Microcalorimetry – versatile applications in biopharma

Natalia Markova, PhD

Challenge 1 - maintain protein stability and function to ensure efficacy and safety of the product.

Physical and chemical integrity of a protein can be affected in multiple ways:

Shear Stress Temperature fluctuationsAgitationHigh Pressure Filtration

OxidationUVpH and salt gradientsFreezing

SizeAggregationFragmentationHigh and low Mw variants

ImpuritiesProduct related impuritiesHost Cell ProteinsPrA leakageCell culture componentsDNAEndotoxinSubvisible particles

Charge heterogeneity and amino acid modificationsAcid forms: deamidated, sialylated and glycatedBasic forms: oxidated and C-terminal lysinevariantsIdentity

Amino acid sequence

Higher order structureSecondary and tertiary structureDisulfide bridgingFree thiolsThermodynamic stability

ActivityTotal concentrationActive concentrationTarget bindingFcR binding

Key aspects and quality attributes

StabilityShelf lifeDegradation profile

BioactivityTargetCDCADCCApoptosis...

GlycosylationGalactosylationSialylationMannosylationAfucosylation

Challenge 2 - determine and control criticalproperties and possible degradation pathwaysof a protein.

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Physical properties:Primary, secondary, ternary and quaternary structuresSolubilitySelf-associationViscosityMolecular weightHydrophobicityGlycosylationsCharge ...

Biological properties:Substarte or receptors affinityIn vitro activityFunction in vivo...

Multiple biophysical methods used for protein characterization › Mass spectrometry › Circular dichroism › Fourier transform infrared spectroscopy› Raman spectroscopy › X-ray crystallography › Nuclear magnetic resonance › Advanced detection SEC › Fluorescence › Light scattering› Differential scanning calorimetry › Isothermal titration calorimetry › Analytical ultracentrifugation › Microscopy Imaging› …..

Microcalorimetry in Life Science.Isothermal Titration and Differential Scanning Calorimeters (DSC and ITC)

Thermal stability of proteins at different conditions (buffers, excipients, adjuvants) Temperature is ramped Thermal denaturation of protein is monitored

A direct measurementof the heat generated or absorbed when molecules interact at constant temperature

ITC DSC

History of MicroCal™ products.Continuous development.

1977 2000

From 50 mg per run. At 5 ml cell volume.Difficult and touchy

The first commercially available

microcalorimeter.

From dozens of g per run. At 0.13 or 0.2 ml cell volume

Modern Malvern MicroCal systems

Advantages of calorimetric assays

Generic and directRobust-nearly any buffer can be usedMinimum assay development No molecular weight limitations Optical clarity does not effect outputsNon-distructive (for ITC)

Diversity of samples studied by calorimetry (ITC and DSC)

CytokinesHormonesPeptidesEnzymesAntibodies Antibody fragmentsAntibody-Drug conjugatesToxinsVaccinesAnticoagulantsFusion proteinsPEGylated proteinsVirus-like particles

How does ITC work?

Reference Cell Sample Cell

SyringeMonitors temperature difference between the sample and reference cells in a course of a titration experiment

Measures voltage across thermopile calibrated in units of thermal power cal/s Cell volume 200 L

Syringe volume 40 L

Compound – in syringeMacromolecule in ITC cell

ITC – Before titration

Ligand in syringeMacromolecule in cellMacromolecule-ligand complex

As the first injection is made, all injected compound is bound to target protein.

Titration begins: First injection

The signal returns to baseline before the next injection.

Return to baseline

As a second injection is made, again all injected compound becomes bound to the target.

Second injection

Signal again returns to baseline before next injection.

Second return to baseline

As the injections continue, the target becomes saturated with compound, so less binding occurs and the heat change starts to decrease.

Injections continue

As the injections continue, the target becomes saturated with compound, so less binding occurs and the heat change starts to decrease.

Injections continue

When the target is saturated with compound, no more binding occurs, and only heat of dilution is observed.

End of titration

Basics of ITC experiment

Integration of heats are used to extract affinity (KD), stoichiometry (N) and binding enthalpy ( H) using appropriate binding model

Universal technique based on heat detection

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Time ->

With isothermal titration calorimetry you can… › Get quick KDs

› Assess protein binding activity

› Confirm specific binding and stoichiometry

› Characterize mechanism of action

› Validate binding data from other assays

› Measure enzyme kinetics

› Novel applications continue to evolve

N=0.35

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Data: EnzSub1R6_RModel: M2 Substrate OnlyChi^2/DoF = 1.159E-9Kcat

A1.86 ±0.057

KmA

4.08 ±0.26H -7000R

ate

(mill

imol

es/l/

sec)

[S] (mM)

More interactions with MicroCal ITC

Kamps, et al, Nature Commun, 6, 8911 (2015)

ITC titrations of 10-mer H3K4me3, H3C4me3 and H3G4 histone peptides binding to the TAF3 PHD domain. N=1 for each binding

KD 790 nM KD 36 MKD 24 nM

Molar Ratio

Kcal

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Protein Quality

Measure active concentrations

Compare protein batches

Fully Active

50%“Fully Active”

Partially Active

Measuring Bioactivity with ITC: Affinity and Stoichiometry

ITC in biotheurapetic drug discovery-Charaterization of multi-specific antibodies.

R.Castoldi, U.Jucknischke, L.P.Pradel, E.Arnold, C.Klein, S.Scheiblich, G.Niederfellner and C.Sustmann(Roche Diagnostics, Germany). PEDS 25, 551–559, 2012

Molecular characterization of novel trispecific ErbB-cMet-IGF1R antibodies and their antigen-binding properties

ITC confirms binding to all three epitopes of tri-specific antibody.

Top panel: soluble receptors titrated into a solution of their corresponding parental mAb in three independent experiments. Bottom panel: the three receptors titrated one after the other into the same solution of TriMab2

Conclusion: ITC showed all 3 receptors could simultaneously bind the tri-specific antibody

Protein quaternary structure by ITC. Self-association of protein X studied by ITC.

0.00 10.00 20.00 30.00 40.00 50.00 60.004.70

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5 mg/ml PrX in PBS 7 mg/ml PrX in SEC 3 mg/ml PrX in Pi

Time (min)

μcal

/sec

Overlays of ITC titrations into and Pi, PBS and high ionic strength phosphate buffers

Protein has propensity to oligomerize. From ITC data supported by SEC -MALS dimerization KD is in the range of 30 – 70

M.

[NaCl], Temperature

PEAQ-ITC system enables fast and detailed characterisation of interactions involving multiple classes of binding sites

The PEAQ-ITC system enables fast and detailed characterisation of interactions between biomolecules involving multiple classes of binding sites. The instrument and software improvements compared to earlier ITC systems significantly increase the throughput - including data evaluation. Furthermore the low volume requirement broadens the applicability of ITC as an orthogonal technique in relation to other interaction techniques.

Anders Dybdal Nielsen, PhDSenior Research ScientistNovo Nordisk A/S

Differential Scanning Calorimetry, DSC

• Thermal stability of proteins at different conditions (buffers, excipients, adjuvants) • Temperature is ramped • Thermal denaturation of

protein is monitored

Protein unfolding

Protein unfolding

Protein unfolding

Protein unfolding

Protein unfolding

Protein unfolding

Protein unfolding

Protein unfolding

Native

Mutant

Phosphorylated

Complexed

Temperature (°C)

Cp

(kJ K

-1m

ol-1

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Compare native, altered and mutant formsMicroCal™ DSC system the universal stability monitor

Tm shift reflects increased stability

Tm and heat of unfolding ( Hcal) in one scan

DSC basics. Analysis of irreversible thermal protein unfolding

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Onset ofunfolding

Tm’s ofminor peaks

Tm ofmain peak

(monoclonal antibody)

› Shifts in Tonset (°C):Higher temperature = more stableLower temperature = less stable

› Shifts in Tm values (°C):Higher temperature = more stableLower temperature = less stable

› Changes in T1/2 values (transition width,°C):Smaller width = more cooperative unfolding usually associated with a compact structureLarger width – less cooperative usually associated with a relaxed, partially unfolded structure

› Lowering of “ H”, area under the curve:Indicates that it takes less energy to unfold the protein due to destabilization (works only if protein concentration values are well known)

Comparing DSC data in the context of irreversible unfolding

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Direct nature of DSC readout allows to address protein complexity Individual domain stabilizationMultiple descriptors of protein stabilityDSC thermogram as a fingerprint,e.g. for biocomparability

Versatile application sof DSC in biopharmaD

iffer

entia

l Sca

nnin

g C

alor

imet

ryProtein engineering

Protein characterization

Process development and manufacturing support

(Pre)Formulation Development

Higher Order Structure Analysis

DSC results can be predictive of relative long-term storage stability

DSC thermograms are used in comparability protocols (comparability of tertiary structure and stability)

DSC thermograms are often included in the characterization package of a CMC section

MicroCal VP Capillary DSC

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1) Sample preparation (96-well plate)

2) Input parameters into software 3) Automated sample measurement

4) Automated sample analysis

) AAuuttomated sample measurement

Multiple Tms. DSC allows to address protein stability on domain level. Protein engineering guided by DSC.

Minor differences in primary sequence can have a big impact on antibody stabilityStability of each domain can be assessedThe least stable Fab variant expressed poorly and quickly formed high MW aggregates. Most stable antibody construct identified based on Tm of the Fab domain.

Demarest et al, Malvern Application note

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Tm3 = 82.0 °C

Tm2 = 73.3 °C

Tm1 = 60.4 °C

The use of DSC to enhance drugability of engineered monoclonal antibodies. Monitoring construct stability,›Use DSC to track stability changes of individal domains

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B: Engineered Protein 1

C: Engineered Protein 2

Park, Malvern Application Note

A: Parent Protein

TM3

TM4

TM1

TM2

TM1

TM1

DSC predictions correlate with industry standard

Eliminate candidates with potential long term stability issues

›DSC data correlates well with SEC-HPLC (accelerated stability studies)

Protein with lowest Tm had greatest aggregation formation

›Early eliminate constructs with potential long-term stability issues

Park, MicroCal Application Note (2008)

How does DSC compare?

›DSC was the most accurate and fastest predictor of suitable formulations

Size ExclusionChromatography

Laser Light Scattering

IsoaspartateFormation CE

DSC Tm

Black: T=0Grey: 2 weeks at 40 CRed: 4 weeks at 40 C

Ollila, MicroCal Application Note (2004)

Results from a pH primary screen of a therapeutic antibody

Tm. Sensitivity of DSC to changes in stability arising from a common chemical degradation pathway, oxidation

Arthur et al. J. Pharm. Sci 104:1548–1554, 2015

Malvern webinar by J. Gabrielson .

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BPase 8.3uM=0.23mg/ml BB293in 20mM TBS + 200mM L-alaninepH 8.0

Data: BpaseTBSLa_cpModel: MN2StateChi^2 = 8813.30Tm1 17.85 ±0.1932

H1 2.19E3 ±181Hv1 1.13E5 ±1.06E4

Tm2 30.40 ±0.6202H2 1.41E4 ±2.8E3Hv2 8.04E4 ±4.94E3

Tm3 34.43 ±0.0458H3 4.01E4 ±2.68E3Hv3 1.25E5 ±2.92E3

Cp

(kca

l/mol

e/o C

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Temperature (oC)

onset at 12ºC

Tm=34.5ºC

DSC: 10ºC upward shift of the low-temperature transition stabilizes Protein X kinetically.

Tonset. Shift in onset of thermal unfolding correlates withprotein stability and aggregation propensity. Case study 3.

Protein X Construct 1

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ca 0.22 mg/ml BPase BB384in 20 mM TBS, pH 8.00.3% DMSO

Data: BB384TBS05_cpModel: MN2StateChi^2 = 7482.82Tm1 23.73 ±0.0840

H1 877 ±93.6Hv1 4E5 ±5.32E4

Tm2 34.30 ±0.1938H2 1.14E4 ±932Hv2 9.88E4 ±4.06E3

Tm3 40.10 ±0.0854H3 2.64E4 ±939Hv3 1E5 ±2.15E3

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Temperature (oC)

onset at 22ºC

Tm=40ºC

Protein X Construct 2

SEC: Protein X homogeneity and stability to aggregation has dramatically increased

SEC

ppppppppppppp (((((((((( )))))))))

SEC

Construct 1 Construct 2

T1/2 and Tm. Multiple metrics of protein thermal stability make buffer optimization /preformulation funnel more efficient. Case study 4.

Rank by Tm

Focus with T1/2

Katherine E. Bowers, Malvern Application Note

12 selected

5 focused on

19 formulations tested

Hcal. Area under DSC curve is an indicator of the content of folded protein material. DSC can be used to assess quality of recombinant proteins. Case study 5.

Malvern Application Note

Hcal/ HvH ratio as indicator of size of cooperative unit for thermal unfolding

› HvH = Hcal – cooperative unit and molecular weight are the same: largely reversible unfolding of one single domain

› HvH < Hcal - cooperative unit is smaller than molecular weight: intermediates

› HvH > Hcal – cooperative unit is bigger than the molecular weight: oligomers or overestimated concentration of folded protein

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Data: Data2_cpModel: MN2StateChi^2/DoF = 99.35Tm 69.15 ±0.021H 3376 ±17.2Hv 1.014E5 ±640

mgst2

Cp

(cal

/o C)

Temperature (Deg. C)

Ratio of calorimetric to van’t Hoff enthalpy H/ HvH, can be indicative of protein oligomerization state. Case study 7.

Ratio of van’t Hoff to calorimetric enthalpy ≈3 indicates that protein unfolds as an oligomer (possibly trimer).

cates that protein unfoldsmer).

…”The high throughput is supported by the analysis

software, which is easy to use and requires no more manual

calculations therefore saving us hours. These time savings have really improved our workflow.”

Dr. Katherine BowersFujifilm Diosynth Biotechnologies- A CRO

MicroCal DSC: The Gold Standard

The Gold Standard in Biopharma

› HOS consortium publishes the ‘value’ of DSC

Gabrielson and Weiss IV, J. Pharm.Sci. 104:1240–1245, 2015

DSC is very or extremely useful in manybiopharma applications

Lot 3

Lot 2

Lot 1

Reference

DSC readout is directly related to protein concentration and conformational state. DSC thermogram is a fingerprint for assessment of conformational equivalence and Higher Order Structure.

›Three lots manufactured at different sites

DSC demonstrates comparability of higher order structure (HOS) for material from different manufacturing processes or manufacturing sites

Malvern Application Note

Assessing modifications in HOS in stressed mAbX samples

In collaboration with Roche, Basel

pH-stress

Met255 oxidized (Fc-CH2)Met431 oxidized (Fc-CH3)

Significant difference in hydrophobic and charge profiles, dimer and oligomer formation

pH 9.0, 37 deg C 7 days

Asn #1 deamidated (Fab-LC, CDR)Asn #2 deamidated (Fab-HC, CDR)Asp isomerized (Fab-LC, CDR)Asn387/392/393 deamidated (Fc-CH3)

Light-stressed

pH-stressed

Forced-oxidized

DSC in chatacterization of protein samples from stress-stability studies: Modifications in stressed mAbXvariants

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Forced-oxidizedTm1(Fc-CH2)

Tm2(Fab)

Wild-type

SampleT1/2, deg C

Tonset, deg C

Tm1, deg C

Tm2, deg C

Wild-type 4,2 64,4 71,2 81,8forced-oxidized 4,2 59,7 69,1 81,9

Decreased thermal stability of CH2 domain offorced-oxidized mAbX detected with DSC

Destabilization of CH2 domain of light-stressed mAbX detected with DSC

Tm2(Fab)

Wild-type

Light-stressedTm1(Fc-CH2)

SampleT1/2, deg C

Tonset, deg C

Tm1, deg C

Tm2, deg C

Wild-type 4,2 64,4 71,2 81,8UV-light stressed 4,4 61,3 70,7 81,9Light-stressed

DSC is the only direct thermal stability assay. Rest of the techniques are phenomenological, i.e. based on observations of secondary effects

Hydrophobic surface exposure

Extent & energeticsof thermal unfolding

We routinely use DSC as the ‘gold standard’ forprotein stability studies and encourage ourresearchers to determine melting points with DSCfirst before using alternative techniques such as CDor Thermofluor. The wide temperature rangeensures that we can see all the transitions in asample and decide which techniques will beappropriate.

Dr. David StauntonFacility ManagerBiochemistry Department’s Molecular Biophysics Suite

DSC as the ‘gold standard’ for protein stability studies

DoE: Impact of extrinsic factors on mAb1 thermal stability scored differently by different techniques

DSF identifies NaCl as VIP factor. Salt might be affecting hydrophobicity of a protein and affecting protein-dye binding.DSF does not relaibly assess effect of Tween 20 on mAb1.

DSC traces are fingerprints of protein HOS. DSC ensured highly reproducible thermal unfolding profiles.

DSF DSC

DSC gives means to assess conformational equivalence/similarity of protein samples

Irreversibility of protein denaturation

N U INative Reversibly

UnfoldedIrreversiblyDenatured

k2

Thermodynamics/EnergeticsFormulations/Functional Stability

K

DSC offers means to assess and optimize kinetic stability of proteins

Tm dependence on the DSC scan rate suggeststhat the denaturation process is kinetically-determined

Scan rate dependence experiments performed on two mAb batches with different glycosylation profiles. VP-capillary DSC scans were collected on 1 mg/mL mAb batch (A) and (B) at multiple scan rates (10, 35, 60, 120, 180, 240ºC per hour).

Morar-Mitrica S. et al. Drug Development. BioPharma Asia. July/August 2013, pp 46-55.

Presence of different oligosaccharides does not affect the thermodynamic or kinetic stability of this mAb.

Tm dependence on the DSC scan rate suggests that the denaturation process is kinetically-determined.The trends in Tm variation with scan rate were found to be equivalent between batches.DSC provided evidence of batch-to-batch comparability.

Morar-Mitrica S. et al. Drug Development. BioPharma Asia. July/August 2013, pp 46-55.

Mode of action

ConcentrationSimilarity of HOS

SolubilityStructure

ActivityBinding to interaction partners

ITC and DSC in chracterization of protein stability and interactions

Stability

DSCITC

DSC

ITC

DSC

DSCITC

Manufacturability DSC

ITC

BindingITC

DSC

DSC in the studies of liposomal DDS, vaccines, viruses and biofluids

Characterization of liposomal preparations with DSC

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Images from wikipedia

Biltonen et al. Chemistry and Physics of Lipids, 64 (1993) 129-142B. Stark et al. European Journal of Pharmaceutical Sciences 41 (2010) 546–555

Fig. 3. Excess heat capacity curves lyophilised/rehydrated liposomes mixed with glucose (2), lactose (3), trehalose (4) and mannitol (5) in a lipid to carbohydrate molar ratio of 1:10.

Fig. 2. Dependence of the transition temperature (Tm) of DPPC unilamellar vesicles on vesicle diameter.Fig. 1. DSC thermogram of DPPC MLVs

Characterization of liposomal drug delivery systems with DSC

Images from wikipedia

Fig. Normalized DSC scans of DPPC bilayers containing losartan (antagonist of AT1 receptor) at different concentrations

B. Stark et al. European Journal of Pharmaceutical Sciences 41 (2010) 546–555

DSC can be used tocontrol overall stabilityof Liposomal DDS

Viruses and VLP in DSC. MDa-size complex samples.

Fig. 1. Differential scanning calorimetry analyses of three different strains of purified active influenza virus. Krell et al. Biotechnol Appl Biochem (2005) 41: 241–246.

Fig. 2. pH-dependence of the thermal unfolding of type I poliovirus ofstrain Mahoney. Krell et al. Biotechnol. Appl. Biochem. (2005) 41, 241–246

“The precision in the determination of the Tm va-lues makes a DSC analysis a very suitable tool to follow the vaccine production process and to assess lot to lot consistency.”

DSC in application to blood plasma samples

Relevance:Extend the use of DSC to more complex samplesInvestigate feasibility to use the profiles for diagnostic purposes

Why DSC:Heat is an extrinsic propertyAllows studies of complex compositionsAccounts also for intermolecular interactions - the “plasma interactome”

Differentiation of diseases by DSC

Reproducibility of experimental serum thermograms from a healthy subject (A) and a diseased patient (B). Two non-consecutive scans with sample refilling were performed for each individual.

DSC thermograms are highly reproducible and serve as fingerprint.

MicroCal DSC in studies of biofluids.

› Comments from Adrian Velazquez-CampoyInstitute BIFI-University of Zaragoza (Spain)

Especially appropriate for running unattended largesets of samples and for minimizing human/accidentalmistakes.Characterized by a remarkable scan reproducibility andan impressive sensitivity for reducing sample usage.Straightforward to program with the control software.Easy to maintain and keep in shape by programmingperiodic cleaning/control scans.

Thank you