In class assignment 5% Home work 1 5% Home work 2 5% Home work 3 15% Final exam 70%

Drug Stability and testing

Comprehensive stability program Understand drug decomposition In pure form In preclinical form In dosage form Monitor Quality control Shelf life Storage conditions Small molecules Proteins Biologicals Stress testing ?

Hair-splitting

Stress testing Harsh conditions Predict possible products Stability testing More relevant to real world conditions Ballpark defined by data from stress testing

Preclinical stability Experimental stability Post experimental stability Production batch stability Pilot stability

Timeline
Rule of thumb

New molecule

Analytical method development Preformulation

Stress testing

Preclinical

Formulation development Marketed product Formulation changes Stability testing

Stress testingWho decides what to do?

No specific rules ICH guidelines Q1A (stability testing) Q1B (photostability) help but not always Discretion of the investigator Company practices Last word-FDA Acidic conditions Which one is applicable 0.1 N HCl 40C for 7 days 0.1 N HCl 105C for 21 days

Application of stress testing data

Analytical methods Formulation and packaging Storage conditions and shelf life Processing parameters ADME Environmental assessment Predictive or Definitive?

Stress testing explores the intrinsic stability of a molecule

Intrinsic stability

Conditions leading to degradation Rate of degradation Major degradation products Pathways of degradation

Conditions leading to degradation

Thermolytic effect of temperature understand conditions leading to artifacts Hydrolytic solid state v/s solution temperature effects Oxidative oxygen levels oxidative agents Temperature effects Solid or solution Photolytic practically applicable to visible range Pure v/s combo

Rates of degradation

Relative rates at different conditions Predict shelf life Select excipients

Major degradation products and degradation pathways

Not implicitly required by the guidelines Detection v/s prediction Useful wherever possible Mass balance, toxic products, carcinogens

Stress testing and the development process

Solid state Humidity Light Solution pH stability Oxidative stability

Stress Test Designwhere to begin

Data gathering Molecular structure pk Solubility Hygroscopicity Enantiometric purity Analytical methods

Preliminary studies
Elicit 5-20% degradation within reasonable limits of stress conditions

Sample condition Solid state at 70C

Time/exposure 1 week 1 week 2-3 times ICH CE 2-3 times ICH CE 1-7 days 1-7 days 1-7 days 1-7 days 1-7 days 1-7 days

Molecular structure pk Solubility Hygroscopicity Enantiometric purity Analytical methods

Solid state at 70C and 75% RH Solid/ simulated sunlight Aqueous solution/ simulated sunlight 0.1 N HCl/ up to 70C Aqueous solution/ up to 70C pH 8, up to 70C 0.1 N NaOH up to 70C Radical initiator at 40C 0.3% peroxide ambient in dark

Stress test screen

Determine upper range of stress test conditions No degradation at these conditions means the drug is stable
Storage condition 70C 70C NaCl ~75%RH Photostress Time 4-6 weeks 28 Days 2-5 times ICH exposure

Design of actual study

Sample preparation
Test samples Solid Liquid Solution Slurry Standards Buffers

Analytical method development

Degraded samples Samples with added impurities Generic v/s compound specific Validation

Analytical methods

Separation Detection Assay based Impurity based

HPLC

Reverse phase Isocratic or gradient elution Normal phase Detection UV Diode array Mass Spec Fluorescence Combination

Other Methods
TLC detection on plate multiple samples in parallel lower sensitivity GC volatiles detection FID MS internal standard for quantitative analysis CE Separation mechanism different from HPLC

10

Stress testing to stability

Degradation Detection measurement/quantification Measurement prediction/extrapolation Occurrence rate

Understanding reaction rate

Rate of a chemical reaction

aA + bB + cC.= P Rate of formation of P AaBbCc Rate = k AaBbCc a+b+c= order Examples Molecularity?

11

Zero order reaction

-dC/dt = k C = C0 - kt Half life=C0/2k

First order reaction

-dC/dt = kC C = C0e-kt Ln C= LnC0-kt LogC = LogC0 kt/2.303 Half life = 0.693/k

12

Second order reaction

-dA/dt = -dB/dt = k[A][B] dx/dt = k(a-x)(b-x) dx/dt = k(a-x)2 x/a(a-x) = kt Half life = 1/ak

Third order reaction

13

Describing a reaction

Basic parameters Rate constant Order Determination of order?

The Arrhenius equation and the effect of temperature on reaction rate

k=Ae-Ea/RT lnk=lnA-Ea/RT In practice it is not always linear over wide ranges of temperature

14

Data
Concentration in M at temperature in C Time (days) 0.5 1 2 3 5 7 14 100 0.029475 0.028959 0.027955 0.026985 0.025145 0.023431 0.0183 95 0.0393 0.038612 0.03727 0.03598 0.0335 0.03124 0.0244 90 0.0393 0.03861 0.03728 0.03599 0.03354 0.031264 0.024436 85 0.04913 0.0482754 0.04661 0.045 0.04195 0.0391 0.03058 80 0.0393 0.03862 0.0373 0.036027 0.0336 0.031336 0.02454 75 0.039309 0.03863 0.0373 0.03603 0.033605 0.03134 0.02456 70 0.03931 0.03863 0.03731 0.036 0.03361 0.03135 0.02457 60 0.03931 0.038639 0.037325 0.036056 0.033645 0.03139 0.02464 55 0.039315 0.03864 0.03733 0.03606 0.03366 0.03141 0.02467 50 0.039317 0.038647 0.0373 0.036077 0.03367 0.031439 0.02471

Determination of k
0.06 100 95 90 0.05 85 80
Concentration (M)

0.04

75 70 60 55 50

0.03

0.02

0.01

0 0 2 4 6 8
Time (days)

10

12

14

16

15

Arrhenius equation
Temperature C 100 95 90 85 80 75 70 60 55 50 k (1/day) 0.0353 0.0353 0.0352 0.0351 0.03487 0.03484 0.0348 0.0346 0.0345 0.0344 Temperature K 373 368 363 358 353 348 343 333 328 323
0.0348 k 0.035 0.0354 y = 0.0421e-65.2x R2 = 0.9782

0.0352

0.0346

0.0344

0.0342 0.0026

0.0027

0.0028

0.0029 1/T

0.003

0.0031

0.0032

K at 25C is 0.033826

T90=(2.303/k) logC0/C90 T90=0.1054/k T90=3.115 days

Curve Fitting Issues

0.03465

0.035 0.0349
y = 0.0417e R2 = 0.9999
-62.349x

0.0346

y = 0.0406e-53.215x R2 = 0.9704

0.0348 0.0347

0.03455

0.0345

0.0346
0.03445

0.0345 0.0344 0.0343 0.0028

0.0344

0.03435 0.00298

0.003

0.00302 0.00304 0.00306 0.00308 0.0031 i/T

0.00312

0.00285

0.0029

0.00295 1/T

0.003

0.00305

0.0031

0.00315

0.03488 0.03487 0.03486 0.03485 0.03484 0.03483 0.03482 0.03481 0.0348 0.03479 0.00282 y = 0.0374e-24.346x R2 = 0.9945

0.00284

0.00286

0.00288

0.0029

0.00292

16

Extrapolation confidence

0.0354

0.0353

C 80 70 60 50 40 30 25

k 0.0354 0.03522 0.0351 0.0349 0.03487 0.03484 0.03482

0.0348
k

0.0352

y = 0.0411e-0.0526x R2 = 0.9927

0.0351

0.035

0.0349

y = 0.0359e-0.0088x R2 = 0.996

0.0347 2.8 2.9 3 3.1 1000/T 3.2 3.3 3.4

T90 extrapolated = ~2 years T90 actual = ~3 years

The Arrhenius equation and the effect of temperature on reaction rate

K=Ae-Ea/RT lnK=lnA-Ea/RT In practice it is not always linear over wide ranges of temperature

Phase transition (abrupt changes, lower than expected reaction rates at high temperature) pH changes Uncontrolled RH Complex reaction mechanisms (curvature, higher than expected reaction rates at high temperature) Modified equations for better curve fitting K=ATne-Ea/RT lnk =- lnm-m 0<n<1 0<<1 m=1/T

17

Non isothermal studies

Increase residence time at temperatures where confidence limits are poor when drug degradation does not follow the Arrhenius equation near ambient conditions. Temperature ramping ovens Programmed heating Linear Reciprocal Logarithmic Exponential

1/T=1/T0 + at

Disadvantages : requires dedicated oven for an experimental cycle Cost of equipment

Eyring Plots
Collision theory v/s transition state theory k=Ae-Ea/RT G=H-TS k=Ze{(S*/R)-(H*/RT)} Z = T/h k= T/h e{(S*/R)-(H*/RT)} k/T= /h e{(S*/R)-(H*/RT)} Boltzmann Planck k/h =2.08 x 1010 k=Ae-G/RT

18

Understanding the variability of k

To what end? Reduce uncertainty in Arrhenius-based experiments Understand factors that affect the reaction rate For simple reactions Temperature Humidity pH Solvents Complex reactions Reversible Parallel Consecutive Saturation

Effect of humidity on reaction rate

Mainly applies to solid dosage forms Humidity as an accelerated condition Drug degradation, dosage form degradation Chemical changes Physical changes Hydrate formations Plasticization Water activity v/s moisture content Thermodynamic term related to Equilibrium Relative Humidity

19

Water Activity

Aw = p/p0 P vapor pressure of water in the substance, and p0 is the vapor pressure of pure water at the same temperature ERH=Aw x 100% Hygroscopic excipient may be stabilizing!

Solvent effects on reaction Rate

Non-ideal solutions and activity coefficients Solubility Rate of a reaction in a given solvent is influenced by the polarity of the products lnk=lnk0 + V/RT(A+B-*) A=1-A

Reactions giving more polar products are accelerated in more polar solvents C2H5OH+(CH3CO)2O = CH3COOC2H5 +CH3COOH Ionic strength Logk = Logk0 + b Dielectric constant Lnk = Lnk= + C/ Solubility v/s stability

20

Reversible reactions

Ln(A0-Aeq)/(A-Aeq)=(kf+kr)t

K= kf/kr=Beq/Aeq Beq=A0-Aeq

Parallel reactions
kb A
kc

B A=A0e-kt C dB/dt=kbA=kbA0e-kt B=A0kb/k(1-e-kt)

21

Series or Consecutive reactions

Non integratable rate laws

Michaelis menten V=Vmax{S/(km+S)} 1/V= 1/Vmax +Km/VmaxS pH

22

What does it all mean?

Arrhenius in the real world

Predicting degradation rate at ambient temperature ASSUMPTION
Mean Temperature (Celcius)

35 30

No Long term changes in temperature No Daily Fluctuations

25 20 15 10 5 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

USP definition of room Temperature?

Impact on extrapolated degradation rate at 25C based on ISOTHERMAL assumption

23

Cyclic testing
T= T1 +T2sin(2t) C = C0-kt C = C0- 0t Z e{-E/(R[T1 +T2sin(2pt)]} Daily cycle 5C fluctuation compared with Isothermal storage

E (kCal/mole) 10 15 20 25 30

% increase in loss 1.9 3.9 7.5 12.1 17.7

Ea<22kcal/mole loss increase <10%

Kinetic mean temperature

35 30
Mean Temperature (Celcius)

25 20 15 10 5 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

T= f(t) C/C0 = Z 012 e[-Ea/Rf(t)]dt

Temperate 21C(45%RH) 25C(60%RH) 30C(70%RH) 30C(35%RH)

Average Conditions

C/C0

Mediterranean Tropical moist Desert

Time

24

Drug Degradation Kineticsadditional complications and interesting situations

Low temperature and freezing Degradation by ice structure formation Degradation due to decrease in micro pH of condensed aqueous phase Propyl paraben Amoxicillin MitomycinC Catalysis product catalysis Solid state degradation Diffusion Auto catalysis and reaction nucleii Reactions forming a liquid product Adsorbed moisture layer

Product Catalysis
k

D=P
P

25

Auto catalysis and reaction nucleii

Rate of product formation is dependent on rate of formation and growth of reaction nuclei Fractional Decomposition Rate = Rate of nuclei formation = In initial stages
probability of propagation probability of termination l,m,n =1,1,0

Rate of nuclei formation =

Prout and Tompkins equation

Kawakita equation Avrami equation

m,n =1,0 l,m =1,0

Ferric oxide decomposition ZnO and BaCO3 Polymorphic transitions

Diffusion controlled reactions

A reacts with B to form a shell that controls the rate of A reaching B

Fractional Decomposition = Where Change of y depending on rate of diffusion of A into B

Jander Equation Spray freeze dried thiamine diphosphate, propantheline bromide in aluminium hydroxide

26

Reactions forming a liquid product

S = solubility of drug in liquid formed ks,kl rate constants in solid and solution state (1-x-Sx) =fraction in solid state Bawn Equation

Decomposition of 5-(tetradecyloxy)-2-furoic acid

Reactions controlled by an Adsorbed moisture layer

Moisture dependent degradation of solid aspirin Leeson Mattocks equation

But [D] did not affect reaction rate

Proposed change to model Still did not fit

Catalytic effect of degradation products is negligible and V is a constant Rate becomes zero order

27

Weibull equation
Empirical equation for cases where derivation of equation is not easy because of complex time dependent physical state changes

Degradation of ascorbic acid

Mass Balance
What went in must come out Referred to in ICH guidelines Perfect mass balance not always achieved The best information money can buy Essential to validate analytical methods All degradation products accounted for Safety

28

Measurement and expression

Mass MP0 MPt = MI0 - MIt Molecular weight Moles NP0 NPt = NI0 - NIt Absolute mass balance deficit (AMBD) = (MP0 MPt) (MI0 MIt) Relative mass balance deficit (RMBD) =100 x {(MP0 MPt) (MI0 MIt)}/ (MP0 MPt)

Keeping track of mass balance

Meaning of analytical data Criteria for degradation Elemental analysis Volatility Method sensitivity

29

Mass Balance problems Positive deficit Something is missing Degradants not eluted Degradants not detected Degradants lost from sample matrix Parent compound lost from sample matrix Co-elution Poor chromatography Change in response factor

Degradants not eluted Modify elution method Spectrophotometric analysis Flow injection analysis Orthogonal separation Degradants not detected Alternative detection

30

Reactivity of Oxygen
Molecular oxygen either in triplet or singlet state is not usually very reactive Reactions with oxygen require initiation RH + In* R* +InH Radiolysis ozonolysis Initiation rate = Ri

Once initiated these reactions tend to self propagating R* + O2 ROO* Propagation rate constant = kp

Unless termination events take place 2ROO* inert products Termination rate constant = kt

Oxidation
Rate = kp[RH]sqrtRi/2kt Independent of O2 saturation Catalyzed by trace impurities Destructive because of propagation Rate limiting step is the initiator Controlling the initiator is more imp than controlling the O2 level Increasing temperature is not necessarily predictive of oxidation at ambient temperature Peroxide radicals stable at ambient temperature but destabilized by increase in temp Degrade to more reactive species that are catalytic

31

Formation of stable degradation products

Termination reaction 2ROO= RO+ROH+O2 Epoxide formation ROO + C=C RO* + epoxide Leads to co oxidation Acid decomposition Protonated hydroperoxide oxonium ion + water alcohol and ketone Decomposition during isolation Base decomposition Redox reactions Reaction with electrophilic substrates PEG is easily oxidized and facilitates the oxidation of otherwise stable molecules

Stress Testing
Objectives Accelerated conditions to simulate mechanisms at storage temperature To discover degradation mechanisms Produce oxidative impurities formed in accelerated and long term degradation Predict sensitivity to oxidation Controlling rate Chain initiator depends on self generating ROO* Propagation limited Better to control supply of ROO* radicals AIBN Azobisisobytyronitrile R-N=N-R 2R* + N2 R* + O2 ROO*

At moderately high temperatures to avoid degradation of hydroperoxide intermediate AIBN is toxic an can explode on heating Max 20 -30% degradation after 48 hours

2R* R2

Azobisdimethylpentanenitrile at 25C equivalent to AIBN at 40C

32

Hydrogen peroxide and peroxy acids Different mechanism of oxidation Ionic reactions not mediated by radical mechanisms Reacts mainly with amines to give N-oxides Or sulfides to give sulfoxides or sulfones Can react more slowly with double bonds to give epoxides Predictive of some minor impurities at long term ambient conditions Useful adjunct to radical chain initiators And for cleaner reactions to produce degradation products for analysis 0.3-3% in water acetonitrile or methanol for 2-7 days at 40C Peracetic acid is faster results in 1 hour Toxic and explosive

Fenton reaction Hydrogen peroxide activated by metal ion catalysis Radical mediated Fe(II) + H2O2 Fe(III)OH + HO* Much more reactive HO* radical Fe(III)OH + H2O2 Fe(II) + HOO* + H2O Heavy metal Salts Directly oxidize substrate through radical or ionic mechanism Activate molecular oxygen by complexation Decompose peroxides Singlet oxygen Dye sensitized oxidation Rose bengal or methylen blue Oxidation of C=C to give =C-C-O-O-H hydroperoxides Oxidation of cholesterol 1mg/ml drug + 0.1mg/ml rose bengal in water or acetonitrile Expose to visible light 10000-10000 lux for 15, 30 and 60 min Dark control and blank rose bengal control

33

Experimental strategy
Structural information? Oxidizable functional groups Known degradation chemistry Accelerated conditions? AIBN Hydrogen peroxide Heavy metal With blanks Amount of degradation? <5% after 48 hours no problem >20% use antioxidant

Photoreactivity and Photostability Light induced reactions Energy must be absorbed Directly or indirectly D + h D* Product RC-CR + h RC-CR* RC-CR* RC. + .CR RC-CR* + O2 RC-CR + 1O2 RC. + O2 RCOO. 1O2 + D Product RCOO. + D products

Absorption spectra indicator of possible light reactions

34

Considerations for photostability testing

Simulate practical conditions Processing Transport/storage Common exposures Daylight Full sun Filtered sunlight Artificial light Wavelengths

Light source

Option 1 Artificial daylight fluorescent lamp, or xenon arc lamp, metal halide lamp Option 2 Cool white fluorescent lamp and near UV fluorescent lamp

D65 equivalence(outdoor daylight ISO standard) Simplicity Availability Reproducibility Stability validation

35

Test container and chamber

Chamber walls Rotation

Light dose

Actinometry Determination of photon flux Thermal detectors Photoelectric detectors Spectral radiometers Chemical actinometers

36

Small molecules v/s proteins

For small molecules Chemical assay usually correlates well with biological activity Simple chemical structure is the functional form Verification of chemical identity is sufficient to correlate to function(biological activity) Proteins have both chemical and physical structure associated with function Protein content (OD 280) Biuret, BCA reduction of Copper ions SDS-PAGE v/s native PAGE 3D structure not just chemical composition is implicit in function

Therefore for proteins Chemical identity does not always imply function Especially for larger polypeptides!!

Understanding protein stability

Physical instability Changes in 3D structure (denaturation) Aggregation Adsorption (loss of active molecule, may also lead to aggregation) Chemical instability Chemical changes at active site Oxidation Deamidation Hydrolysis/Peptide bond cleavage

Consequences of protein instability Loss of activity Toxicity (embolism from aggregates) immunogenicity

37

Detecting instability

Circular dichroism (structure) FTIR (structure) DSC (structure) RP-HPLC (hydrophobicity) Ion exchange chromatography (charge) Mass spectrometry (MW) PAGE (MW) Immunoblotting (conformation) Analytical Ultracentrifugation (sedimentation coeff or sed equilibrium molecular conformation info) No one method (physical or chemical) is totally indicative of protein function Therefore..Subvert the small molecule assumption Bioassays provide the only definitive answer regarding the activity of the protein

Physical stability studies on protein

Designed to provide information on the following: Effect of pH Effect of electrolytes Interaction with surfaces and interfaces(adsorption)

Adsorption Container and device surfaces Air liquid interfaces (tend to expose hydrophobic sites and lead to aggregation) Can be overcome by Addition of albumin which competes for adsorption sites Or low concentrations of surfactants (poloxamers) Minimizing exposure to air Cosolvents (PEG, glycerol) improve protein hydration and compact protein in solution reducing aggregation or may act by adsorbing to protein molecule and protecting it from unfolding

38

Assessment of common modes of chemical instability in proteins

Oxidation (change hydrophobicity) Methionine cysteine tryptophan tyrosine side chains Can be overcome by Low temperature storage Removal of air or inert gas filling Antioxidants(radical scavengers or metal chelators) Deamidation (change charge) Glutamine or asparagine (more susceptible) hydrolyzed to acid Acid base catalysed Overcome by Adjustment of pH if possible Hydrolysis (change MW) Asparagine-proline asparagine-tyrosine Overcome by Adjustment of pH if possible Bottom line Water is actually the biggest problem!!

General approach to stabilizing protein formulations: Considerations for the removal of water
Evaporation (not really advisable, even under reduced pressure) Heat exposure Bubbling Progressive increase in concentrations of electrolytes Progressive removal of water causes changes in concentration and pH SolutionRemove water in one go! Freeze drying or lyophilization Water removed by sublimation Basic steps Sample frozen at a controlled rate to a temperature below Tg Rapid cooling rate facilitates the formation of small ice crystals minimizes pH shifts High vacuum is drawn. Ice sublimates Additional considerations Heat is usually supplied to prevent additional drops in temperature due to energy demand of sublimation Temperature should not be allowed to rise above Tg or cake will collapse

39

Lyophilization excipients

Excipients Bulking agents for cake formation and prevention of blowout Albumin, Glycine Collapse temperature modifiers Lyoprotectants Hold residual moisture, replace stabilization by water of hydration Sugars glucose (reducing sugar can cause discoloration of cake) Sucrose, trehalose (non reducing sugars)

General approach to stabilizing protein formulations: Considerations for the removal of water
Evaporation (not really advisable, even under reduced pressure) Heat exposure Bubbling Progressive increase in concentrations of electrolytes Progressive removal of water causes changes in concentration and pH SolutionRemove water in one go! Freeze drying or lyophilization Water removed by sublimation Basic steps Sample frozen at a controlled rate to a temperature below Tg Rapid cooling rate facilitates the formation of small ice crystals minimizes pH shifts High vacuum is drawn. Ice sublimates Additional considerations Heat is usually supplied to prevent additional drops in temperature due to energy demand of sublimation Temperature should not be allowed to rise above Tg or cake will collapse

40

Lyophilization excipients

Excipients Bulking agents for cake formation and prevention of blowout Mannitol, Glycine Collapse temperature modifiers Dextran,albumin Lyoprotectants Hold residual moisture, replace stabilization by water of hydration Sugars glucose (reducing sugar can cause discoloration of cake) Sucrose, trehalose (non reducing sugars preferred)

Understanding protein stability for formulation development

Summary Understand prevalent mechanisms of degradation Use proper detection techniques Use appropriate excipients Get rid of water during storage

41

42