolysis 14

15  Deamidation is hydrolysis of side chain of glutamine or asparginase.  Rate is a function of nature of amino acid residues adjacent to amide group and properties of solvent such as pH, ionic strength.  Deamidation strongly influences the noncovalent self-association of proteins.

16  β elimination  Degradation in alkaline solution is β elimination involving cystinyl, seryl, theronyl residues.  Can be studied using increasing UV absorbance at 241 nm  Rate is dependant upon pH with a rate directly proportional to hydroxide ion conc.  Presence of cations like Ca, Na also influence rate of β elimination

Effect of formulation exipient  Physical  Chemical  Physiological/Biopharmaceutical.  Excipient interactions can have implications for  drug stability,  product manufacture,  drug release (dissolution; both in vitro and in vivo),  therapeutic activity, and  side effect profile. 17

 Physical interactions do not involve chemical change. The components retain their molecular structure.  Physical interactions are frequently used in pharmaceutical science are  for example, to aid processing and to aid or modify drug dissolution (such as oral modified release) or distribution in the body (such as with the use of a parenteral modified release product).  Physical interaction between an API and an excipient is that between certain primary amine drugs and microcrystalline cellulose.  When dissolution is carried out in water a small percentage of the drug may be bound to the microcrystalline cellulose and not released. 18

 Chemical interaction  involve chemical reactions; i.e., a different molecule (or molecules) is (are) created.  Primary amines will undergo a Maillard reaction with reducing sugars.  Secondary amines may also interact with reducing sugars 19

Formulation of protein and peptide 20

Formulation principles  pH  A pH should be selected in such way that sum of various degradation reaction is minimum  Peptides are usually formulated at slightly acidic pH (3-5).  At acidic pH major concern are deamidation of asparagine, glutamine and C terminal amide, hydrolysis of peptide backbone.  At neutral pH(6-7)oxidation of cystein and methionine in addition to deamidation and of peptide hydrolysis 21

22  Above pH 8 disulphide exchange of cystein, transpeptidation of aspartic acid glutamic acid occurs  Aggregation is concern for more complex proteins  Formulation pH is generally away from isoelectric point

Effect of salt  At low concentrations of divalent ions such as calcium, magnesium and zinc thermal stability may be enhanced  At high concentration,1M or higher salts may salt in(increase protein solubility) or salt out (decrease solubility)  Hydrophobic residues are salted out, they are compressed into the interior space of protein molecule, thus proteins becomes more resistant to thermal unfolding and increases thermal stability.  The stabilizing effect of cation and anions are in the following order  (CH 3 ) N + >NH 4 + >K + >Na + >Mg +2 >Ca +2 >Ba +2  So 4 -2 >Cl - >Br - >NO- 3 > ClO4 - > SCN - 23

Common stabilizers  Serum albumin  Amino acids  Surfactants  Fatty acids 24

 Serum albumin  It can withstand heating to 60 ⁰ C for 10 hours.  At pH 1 to 2 albumin molecule expands and elongates but can return to native confirmation reversibly.  It has good solubility. Soluble in conc. Salt solution.  Mechanism for stability by albumin may be one of the following :  Inhibition of surface adsorption or  Substitution for a nascent complex protein  Dispersion of small protein in the interstitial space of thermally resistant albumin  Cryoprotection  Used in conc. Of 0.1 – 1% range depending upon protein conc. to be stabilized. 25

Amino acids  Mechanism of action of amino acids as stabilizers may be one of the following  Reduce surface adsorption  Inhibit aggregate formation  Stabilize protein against heat denaturation  Glycine is most commnly used amino acid  Other are alanine, arginine. 26

Surfactants  Frequently cause denaturation of proteins by hydrophobic disruption.  Even at low concentrations exert potent denaturizing activity.  judicious use of surfactants can protect proteins from other denaturants.  Proteins have tendency to concentrate at liquid/liquid or liquid/air interface. Due to this proteins may adopt non native confirmation and such confirmation is having less solubility. Protein precipitate out of solution. 27

 Surfactant either reduce the interfacial tension, reducing propensity for protein unfolding, or solubalize the protein to reduce the amount of protein at the interface.  Optimal concentration of surfactants for stabilization should be greater than cmc.  Ionic surfactants are more effective stabilizers than non ionic surfactants because of their electrostatic and hydrophobic binding.  Various surfactants used are : poloxamer 188( pluronic 68), polysorbate 28

Polyhydric Alcohols and Carbohydrates  They contain same feature in their backbone –CHOH- CHOH- groups which are responsible for stabilizing proteins.  Polyhydric alcohol include sorbitol, mannitol, glycerol and polyethylene glycols ( PEG) these are straight chain molecules  Carbohydrates such as sucrose, glucose and lactose are cyclic molecules that contain ketone or aldehyde group  They stabilize proteins against denaturation caused by elevated temperature or by freeze drying cycles. 29

 Many important therapeutic proteins and peptides are derived from blood such as immune globulin, coagulation factors.  For viral destruction pasteurization at 60 ⁰ C for 10 hours is needed. Hence thermal stability is needed.  For this reason 54% sucrose or 10% maltose is added.  The mechanism of stabilization is through the effect on the structure of surrounding water molecules which in turn strengthens the hydrophobic interaction in protein molecule. This hydrophobic interactions are generally considered to be the major force stabilizing the three dimensional structure of proteins.  For freeze drying process stabilizers are about 0.1M where as for thermal denaturation 1or 2M. 30

ANTI-OXIDANTS  Thiol compounds are capable of inhibiting disulphide bond formation ( two cysteines oxidises to one).  Thiol compounds such as thioacetic acid, thioethanolamine, reduced glutathione used as antioxidant  Metal chelants such as EDTA are also used as antioxidants. 31

MISCELLANEOUS  Proteins which has specific binding site for substrate can be stabilized by compound resembling their substrate. e.g. Glucose stabilizes glucoamylase while aspargine stabilizes asparginase.  Compounds forming stable complex through ionic or hydrophobic interaction with proteins, can stabilize proteins. Eg. Heparin sulfate stabilizes fibroblast growth factor, tryptophan stabilizes serum albumin.  Calcium is essential for thermal stability of certain amylases or proteases. The bridging function of Ca within polypeptide chain reduces the flexibility of polypeptide backbone, thus enhancing stability. 32

33  Aggregation  Aggregation due to heat, shaking, freezing is prominent.  Aggregation prevented by addition of mild surfactant.