physical biochemistry, metrology, and accelerated - mbsw online

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Physical Biochemistry, Metrology,and Accelerated Degradation

Experiments

William R. Porter, PhD

Peak Process Performance Partners

Physical Biochemistry, Metrology &Accelerated Degradation: MBSW 2013

Biologics Manufacturing

The three most important things required fordeveloping a biologics product have been:

Analytical methods, analytical methods and analyticalmethods.

• (i.e., location, location and location—just as in real estateinvestment—but scale [dispersion] is even more important.)

• The major source of variability in product performance hasbeen traceable to the methods used to monitor quality,particularly bioassays.

Copyright 2013 W. R. Porter 2

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CHEMICAL STABILITY OF PROTEINPHARMACEUTICALS



Why worry about proteins?

Most biologics currently marketed areproteins.

Protein stability depends on both chemicalstability and physical stability.

These same two factors are responsible forstability concerns with other biologics.


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Chemical degradation of proteins

Deamidation of Asn (asparagine) and Gln(glutamine) side-chain amides.

Asp ↔ iso-Asp interconversion/isomerization.

Racemization.

Proteolysis.

Oxidation:

Metal-catalyzed oxidation.

Photooxidation.

Free radical cascade oxidation.



Chemical degradation of proteins (2)

Disulfide exchange.

DKP (diketopiparazine) formation from N-terminalamino acids.

Condensation reactions.

pGlu (pyroglutamic acid) formation.

Hinge region hydrolysis.

Trp (tryptophan) hydrolysis → kynurenine.

Glycation (Maillard reaction with reducing sugars).


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Chemical degradation of proteins (3)

See backup slides for details…

More than one chemical degradationpathway can be important!

Degradation resulting in loss of efficacy orreduction in safety may reflect a combinationof parallel degradation reactions influenced bydifferent environmental factors

• So, factorial experiments are needed!



PHYSICAL STABILITY OF PROTEINPHARMACEUTICALS


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Physical stability of biologics

Physical instability refers to changes inmacromolecular structure or function without anychemical changes in structure:

Denaturation.• Loss of globular or 3-dimensional structure; unfolding.

• Can cause loss of secondary or tertiary structure (or both).

Surface adsorption.

Aggregation (formation of soluble aggregates)• Often reversible.

Precipitation (formation of insoluble aggregates)• Usually irreversible.



Thermal denaturation

All macromolecules in aqueous solution undergounfolding when heated.

Plot of fraction unfolded vs. temperature is sigmoidal;inflection point is Tm (temperature of ‘melting’).

Partial unfolding may be reversible.• May result in some aggregation.

More complete unfolding usually leads to irreversibleaggregation, precipitation.

• Example: Cooking an egg. Once the albumin in the eggwhite is heated above a certain temperature, the albumin isphysically denatured to the extent that it cannot redissolve.


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Cold denaturation

Aggregation at low temperature is the resultof solidification to form a glass.

Occurs when protein is cooled below glasstransition temperature Tg, then reheated.

Internal nonpolar groups become hydrated atlow temperature due to freezing of water → ‘freeze-fracturing’ induced unfolding. Whenthe ice melts, the protein may not refoldproperly.



Excipient-induced unfolding

Chaotropic agents that disrupt hydrogenbonding of water molecules (e.g., urea,guanidinium HCl, polar aprotic solventssuch as DMSO, N-methylpyrrolidone)destabilize water cage around protein → unfolding.


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Pressure-induced denaturation

Very high pressures (> 2000 atmospheres)can cause unfolding.

May be mechanism for sheer force induceddenaturation.

Intermediate pressures (~1000atmospheres) can sometimes dissociateaggregates, leading to refolding andpossible renaturation when pressure isremoved.



Solid state denaturation

Occurs above glass transition temperaturewhen dry protein is heated.

Highly dependent on moisture content of driedmaterial.


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Aggregation

Five distinct mechanisms have been proposed:

Aggregation of monomers to form higher orderstructures (usually reversible, and has little effect onactivity).

Aggregation of conformationally altered monomers(→ precipitation).

Aggregation of chemically modified monomers(resulting from chemical degradation).

Nucleation-controlled aggregation (needs a seed).

Surface-induced aggregation.



Aggregation steps


Partially unfoldedprotein

Native protein Denatured protein

Partiallydenatured protein

Partiallyaggregated form

Precipitatedprotein

Soluble aggregate

Misfolding

Misfolding

Refolding

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Hydrophilic colloids

Biologics are hydrophilic colloids.

Colloids can aggregate reversibly (flocculate) orirreversibly (caking, precipitation).

Aggregation of colloids is sensitive to shear forces(vibration, stirring, shaking, turbulent flow throughorifices).

Aggregation is sensitive to thermal effects(freeze/thaw, Tm)

Aggregation is sensitive to ionization state (pH),which affects surface charge.

Aggregation is sensitive to interfacial surface area.



Surface adsorption

Biologics can adsorb to interfaces:

Solution/air• Agitation-induced

• Surfactants

Solution/solid• Ice/water

• Container wall/plumbing/filters

Solution/solution (e.g., oil droplets)

Solid/air (for lyophilized products)

Partially unfolded proteins adsorb more readily.


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METROLOGY ISSUES



Measurement uncertainty

All measurements are uncertain; there are none, whichare not uncertain. All measurements are wrong, but some are useful.

• (with apologies to G. E. P. Box)

Only a quantitative estimate of uncertainty distinguishes usefuldata from unbelievable raw numbers .

• In Bayesian terms, numbers without informative prior distributions cannotqualify as data. Useful measurements always have informative priors.

Beginning in the 1990’s, mainly in Europe, efforts to formalizeevaluation of measurement uncertainty were undertaken.

• GUM: Guide to the expression of uncertainty of measurement (1995, 2008).

• EURACHEM/CITAC Guide: Use of uncertainty information in complianceassessment (2007).


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Known knowns, known unknowns andunknown unknowns

“Reports that say that something hasn'thappened are always interesting to me becauseas we know, there are known knowns. Theseare things we know that we know. There areknown unknowns. That is to say, there arethings that we know we don't know. But thereare also unknown unknowns. There are thingswe don't know we don't know.”

Donald Rumsfeld (U.S. Secretary of Defense,February 2002) [emphasis added]



Unknown unknowns, known unknownsand known knowns

At the outset of any stability experiment, Critical-to-Stability(CtS) parameters are at some initial value.

With time, CtS parameters change for the worse. At early times, the changes in CtS parameters are too small to

detect.

• These changes are unknown unknowns.

At slightly later times, the changes in CtS parameters, whiledetectable, are too small to quantify with acceptableuncertainty.

• These changes are known unknowns.

Eventually changes in CtS parameters become large enough toquantify with acceptable uncertainty.

• Only then do these changes become known knowns.


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Sources of variation in biologics

Assay variation is the dominate factor in controlling thequality of biologics, and batch variation takes a backseat. Biologics are typically compared, batch by batch, to a certified

reference standard.

From personal experience, variation between testing laboratoriesparticipating in round-robin certification of new global standardsgrossly exceeds in-house assay variability.

• Batch variation is swamped by measurement uncertainty.

• ICH recognized the need for a separate guideline (Q5C).

Sampling plans are poorly defined.• Biologics traditionally were homogeneous solutions, so sampling

was not considered to be an issue.



Example bioassay data


Wolfenson C, Groisman J, Couto AS, Hedenfalk M, Cortvrindt RG, Smitz JE, Jespersen S. Batch-to-batch consistency of human-derived gonadotrophin preparations compared with recombinant preparations. Reprod Biomed Online. 2005 Apr;10(4):442-54.

The relative standard deviation is expressed as a percentage and is obtained by multiplying the standard deviation (perbatch) by 100 and dividing this value by the average (per batch).

Product Drug FSH LH HCG

product immunoactivity immunoactivity immunoactivity

batch no. lU/vial (relative lU/vial (relative lU/vial {relative

SD. n = 5) SD. n = 5) SD. n = 5)

Pergonal 0331206B 58.77 (2.2) 13.49(3.6) 3.39(1.7)

Humegon 43905119 65.12(1.7) 5.77(1.0) 6.86(1.8)

2nd WHO standard (FSH 54/LH 46) 77.72(5.0) 7.39 (2.4) 7.22 (4.8)

4th WHO standard (FSH 72/LH 70) 86.14(5.3) 3.82(1.8) 10.10(5.1)

Menopur 32509 74.17(1.9) 0.29 (5.2) 9.61 (2.3)

Menopur 32307 73.44 (3.9) 0.48(1.7) 9.05 (3.3)

Menopur 34104 82.62(1.3) 0.39(3.1) 11.06 (1.8)

Table 4. FSH. LH and HCG immunoactivity in different HMG preparations.

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Bioassay variation

Two generations of the WHO standard were tested.

The 2nd generation standard is certified to contain FSH 54 IU/ampouleand LH 46 IU/ampoule. The 4th generation standard is certified tocontain FSH 71.9 (69.0-74.9 [95% fiducial limits]) IU/ampoule and LH70.2 (61.7-80.0 [95% fiducial limits]) IU/ampoule. The values reportedare in comparison with the standards provided by the test kit vendor.

The reported values for LH differ grossly from the certified values; theFSH values are systematically high.

The reported values are overly precise; the last two digits in thefour-digit reported values are meaningless. The ‘uncertainty’ reported is the within day within analyst repeatability,

and does not include inter-day, inter-analyst, or most importantly,interlaboratory uncertainty.

There is insufficient evidence that any of the batches are different.



Assay repeatability, reproducibility

Assays used for studying biologics are often complex; it’sdifficult to achieve reproducibility over multiple sampleseven in short time periods. Frequent comparison with positive and negative control samples

may be needed.

Calibration curve assay designs may be difficult to implementbecause of repeatability problems.

• May be OK for chromatography, electrophoresis, immunoassays.

Consider parallel line or slope-ratio assay designs.

• More common for bioassays.


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Assay intermediate precision

Day-to-day, analyst-to-analyst, instrument-to-instrument, lab-to-lab variation can bemajor problems.

Use positive and negative controls!

Report results as % of control (normalize).

Immunoassays in microtiter plates: outerrows and columns show “edge effects.”



Method transfer

Transfer of methods between labs is aMAJOR problem.

Frequently, unidentified factors (lurkingvariables) have major impact.

Have experienced personnel train newbies,verify comparable results for same samplesplit between analysts.

Water purity (trace metals), lab lighting,reagent sourcing can be issues.


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Components of variance

Assess sources of variability using nesteddesigns.

Between labs.

Between analysts within labs.

Between days within analysts with labs.

Etc., etc. etc.

Verify linearity under all above conditions.

Use both High and Low and “reagent blank”test samples as controls.



ACCELERATED DEGRADATIONEXPERIMENTS


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Quantifying the known unknowns

Well, of course, you can’t—unless youconvert known unknowns into knowableknowns by using STRESSDEGRADATION EXPERIMENTS tospeed up degradation.

Find an elephant to step on yourproduct to make it fail!

Nelson W. Accelerated Testing:Statistical Models, Test Plans, andData Analysis. New York: John Wiley& Sons (1990).

Meeker WO, Escobar LA StatisticalMethods for Reliability Data, NewYork: John Wiley & Sons (1998).



Arrhenius kinetics

The effect of temperature on the rate at which achemical reaction proceeds is well-approximatedby the Arrhenius kinetic model:

where αt,T is the fraction of material degraded at time t after storageat temperature T, f(αt,T) is the reaction model (which varies withreaction mechanism), k is the reaction rate at any particulartemperature T, A is the Arrhenius frequency factor (the rate at infiniteT), Ea is the Arrhenius activation energy and R is the universal gasconstant (8.314 J mole–1 K–1).


RT

EAfkf

ta

TtTTt

Ttexp,,

,

Tt

TtS

SS

0

,0

,

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Arrhenius models for biologics

The Arrhenius model adequately predicts thekinetics of degradation of mostbiotechnological/biologic drug substances anddrug products over a restricted temperaturerange. The lower limit is usually determined bythe freezing point of the formulated drugsubstance, while the upper limit depends uponthe temperature at which physicalunfolding/denaturation occurs.

Typical range = 0–50 °C.



Chemical kinetics


In solution:

Traditionally based onkinetic models in whichconcentration (C) ismonitored as a function oftime.

Reaction order conceptdominates model choices.

• In stability experiments, mostkinetic models can be replacedby a zero-order model with littleloss of accuracy.

• Stress experiments are usuallyrun under conditions such thateither zero- or first-order kineticmodels can be fitted to thedata.

In solids:

Based on models in which fractiondegraded (α) is monitored as afunction of time.

Reaction order models lesscommon.

• Diffusion models

• Geometric contraction models

• Reaction order models

• Induction models

– Power law growth…

» You see nothing,nothing,

nothing…

» then WHAM! you getexplosive growth, and yourproduct fails catastrophically.

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Arrhenius kinetics for solids

Genton & Kesselring* proposed thismodified Arrhenius model:


BhRT

EAk a

RHT lnln ,%

RT

EBhAk a

RHT exp,%

*Effect of temperature and relative humidity on nitrazepam stability insolid state. J Pharm Sci 66: 676–680 (1977)

The fraction degraded at time t for small α is:

rrRTEBhg teAct a

0lim

Humidity sensitivity

Relative humidity


Isoconversional models

Vyazovkin & Wight* demonstrated thatisoconversional methods gave consistentresults and were model-free.

Isoconversional (e.g., time-to-failure) methodsrequire allowing degradation to proceed to thesame extent under different conditions.


BhRT

EACk

ct a

ghT

rfail

fail

lnlnlnln ,

1

Model-dependent terms

* Vyazovkin A & Wight CA. Kinetics in Solids Annu Rev Phys Chem. 48:127-1128 (1977)

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Rates vs. isoconversional approaches

Traditional small molecule accelerated stabilitystudies usually focus on determining reactionrates.

This is short-sighted, and put huge demands on theanalytical method to precisely determine smallchanges (unknown unknowns).

Time-to-failure experiments (which measureknown knowns) are mathematically equivalent,and less demanding on analytical precision.



Thermal design space for storage ofbiologics

Physical changes in state (freeze/thaw, thermalunfolding or “melting”) provide lower and upperboundaries of thermal design space for storageof biologics.

Inside the thermal design space, degradation isentirely due to chemical processes that followArrhenius kinetics.

Plot ln(time-to-failure) vs. reciprocal absolutetemperature get Arrhenius activation energy fromslope (Ea/R).


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But WHICH property should wemeasure?

Multiple critical-to-quality properties ofbiologics may beinfluenced bytemperature.



Multiple Dependent Variables

Biologics are difficult to characterize by asingle measurement technique.

Multiple assay methods are used tomeasure different properties.

Multiple properties may change over time asbiologics degrade.

Which is the most sensitive stability-indicatingmeasurement?


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Example: Analytical control strategiesfor antibody drug conjugates

Observed Apply to:Appearance and Description mAb/DS/DPpH mAb/DS/DPReconstitution Time (for Lyo) DP OnlyResidual Moisture (for Lyo) DP OnlyParticulate Matter (Subvisualparticles)

DP Only

Content uniformity DP OnlyProtein content (UV) mAb/DS/DPCharge profile (ICE) mAb/DS/DPImpurity profile (SE+HPLC) mAb/DS/DPImpurity profile (reduced CGEor SDS-CE)

mAb/DS/DP

Impurity profile (Non-reducedCGE or SDS-CE)

DS/DP

Impurities (free drug related) DS/DP

Observed Apply toImpurities: Residual host cellproteins (HCP ELISA)

mAb only

Impurities: Residual host cellDNA (cPCR DNA analysis)

mAb only

Impurities: Residual rProtein A(ELISA)

mAb only

Peptide mapping mAb onlyN-linked oligosaccharide profile(Glycan fingerprint)

mAb only

Drug loading & drug profile(HIC, UV or HPLC)

DS/DP

Osmolarity DS/DPBiological activity (BindingELISA)

mAb/DS/DP

Biological activity (Cytotoxicity) DS/DPSafety (Bioburden) mAb/DSSafety (Endotoxicn) mAb/DS/DPSafety (Sterility) DP only


Li H. Analytical characterization and controlstrategies for antibody drug conjugates. DIA CMCWorkshop, Washington DC, April 16, 2013.


Identifying CtQ parameters

Extensive data mining may be required toidentify parameters that change withtemperature and follow Arrhenius model.

Confirm by experiment!


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Stress degradation for Arrheniuskinetics

Use multiple temperatures—at least 3.

Attempt to achieve same extent of degradation ateach temperature by varying exposure time.

Assume an activation energy and calculate how muchlonger it will take to achieve the same fractionaldegradation at lower temperature based on results forhigher temperature.

High temperature should be below thermal “melting”temperature.



pH and buffer catalysis

Since most chemical degradation is sensitive to pHand buffer catalysis, experiments should usemore than one pH (at least 3, maybe more—typically ~2–3, ~4–5, ~6–7, ~8–9, ~12–13*)

*High pH typically used for cleaning equipment.

Use low buffer strength; check for buffer catalysisby increasing buffer concentration ×2, ×5.

Phosphate vs. citrate and same pH, e.g. 6.5.


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Oxidation

Test effect of antioxidants.

Radical blockers (esp. Met oxidation)

Important for mAbs.

Metal chelators (e.g., EDTA) can inhibit metal-catalyzed oxidation.

His, Cys Trp sensitive

Disulfide protectors (reducing agents)

UV light (esp. Trp)

polysorbate excipients accelerate.



Multifactorial stress degradationexperiments

Typical experiments combine multiplefactors:

Temperature (3 or more)

Solution pH (3 or more)

Buffer concentration (2 or more)

Excipients

Don’t try to cover all of factor space!

Use screening designs to identify key factors.


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Physical changes: reversibility

Changes in physical state (freezing/thawing,thermal “melting” often have partiallyreversible effects on stability.

Cycling experiments are one way to assessirreversible losses.

Expose material to high stress then low stressand take sample for analysis.

Repeat enough times (3? 5?); plot data & lookfor trend.



Cycling experiments

Freeze/thaw

Lyophilize/reconstitute

Thermal “melting” (e.g. Tm – 5°C to Tm +5°C)

High shear/low shear (or no shear)

How many cycles? Enough to show trend!


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Physical changes: thermal effects

Structural changes (denaturation, aggregation)also follow Arrhenius kinetics.

Ea for unfolding >> Ea for chemical degradation(typically ×3–×6)

• Abrupt changes in Arrhenius plot at design spaceboundaries.

• Excipients affect Tm. So does pH.

Important to confirm Tm for chemical degradationexperimental conditions!



Adsorption losses

Place biologic in container #1, equilibrate for fixedtime, take sample for assay and transferremainder to container #2.

Repeat through at least 5 containers.

Look for trend in loss of potency.

If nonadsorptive container is found, can use to testexposure to other materials by adding materialwith known surface area (coupons, balls).


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Physical stability experiments are alsomultifactorial

Do not attempt to cover factor space on firsttrial.

Use screening designs to identify mostimportant factors.

Avoid one-factor-at-a-time experiments; theycan be seriously misleading.



K.I.S.S.

Accelerated degradation experiments onbiologics can easily become overlycomplex.

Use screening experiments to zero in on themost critical-to-quality parameters thatlimit shelf life.

Focus on these CtQ parameters!


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Questions?

For more informationcontact:

William R. Porter, PhD

[email protected]




References

Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS.Stability of protein pharmaceuticals: an update. Pharm Res 27(4)544-575 (2010).

Chang BS, Yeung B. Physical stability of protein pharmaceuticals. In:Jameel F, Hershenson S, eds. Formulation and ProcessDevelopment Strategies for Manufacturing Biopharmaceuticals.John Wiley & Sons Inc., pp. 69–104(2010).

Porter WR. Stability by design. J Val Technol 17(3) 82-96 (2011).

Porter WR. Thermally accelerated degradation and storagetemperature design space for liquid products. J Val Technol 18(3)73-92 (2012).

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Understanding the chemistry:Solvolysis, oxidation/reduction, photolysis

Some books to read in your copious spare time… If (heaven forbid!) you have never taken a course in organic

chemistry…

• Winter A. Organic Chemistry 1 for Dummies. John Wiley & Sons(2005).

If you have forgotten organic chemistry…

• Smith MB, March J. March's Advanced Organic Chemistry :Reactions, Mechanisms, and Structure. John Wiley & Sons (2007).

If you have forgotten chemical kinetics and physical chemistry…

• Moore JW, Pearson RG. Kinetics and Mechanism. John Wiley &Sons (1981).



Understanding the chemistry:Solvolysis, oxidation/reduction, photolysis

Some books to read in your copious spare time… If you want to focus on stability of pharmaceuticals…

• Baertschi SW. Pharmaceutical Stress Testing: Predicting DrugDegradation. Taylor & Francis (2005).

• Yoshioka S, Stella VJ. Stability of Drugs and Dosage Forms. KluwerAcademic/Plenum Publishers (2000)

• Carstensen JT. Drug Stability Principles and Practices (2nd Ed).Marcel Dekker (1995)

• Zhou D, Porter WR, Zhang GZZ. “Drug Stability and DegradationStudies.” Chapter 5 in: Qiu Y, Chen Y, Zhang GGZ, Liu L, PorterWR. Developing Solid Oral Dosage Forms: Pharmaceutical Theoryand Practice. Elsevier/Academic Press (2009).

• Porter WR. “Residues and Cleaning Chemistry,” Chapter 9 in PlutaP. Cleaning and Cleaning Validation, Vol 1. PDA/Davis HealthcareInternational Publishing LLC (2009).


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CHEMICAL DEGRADATION OFPROTEINS

Further details for self-study…



Deamidation

Deamidation of Asn (and, toa lesser extent, Gln)residues in proteins andpeptides is the mostcommon degradationreaction observed. Asp ↔ iso-Asp

interconversion /isomerization goes throughsame intermediate.

Widely studied; majordegradation pathway inmAbs.


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Deamidation

Sensitive to sequence effects; Asn-Gly especiallyreactive.

V-shaped pH-degradation rate profile: minimumbetween pH 3 and 6.

Dominates effect of pH on protein/peptidedegradation.

Rate can be modified by conformational changes.

Nonaqueous solvents, addition of sugars, polyols.

Buffer catalysis: phosphate vs. citrate.



Racemization

Racemization and β-elimination occur through the same intermediate.

Most common at Asp.

At high temperatures, Cys is affected.

Usually very slow, and has been used as adating tool for archeological samples.

• Seldom important for limiting shelf-life.


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Proteolysis

Chemically induced by extreme pH.

At neutral pH due to contaminating enzymesor autolysis (if biologic is a proteolyticenzyme).

Autolysis is pH-sensitive and temperaturesensitive.

• Can be controlled by changing pH, storing at lowtemperature.

• Major problem for therapeutic enzymes.



Oxidation

Met, Cys, Trp, His, Tyr are main targets.

Metal-ion catalyzed oxidation important forCys, His.

Can involve peroxides.

Fe+3, Cu+2 major culprits.

• Source: leaching from corrosion of equipment,plumbing or from impure feed water is moreimportant than from added reagents.


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Disulfide exchange

Disulfide exchange results inchanges in the secondarystructure of proteins. Disulfide bonds help to stabilize the

folding of proteins containing onlyone polypeptide chain.

Disulfide bonds link togethermultiple polypeptide chains inproteins and peptides that havemore than one chain.

Scrambling → misfolding → loss of activity.



DKP (diketopiparazine) formation fromN-terminal amino acids

Proteins and peptideswith underivatizedN-terminal aminoacids canspontaneouslycyclize to form adiketopiparazineand shorter chainthat may havealtered activity.


O

R1

NH

NH2

R2

O

NH

R3

O

R4

O

R1

NH

NH

R2

O

NH2

R3

O

R4+

Diketopiperazine Shortened peptide

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Condensation reactions

Condensation reactions are the opposite ofproteolysis reactions.

Polypeptide chains can be linked together inunusual ways by reactions between freecarboxylic acids (Asp, Glu) and free alcohol(Ser) or thiol (Cys) groups, resulting in loss ofwater and a change in secondary structureand misfolding.

Possible reaction during lyophilization,exposure to dehydrating conditions.



pGlu (pyroglutamic acid) formation

pGlu (pyroglutamic acid)formation can occuranywhere there are free Gluresidues in a protein.

Results in changing 3-dimensional geometry ofpeptide chain.

Misfolding → change in activity.


O O

NHOH

NH

R2

R1

Glutamic Acid(Glu)

O

O

NH

N

R2

R1

pyroGlutamic Acid(pGlu) peptide

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Hinge region hydrolysis

Hinge regions (where peptide chain foldsback upon itself) are especially likelylocations for proteolysis to occur.

Result is clipping to form two chains.

• May result from enzymatic cleavage (e.g., pepsin).

May unfold or misfold or totally lose onechain.

• Reported degradation product for many mAbs.



Trp (tryptophan) hydrolysis → kynurenine

Trp (tryptophan) hydrolysis toform kynurenine undersome conditions.

This may also happen duringTrp oxidation.

• Has been observed indegradation of mAbs.


Tryptophan(Trp)

O

NH

NH2 NH

R2

R1

O

O

NH

NHNH

R2

R1

Kynurenine peptide

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Glycation

Glycation (Maillard reaction) occurs whenproteins containing free amino groups(e.g., Lys, N-terminal aa) are incubatedwith reducing sugars (e.g., glucose,lactose, fructose, maltose).

Can occur in solid state or in solution.

• A typical brown color is formed.

• Sucrose + H2O → glucose + fructose.


physical biochemistry, metrology, and accelerated - mbsw online

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