Malin Suurkuusk, PhD TA Instruments, Sweden · • For the study of solid state drug stability • For the characterization of the thermal stability of proteins and other macromolecules

Isothermal Microcalorimetry for Pharmaceutical Stability Assessment

Malin Suurkuusk, PhDTA Instruments, Sweden

SOS 2019 Amsterdam

TAINSTRUMENTS.COMTAINSTRUMENTS.COM

Calorimetry – a universal technique

Virtually all chemical and physical processes result in either heat production or heat consumption.

A heat flow calorimeter measures heat flow

dQ/dt

Heat flow is directly related to the heat production (or consumption) rate in a sample

P

P and dQ/dt is measured in

W = J/s

A microcalorimeter is a calorimeter that can measure heat production in the µW range


Microcalorimetric techniques and applications

Ampoule

calorimetry

Perfusion

calorimetry

Solution

Calorimetry

Stability Compatibility Amorphicity

Interactions in

solution

Effect of

atmosphere

Air/N2 and/or RH

Polymorphism


Calorimetric techniques

Differential Scanning Calorimetry –DSC

• For the study of solid state drug stability

• For the characterization of the thermal stability of proteins and othermacromolecules in solution

Isothermal Microcalorimetry – IMC

For stability, compatibility, safety assessment, microorganism growthstudies, curing, sorption, interactions, polymorphism, amorphicity, metabolismetc.


Scanning and Isothermal Calorimetry

A (s) →B

Unstable System

dQ/dt 0 W at T= const

Kinetic:

Thermodynamic:

Stable System

dQ/dt = 0W at T=const


Isothermal microcalorimetry

An isothermal microcalorimeter measures heat produced or

consumed during a physical process or a chemical reaction

in terms of heat flow at a constant temperature with

microwatt sensitivity or better.


Microcalorimetry with and without humidity


Benefits of using IMC for stability assessment

• Highly sensitive – no need for accelerated conditions

• Can detect both chemical and physical changes.

Physical changes can be related to

◆ Polymorphism

◆ Amorphicity

◆ Hydrate- and Solvate-state.

• Non-specific

• Non-destructive and

real-time data


Decomposition kinetics - ASA

Decomposition of ASA at 25, 30 and 35C.

Upper graph: Isothermal heat flow vs time

curves: data as obtained from the

calorimeter.

Degree of conversion:

𝛼 = 𝑐𝑢𝑚𝑚𝑢𝑙𝑎𝑡𝑖𝑣𝑒 ℎ𝑒𝑎𝑡 /Δ𝑟𝐻


Ampoule calorimetry and stability

Otsuka T., Yoshioka S., Aso Y. and Terao T.,

Chem. Pharm. Bull., 42(1) 1994


Willson, R.J., A.E. Beezer, J.C. Mitchell, and W. Loh, Determination of thermodynamic and kinetic parameters from

isothermal heat conduction microcalorimetry: applications to long-term-reaction studies. J. Phys. Chem., 1995.

99(7108-7113).

Fitting kinetics models to calorimetric data

For a single component reaction:

• Zero order kinetics – Heat flow is constant as a function of time. Rate constant from P=DH · k · [A]

• First order kinetics – Ln(Heat flow) is linear. Rate constant from the slope

• Second order kinetics – (Heat flow)-0.5 is linear. Rate constant from the slope


Stability and compatibility tests

•Stress tests can be performed for screening with increased temperature

and/or relative humidity

•Can be performed both qualitatively (Yes/No) and quantitatively

(understanding the kinetics of the process)

•On the API alone and in combinations with excipients – Compatibility

•On the formulated product

•With packaging material


Primary screen of stability

Measure heat

flow over time of a

known amount of

API

Assume stable

Assume unstable

No detectable

heat flow

Measurable heat flow

Discard as API

Conduct a quantitative

analysis to determine

the rate constant

Flow chart redrawn from Pharmaceutical Isothermal calorimetry by S. Gaisford &

M.A.A O´Neil. Concept from Pikal, Themometric Application Note 335


Primary screen of binary mixture

Measure heat flow

of API

Assume compatible

Assume

unstable/incompatible

Difference in data

Discard API/excipient

combination

Conduct a quantitative analysis

to understand the interaction

Measure heat flow

of excipient

Calculate

theoretical heat flow

Measure actual

heat flow of mixture

Compare the

theoretical and

measured heat flow

No difference

in data

Flow chart redrawn from Pharmaceutical Isothermal

calorimetry by S. Gaisford & M.A.A O´Neil. Concept

from Pikal, Themometric Application Note 335


Compatibility Experiment with TAM

Phipps, M.A.; Mackin, L.A. Pharm. Sci. Tech. Today, 3(1), 9-17 (2000)

TAM Temperature: 50 °C

Relative Humidity: 75%


Hydrate formation in Ethinyl Estradiol

0 20 40 60 80 100 1200

200

400

600

P/m

/uW

g-1

Time /h

-5 0 5 10 15 20 25 30-200

0

200

400

600

P/m

/uW

g-1

q/m /J g-1

Measuring

temperature: 45ºC

•Blue trace: 100 %RH

•Red trace: 95 % RH

•Green trace: 88 %RH

)/()( HqfTkP D=

Rate equation:

Peter Vikegard, J&J – Cilag Ag, Switzerland


Avrami’s model

-5 0 5 10 15 20 25 30-200

0

200

400

600

P/m

/uW

g-1

q/m /J g-1

82 84 86 88 90 92 94 96 98 1000

1

2

3

4

5

x 10-4

Avr

am

i ra

te c

onsta

nt

Relative humidity (%)

•Blue trace: experimental

data

•Black trace: fitting

equation (k=0.0005 s-1)

•Rate constant as a

function of relative

humidity

RHcritical

2/1)]/1ln([)/1(2)/(

)/()(

HqHqHqf

HqfTkP

D−−D−=D

D=


Summary stability and compatibility

• Multichannel capability for parallel analysis

• High sensitivity gives the ability to detect instability and

interactions at or close to ambient temperatures

• Samples are reusable if no reaction is detected

• Ability to expose the sample to relative humidity

• Experiments may be long, but not as long as accelerated

aging studies.


BIOPHARMACEUTICAL STABILITY


Biopharmaceutical Stability Assessment

Proteins, mAbs, as pharmaceuticals is the fastest growing

field in the biopharmaceutical industry.

• Usually requires high concentrations (100 mg/mL or more)

• High concentration Mab formulations concerns

▪ Denaturation

▪ Aggregation

Rapidly determine the best buffer and excipient

conditions that maximize stability and minimize

protein aggregation for at least one year at

required high concentrations


Stability method challenge

•Traditional methods for the characterization and analysis of protein stability

and/or aggregation often require other physical or chemical conditions than

those in a formulation and therefore provide little or no predictive power,

necessitating long incubation times for shelf life determination.

•Methods for structural stability and aggregation include DSC, DSF, ICD, SEC

and light scattering. These methods will require dilution of formulations and

long term incubations prior to analysis

•Desired to find a methodology to be used on the actual formulation that will

give fast and accurate stability data from the rates of denaturation/aggregation


Protein denaturation temperature as a stability indicator

•Excipient to stabilize against chemical and physical degradation

•Choice of an additive or a formulation is generally determined empirically

•DSC is the fastest way of evaluating

additives effect on Tm, reversibility

•Techniques for studying denaturation

and not aggregation:

▪pH far from isoelectric point

▪Dilute solutions

▪Reversibility

Olsen et al., Thermochimica Acta, 484, (2009), 32-37

Native Denatured Irreversibly

denatured


Denaturation

Precedes

Aggregation

Denaturation,

Aggregation Occur

Simultaneously

ku kagg

ku >> kagg

ku << kagg

k

-100

-50

0

50

100

150

50 55 60 65 70 75 80

Cp

Temp deg C

mAb5

HEWL

Two Different Mechanisms of Protein Denaturation/Aggregation

Schön, A. et al. “ Temperature Stability of Proteins: Analysis of Irreversible

Denaturation Using Isothermal Calorimetry” Proteins. (2017)


DSC of the Irreversible Denaturation of HEWL

Schön, A. et al. “ Temperature Stability of Proteins: Analysis of Irreversible

Denaturation Using Isothermal Calorimetry” Proteins. (2017)


-40

-20

0

20

40

60

0 1 2 3 4 5 6

pH 9.0 59deg CH3pH 9.0 59deg CH5pH 9.0 58deg CH3pH 9.0 58deg CH5pH 9.0 57deg CH3pH 9.0 57deg CH5

dQ

/dt k

ca

l/D

ay/m

ol

Time (Days)

IMC of HEWL pH 9: Endotherm Occurs Before Exotherm

Denaturation Precedes

Aggregation

Endotherm

Exotherm

IMC of the two events HEWL denaturation


Non-linear Least Squares Fit of HEWL pH 9 Heat Flow

Heat Flow is Proportional to the Amount of

Denatured and Aggregated Protein Formed

59 °C

58 °C

57 °C𝑑𝑄/𝑑𝑡= 𝑘𝑢∆𝐻𝑢𝑒

−𝑘𝑢𝑡 + 𝑘𝑎𝑔𝑔𝑄𝑎𝑔𝑔𝑒−𝑘𝑎𝑔𝑔𝑡

+ 𝑘𝑢𝑄𝑎𝑔𝑔𝑒−𝑘𝑢𝑡 − ሺ𝑘𝑢

+ 𝑘𝑎𝑔𝑔)𝑄𝑎𝑔𝑔𝑒−ሺ𝑘𝑢+ 𝑘𝑎𝑔𝑔)𝑡


Results of IMC Characterization of Proteins

This data can be plotted according

to the Arrhenius equation (lnk vs 1/T)

Extrapolating to room temperature

will give a rate of 0.00285 days-1 at

25 °C which corresponds to about

350 days.


Long term stability of HIV-1 neutralizing mAb

DSC

IMC

SEC after

12 weeks

at 25 C

B.R. Clarkson et al. Long term stability of a HIV-1 neutralizing monoclonal

antibody using isothermal calorimetry, Analytical Biochemistry (2018)


Results

Calculated from results at 60 C




Comparing SEC with IMC

Correlation between %mAb

aggregates measured by

SEC (ACQUITY UPLC)

after 10 weeks’ incubation

at 25°C and the

denaturation/aggregation

rates measured by TAM,

10 day test.

D

A

Also correlated with 5⁰C data at 0.98 & 40 ⁰C data at 0.86




IMC stability measurements of biopharmaceuticals

•Can detect denaturation/aggregation kinetics at temperatures below the Tm

•Accurate stability assessment in 3-5 days, compared to weeks for storage tests

•Provides sensitivity to detect heat signal in small volume (<1 mL), high protein concentrations (>100 mg/mL)

•Provides adequate sample throughput (1-48 samples) to assess multiple buffers and excipient conditions simultaneously

•IMC can much quicker then conventional methods identify mAbs and their formulations that have best long term stability

•In addition, methods as SEC, AUC and DLS measure the existence of aggregates after storage, while IMC measures rates of aggregation andtherefore have the potential to predict time of storage

Thanks!

Documents

Malin Suurkuusk, PhD TA Instruments, Sweden · • For the study of solid state drug stability • For the characterization of the thermal stability of proteins and other macromolecules