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

Comprehensive stability program

Understand drug decompositionIn pure formIn preclinical formIn dosage form

MonitorQuality controlShelf lifeStorage conditions

Small moleculesProteins Biologicals

Drug Stability and testing

Stress testing ?

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Stress testingHarsh conditionsPredict possible products

Stability testingMore relevant to real world conditions“Ballpark” defined by data from stress testing

Hair-splitting

Preclinical stabilityExperimental stabilityPost experimental stabilityProduction batch stabilityPilot stability

Stress testing

Preclinical

Formulation development

Analytical method development

Timeline

Rule of thumb

Formulation changes Stability

testing

New molecule

Marketed product

Preformulation

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No specific rules

ICH guidelines Q1A (stability testing)Q1B (photostability)

help but not always

Stress testing…Who decides what to do?

Acidic conditions

Which one is applicable

0.1 N HCl 40ºC for 7 days0.1 N HCl 105ºC for 21 days

Discretion of the investigator

Company practices

Last word-FDA

Analytical methodsFormulation and packagingStorage conditions and shelf lifeProcessing parametersADMEEnvironmental assessment

Predictive or Definitive?

Stress testing explores the intrinsic stability of a molecule

Application of stress testing data

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Conditions leading to degradation

Rate of degradation

Major degradation products

Pathways of degradation

Intrinsic stability

Thermolyticeffect of temperatureunderstand conditions leading to artifacts

Hydrolyticsolid state v/s solutiontemperature effects

Oxidativeoxygen levels oxidative agentsTemperature effectsSolid or solution

Photolyticpractically applicable to visible rangePure v/s combo

Conditions leading to degradation

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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

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Stress testing and the development process

Solid stateHumidityLight

SolutionpH stabilityOxidative stability

Stress Test Design…where to begin

Data gatheringMolecular structurepkSolubilityHygroscopicityEnantiometric purityAnalytical methods

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Preliminary studies

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

1-7 daysRadical initiator at 40ºC

1-7 daysAqueous solution/ up to 70ºC

1-7 dayspH 8, up to 70ºC

1-7 days0.1 N NaOH up to 70ºC

2-3 times ICH CEAqueous solution/ simulated sunlight

2-3 times ICH CESolid/ simulated sunlight

1-7 days0.1 N HCl/ up to 70ºC

1-7 days0.3% peroxide ambient in dark

1 weekSolid state at 70ºC and 75% RH

1 weekSolid state at 70ºC

Time/exposureSample condition

Molecular structurepkSolubilityHygroscopicityEnantiometricpurityAnalytical methods

Stress test screen

Determine upper range of stress test conditionsNo degradation at these conditions means the drug is stable

2-5 times ICH exposurePhotostress

28 Days70ºC NaCl ~75%RH

4-6 weeks70ºC

TimeStorage condition

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Design of actual study

Test samplesSolid

LiquidSolutionSlurry

Standards

Buffers

Sample preparation

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Analytical method development

Degraded samples

Samples with added impurities

Generic v/s compound specific

Validation

Analytical methods

SeparationDetection

Assay basedImpurity based

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Reverse phaseIsocratic or gradient elution

Normal phase

DetectionUVDiode arrayMass SpecFluorescenceCombination

HPLC

TLCdetection on platemultiple samples in parallellower sensitivity

GCvolatilesdetection FID MSinternal standard for quantitative analysis

CESeparation mechanism different from HPLC

Other Methods

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Stress testing to stability

DegradationDetection

measurement/quantification

Measurement prediction/extrapolation

Occurrence rate

Understanding reaction rate

Rate of a chemical reaction

aA + bB + cC….= P

Rate of formation of P α AaBbCc…

Rate = k AaBbCc…

a+b+c…= order

Molecularity?

Examples

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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

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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

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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

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Data

0.024710.024670.024640.024570.024560.024540.030580.0244360.02440.018314

0.0314390.031410.031390.031350.031340.0313360.03910.0312640.031240.0234317

0.033670.033660.0336450.033610.0336050.03360.041950.033540.03350.0251455

0.0360770.036060.0360560.0360.036030.0360270.0450.035990.035980.0269853

0.03730.037330.0373250.037310.03730.03730.046610.037280.037270.0279552

0.0386470.038640.0386390.038630.038630.038620.04827540.038610.0386120.0289591

0.0393170.0393150.039310.039310.0393090.03930.049130.03930.03930.0294750.5

505560707580859095100Time (days)

Concentration in M at temperature in C

0

0.01

0.02

0.03

0.04

0.05

0.06

0 2 4 6 8 10 12 14 16

Time (days)

Con

cent

ratio

n (M

)

100959085807570605550

Determination of k

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0.034450

0.034555

0.034660

0.034870

0.0348475

0.0348780

0.035185

0.035290

0.035395

0.0353100

k (1/day)Temperature C

323

328

333

343

348

353

358

363

368

373

Temperature K

y = 0.0421e-65.2x

R2 = 0.9782

0.0342

0.0344

0.0346

0.0348

0.035

0.0352

0.0354

0.0026 0.0027 0.0028 0.0029 0.003 0.0031 0.0032

1/T

kK at 25ºC is 0.033826 T90=(2.303/k) logC0/C90

T90=0.1054/k

T90=3.115 days

Arrhenius equation

y = 0.0417e-62.349x

R2 = 0.9999

0.03435

0.0344

0.03445

0.0345

0.03455

0.0346

0.03465

0.00298 0.003 0.00302 0.00304 0.00306 0.00308 0.0031 0.00312

i/T

K

y = 0.0406e-53.215x

R2 = 0.9704

0.0343

0.0344

0.0345

0.0346

0.0347

0.0348

0.0349

0.035

0.0028 0.00285 0.0029 0.00295 0.003 0.00305 0.0031 0.00315

1/T

k

Curve Fitting Issues

y = 0.0374e-24.346x

R2 = 0.9945

0.03479

0.0348

0.03481

0.03482

0.03483

0.03484

0.03485

0.03486

0.03487

0.03488

0.00282 0.00284 0.00286 0.00288 0.0029 0.00292

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Extrapolation confidence

0.0348225

0.0348430

0.0348740

0.034950

0.035160

0.0352270

0.035480

kC

y = 0.0359e-0.0088x

R2 = 0.996

y = 0.0411e-0.0526x

R2 = 0.9927

0.0347

0.0348

0.0349

0.035

0.0351

0.0352

0.0353

0.0354

2.8 2.9 3 3.1 3.2 3.3 3.4

1000/T

k

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

Modified equations for better curve fitting

K=ATne-Ea/RT

lnk =α-β lnm-γm 0<β<1

0<n<1

m=1/T

Phase transition (abrupt changes, lower than expected reaction rates at high temperature)pH changesUncontrolled RHComplex reaction mechanisms (curvature, higher than expected reaction rates at high temperature)

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Non isothermal studies

Temperature ramping ovensProgrammed heating

LinearReciprocalLogarithmic Exponential

when drug degradation does not follow the Arrheniusequation near ambient conditions.

Disadvantages : requires dedicated oven for an experimental cycleCost of equipment

Increase residence time at temperatures where confidence limits are poor

1/T=1/T0 + at

Eyring Plots

Collision theory v/s transition state theory

ΔG=ΔH-TΔS

k=Ae-Ea/RT

k=Ze{(ΔS*/R)-(ΔH*/RT)}

Z = κT/h BoltzmannPlanck k/h =2.08 x 1010

k= κT/h e{(ΔS*/R)-(ΔH*/RT)}

k/T= κ/h e{(ΔS*/R)-(ΔH*/RT)}

k=Ae-ΔG/RT

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Understanding the variability of k

To what end?Reduce uncertainty in Arrhenius-based experiments

Understand factors that affect the reaction rate

For simple reactionsTemperatureHumiditypHSolvents

Complex reactionsReversibleParallelConsecutiveSaturation

Effect of humidity on reaction rate

Mainly applies to solid dosage forms

Humidity as an accelerated condition

Drug degradation, dosage form degradationChemical changesPhysical changes

Hydrate formationsPlasticization

Water activity v/s moisture content

Thermodynamic term related to Equilibrium Relative Humidity

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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

SolubilityRate of a reaction in a given solvent is influenced by the polarity of the products

lnk=lnk0 + V/RT(ΔδA+ΔδB-Δδ*) ΔδA=δ1-δA

Reactions giving more polar products are accelerated in more polar solvents

C2H5OH+(CH3CO)2O = CH3COOC2H5 +CH3COOH

Ionic strength

Logk = Logk0 + bμ

Non-ideal solutions and activity coefficients

Dielectric constant

Lnk = Lnkε=∞ + C/ε

Solubility v/s stability

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Reversible reactions

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

Beq=A0-Aeq

Parallel reactions

A

B

C

kb

kc

A=A0e-kt

dB/dt=kbA=kbA0e-kt

B=A0kb/k(1-e-kt)

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Series or Consecutive reactions

Non integratable rate laws

Michaelis menten

V=Vmax{S/(km+S)}

1/V= 1/Vmax +Km/VmaxS

pH

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What does it all mean?

Arrhenius in the real world

0

5

10

15

20

25

30

35

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Mea

n Te

mpe

ratu

re (C

elci

us)

USP definition of room Temperature?

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

Predicting degradation rate at ambient temperature

ASSUMPTION

No Long term changes in temperatureNo Daily Fluctuations

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Cyclic testing

T= T1 +T2sin(2πt)

C = C0-kt

C = C0- 0∫t Z e{-E/(R[T1 +T2sin(2pt)]}

7.520

12.125

17.730

3.915

1.910

% increase in loss

E (kCal/mole) Ea<22kcal/mole

loss increase <10%

Daily cycle ± 5C fluctuation compared with Isothermal storage

Kinetic mean temperature

0

5

10

15

20

25

30

35

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Mea

n Te

mpe

ratu

re (C

elci

us)

30C(35%RH)Desert

30C(70%RH)Tropical moist

25C(60%RH)Mediterranean

21C(45%RH)Temperate

Average Conditions

C/C0

Time

T= f(t)

C/C0 = Z 0∫12 e[-Ea/Rf(t)]dt

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Drug Degradation Kinetics…additional complications and interesting situations

Catalysisproduct catalysis

Solid state degradationDiffusionAuto catalysis and reaction nucleiiReactions forming a liquid productAdsorbed moisture layer

Low temperature and freezing

Degradation by ice structure formationDegradation due to decrease in micro pH of condensed aqueous phase

Propyl parabenAmoxicillinMitomycinC

Product Catalysis

D = Pk

P

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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

Rate of nuclei formation = α probability of propagationβ probability of termination

Prout and Tompkins equation

Kawakita equation

Avrami equation

l,m,n =1,1,0

m,n =1,0

l,m =1,0 ZnO and BaCO3Polymorphic transitions

Ferric oxide decomposition

Diffusion controlled reactions

Jander Equation

Fractional Decomposition =

Where

Change of y depending on rate of diffusion of A into B

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

Spray freeze dried thiamine diphosphate, propantheline bromide in aluminium hydroxide

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Reactions forming a liquid product

S = solubility of drug in liquid formedks,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

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

Still did not fit

But [D] did not affect reaction rate

Proposed change to model

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Weibull equation

Empirical equation for cases where derivation of equation is noteasy 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

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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

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Mass Balance problems

Positive deficit

Something is missing

Degradants not elutedDegradants not detectedDegradants lost from sample matrixParent compound lost from sample matrixCo-elutionPoor chromatographyChange in response factor

Degradants not eluted

Modify elution method

Spectrophotometric analysis

Flow injection analysis

Orthogonal separation

Degradants not detected

Alternative detection

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Reactivity of Oxygen

Reactions with oxygen require initiation

Once initiated these reactions tend to self propagating

Unless termination events take place

Initiation rate = Ri

Propagation rate constant = kp

Termination rate constant = kt

Radiolysisozonolysis

RH + In* R* +InH

R* + O2 ROO*

2ROO* inert products

Molecular oxygen either in triplet or singlet state is not usually very reactive

Oxidation

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

Rate = kp[RH]sqrtRi/2kt

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Formation of stable degradation products

Termination reaction2ROO= RO+ROH+O2

Epoxide formationROO + C=C RO* + epoxideLeads to co oxidation

Acid decompositionProtonated hydroperoxide oxonium ion + water alcohol and ketone

Decomposition during isolationBase decompositionRedox reactionsReaction with electrophilic substratesPEG is easily oxidized and facilitates the oxidation of otherwise stable molecules

Stress TestingObjectives

Accelerated conditions to simulate mechanisms at storage temperature

To discover degradation mechanismsProduce oxidative impurities formed in accelerated and long term degradationPredict sensitivity to oxidation

Controlling rateChain initiator depends on self generating ROO*Propagation limited

Better to control supply of ROO* radicals

AIBN AzobisisobytyronitrileR-N=N-R 2R* + N2

R* + O2 ROO*

At moderately high temperatures to avoid degradation of hydroperoxide intermediateAIBN is toxic an can explode on heating

2R* R2Max 20 -30% degradation after 48 hours

Azobisdimethylpentanenitrile at 25C equivalent to AIBN at 40C

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Hydrogen peroxide and peroxy acids

Different mechanism of oxidationIonic reactions not mediated by radical mechanismsReacts mainly with amines to give N-oxidesOr sulfides to give sulfoxides or sulfonesCan react more slowly with double bonds to give epoxides

Predictive of some minor impurities at long term ambient conditionsUseful adjunct to radical chain initiatorsAnd for cleaner reactions to produce degradation products for analysis

0.3-3% in water acetonitrile or methanol for 2-7 days at 40C

Peracetic acidis faster results in 1 hourToxic and explosive

Fenton reactionHydrogen peroxide activated by metal ion catalysisRadical mediatedFe(II) + H2O2 Fe(III)OH + HO*

Much more reactive HO* radical Fe(III)OH + H2O2 Fe(II) + HOO* + H2O

Heavy metal SaltsDirectly oxidize substrate through radical or ionic mechanismActivate molecular oxygen by complexationDecompose peroxides

Singlet oxygenDye sensitized oxidationRose bengal or methylen blueOxidation of C=C to give =C-C-O-O-H hydroperoxidesOxidation of cholesterol

1mg/ml drug + 0.1mg/ml rose bengal in water or acetonitrileExpose to visible light 10000-10000 lux for 15, 30 and 60 minDark control and blank rose bengal control

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Experimental strategy

Structural information?Oxidizable functional groupsKnown degradation chemistry

Accelerated conditions?AIBNHydrogen peroxideHeavy metalWith blanks

Amount of degradation?<5% after 48 hours no problem>20% use antioxidant

Photoreactivity and Photostability

Light induced reactions

Energy must be absorbedDirectly or indirectlyD + hν D* Product

RC-CR + hν RC-CR*RC-CR* RC. + .CRRC-CR* + O2 RC-CR + 1O2RC. + O2 RCOO.

1O2 + D ProductRCOO. + D products

Absorption spectra indicator of possible light reactions

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Considerations for photostability testing

Simulate practical conditionsProcessingTransport/storage

Common exposuresDaylight

Full sunFiltered sunlight

Artificial light

Wavelengths

Light source

Option 1Artificial daylight fluorescent lamp, or xenon arc lamp, metal halide lamp

Option 2Cool white fluorescent lamp and near UV fluorescent lamp

D65 equivalence(outdoor daylight ISO standard)SimplicityAvailabilityReproducibilityStabilityvalidation

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Test container and chamber

Chamber walls

Rotation

Light dose

Actinometry

Determination of photon flux

Thermal detectorsPhotoelectric detectorsSpectral radiometers

Chemical actinometers

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Small molecules v/s proteins

For small molecules…Chemical assay usually correlates well with biological activitySimple chemical structure is the functional formVerification of chemical identity is sufficient to correlate to function(biological activity)

Proteins have both chemical and physical structure associated with functionProtein content (OD 280)Biuret, BCA reduction of Copper ionsSDS-PAGE v/s native PAGE3D structure not just chemical composition is implicit in function

Therefore for proteins…Chemical identity does not always imply functionEspecially for larger polypeptides!!

Understanding protein stability

Physical instabilityChanges in 3D structure (denaturation)Aggregation Adsorption (loss of active molecule, may also lead to aggregation)

Chemical instabilityChemical changes at active site

Oxidation DeamidationHydrolysis/Peptide bond cleavage

Consequences of protein instabilityLoss of activityToxicity (embolism from aggregates)immunogenicity

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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)

Therefore..Subvert the small molecule assumption

Bioassays provide the only definitive answer regarding the activity of the protein

Detecting instability

No one method (physical or chemical) is totally indicative of protein function

Physical stability studies on protein

Designed to provide information on the following:Effect of pHEffect of electrolytesInteraction with surfaces and interfaces(adsorption)

AdsorptionContainer and device surfacesAir liquid interfaces (tend to expose hydrophobic sites and lead to aggregation)Can be overcome by…

Addition of albumin which competes for adsorption sitesOr 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

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Assessment of common modes of chemical instability in proteins

Oxidation (change hydrophobicity)Methionine cysteine tryptophan tyrosine side chainsCan be overcome by…

Low temperature storageRemoval of air or inert gas fillingAntioxidants(radical scavengers or metal chelators)

Deamidation (change charge)Glutamine or asparagine (more susceptible) hydrolyzed to acidAcid base catalysedOvercome by…

Adjustment of pH if possible

Hydrolysis (change MW)Asparagine-proline asparagine-tyrosineOvercome 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 exposureBubblingProgressive increase in concentrations of electrolytesProgressive removal of water causes changes in concentration and pH

Solution…Remove water in one go!

Freeze drying or lyophilizationWater removed by sublimationBasic 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 sublimationTemperature should not be allowed to rise above Tg or cake will collapse

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ExcipientsBulking agents for cake formation and prevention of blowoutAlbumin, Glycine

Collapse temperature modifiers

LyoprotectantsHold residual moisture, replace stabilization by water of hydrationSugars glucose (reducing sugar can cause discoloration of cake)Sucrose, trehalose (non reducing sugars)

Lyophilization excipients

General approach to stabilizing protein formulations:Considerations for the removal of water

Evaporation (not really advisable, even under reduced pressure)Heat exposureBubblingProgressive increase in concentrations of electrolytesProgressive removal of water causes changes in concentration and pH

Solution…Remove water in one go!

Freeze drying or lyophilizationWater removed by sublimationBasic 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 sublimationTemperature should not be allowed to rise above Tg or cake will collapse

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ExcipientsBulking agents for cake formation and prevention of blowoutMannitol, Glycine

Collapse temperature modifiersDextran,albumin

LyoprotectantsHold residual moisture, replace stabilization by water of hydrationSugars glucose (reducing sugar can cause discoloration of cake)Sucrose, trehalose (non reducing sugars preferred)

Lyophilization excipients

Understanding protein stability for formulation development

SummaryUnderstand prevalent mechanisms of degradationUse proper detection techniquesUse appropriate excipientsGet rid of water during storage

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