DSC Case Study: Pfizer ADC mimics...

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DSC Case Study:

Pfizer ADC mimics application

Colette Quinn, Henry Shion, Weibin Chen


Antibody Structure, Regions

�Antibodies demonstrate a strong structure-function relationship. Because of this relationship, the unfolding or denaturation temperature measured by DSC is associated with functional differences.


Information from DSC

�Heat capacity change (∆∆∆∆Cp)

� Typically ∆Cp is positive.

� Increase in flexibility, degeneracy,

� Hydrophobic exposure

�Enthalpy

�∆ H: e2 non-covalent interactions –

hydrophobic & electrostatic, H+ bonding

�Tm

� macromolecular stability

Entropically Favorable

Enthalpically Favorable

�Folding and unfolding of a small protein, a domain, or a subunit, is ‘cooperative’ (once started, it goes to completion).


Antibody Thermograms


Antibody Drug Conjugates

https://circa71.wordpress.com/2009/03/16/lion-sidecar-wall-of-death/

2016 –2 licensed ADCs brentuximab vedotin(Adcetris) and ado-trastuzumab emtansine(Kadcyla) on market

Global market for antibody drug conjugates was valued at $1.3 billion in 2016 with +20% growth until 2021 -BBC Research, pub in ADC Review

In 2015 there were 17 novel ADCs entering clinical testing for the first time, with a similar trend identified in 2016.

Beacon, Hanson Wade


Antibody Drug Conjugates

Pharma interest surges in antibody drug conjugatesWebb, S. Nature Biotechnology 29, 297–298 (2011)

Figure 1: Structure of trastuzumab emtansine (T-DM1). Discovery Medicine, A. Beck et al., Discov. Med. 10 (53), 329–339 (2010).


Conjugation Type-1 ADC Series, 0.5 mg/mL

Type-1 SERIES AVERAGES FWHM Tmax Tonset Tmax-Tonset ∆Hcal

% Enthalpy Compared to

Native

Type-1 Nat 5.1 76.7 69.0 7.7 4281

Type-1 Low 4.8 76.5 69.2 7.3 3321 78

Type-1 Mod 4.9 76.4 69.9 6.5 3461 81

Type-1 High 4.5 76.2 70.5 5.7 2941 69

Type-1 High Baseline Substracted (KJ/mol·K) Type-1 Low Baseline Substracted (KJ/mol·K)

Type-1 Mode Baseline Substracted (KJ/mol·K) Type-1 Native Baseline Substracted (KJ/mol·K)


Native

Mod High

Low

Fitted Conjugation Type-1 Data


Comments on Fitted Conjugation Type-1 Data

%Q1 %Q2+3 %Q4

Native 0 88 12

Low 2 90 8

Mod 4 89 7

High 9 77 15

� Deconvolution of the data and subsequent fitting with the pseudo van’t Hoff model allows assignment of percentage of heat, Q, coming from each feature(Q = ∆HVH*Aw).

� Normalizing the heat allows a comparison across the series.

� The majority of the heat originates from the middle temperature transition.

� The low temperature event (Q1) increases from non-existent at Native to 9% at high.

� The magnitude of heat values tend to trend with the quantity therefore it is reasonable to assume that the population of the this less stable species increased by 9%.

� The increase in Q1 is linear.


High

Fitted Conjugation Type-1 Data, relation to antibody regions

Q4, CH3

Q3, Fab

Q2, CH2

Q1, CH2?


Comments on Conjugation Type-1 Data

� Tmax decreased from Native to High� Native form is more stable than the modified forms. � > 0.3 ⁰C is significant.

� The width of peak (FWHM) decreases from Native to High� The increase is also observed in a lower Tonset and a

slightly larger Tmax.� The modified forms have a low temperature peak that is

absent in the Native form. The peak’s magnitude increases from low to high.

� Enthalpy decreased from Native to High.� The enthalpy comes from the direct breaking of

hydrogen bonds or electrostatic interactions. Either these bonds are not formed in the modified forms or a smaller percent of the correctly folded molecule remains after modification.


Multiple Platform Analysis: LC/UV Hydrophobic

Interaction Chemistry (HIC) and LC/LC/QTOF-MS

“A rapid on-line method for mass spectrometric confirmation of a cysteine-conjugated antibody-drug-conjugate structure using multidimensional chromatography” mAbs Volume 7, 2015 - Issue 6 Birdsall, R.; Shion, H.; Kotch, F.; Wu, A.; Porter, T.; Chen, W.

HIC UV


Conjugation Type-2 Series

Type-2 SERIES

AVG FWHM Peak 1 Tmax1 %Area peak 1 FWHM Peak 2 Tonset2 Tmax2 ΔHcal (kJ/mol) Tmax2-Tonset2

Type-2 Nat 6.7 71.8 21 4.8 75.4 79.9 3989 4.5

Type-2 Low 7.0 72.0 15 4.8 75.5 80.0 3370 4.6

Type-2 Mod(1) - - - 4.7 74.9 79.9 2811 5.0

Type-2 High(1) - - - 4.9 74.3 79.6 2728 5.3

Peak 1

Peak 2

Type-2 High Baseline Substracted (KJ/mol·K) Type-2 Low Baseline Substracted (KJ/mol·K)

Type-2 Mode Baseline Substracted (KJ/mol·K) Type-2 Native Baseline Substracted (KJ/mol·K)


LYS Series

NativeLow

Mod High

Fitted Conjugation Type-2 Data


Comments on Fitted Conjugation Type-2 Data

%Q1 %Q2 %Q3

Native 21 76 4

Low 15 82 3

Mod 0 98 2

High 0 97 3

� The majority of the heat originates from Q2 with an increase the %Q2 as the extent of modification increased.

� This increase was concurrent with an increase in the width which could indicate that the extent of Conjugation Type-2 modification actually stabilized this population.

� For certain antibodies the initial peak can indicate flexibility of the hinge region. It is possible that this flexibility decreases as the modification increases.


Comments on Conjugation Type-2 Data

� Tmax decreased from Native to High is insignificant at 0.3 ⁰ C.

� The majority of the species are similar in stability.

� The width of peak increases from Native to High� his could indicate a greater level of homogeneity in the

Native sample or it could come from the stability of the low temperature species increasing and overlapping with the stability of the middle species.

� The first integratable peak on the Native form is absent in the modified forms and these lower stability species are even less stable for the modified forms with an integratable peak than increases from low to high.

� Enthalpy decreased from Native to High.� The enthalpy comes from the direct breaking of hydrogen

bonds or electrostatic interactions. Either these bonds are not formed in the modified forms or a smaller percent of the correctly folded molecule remains after modification.


ADC Recap

Type 1 and Type 2 have different trends with increasing saturation levels.

Deconvolution and simple heat (Q) assignment describe the systems.

DSC for QA and QC


Thank You

The World Leader in Thermal Analysis, Rheology, and Microcalorimetry


Denaturation

Unfolding describes the natively folded protein.

DenaturationChange in solubilityLoss of Compactness (increased viscosity)Disorder in conformation (decreased ellipticity)Increase in H+ exchange rate

Study denaturation and not aggregation1. pH far form isoelectric point2. Dilute solutions3. Reversibility


Gaussian Model

Max

½ Max

�Folding and unfolding of a small protein, a domain, or a subunit, is ‘cooperative’ (once started, it goes to completion).


What does a DSC scan tell us?

�If the native (N) and unfolded (U) state are in equilibrium �Assume Two-state

N U

�At any T:�Keq = [U]/[N]�∆G = -RTlnKeq

�∆G = ∆H – T∆S

�At Tm, ½ unfolded, ∆G = 0 then ∆S = ∆H/Tm and [N] = [U]

�The measured values, are used to calculate entropy (∆S; a measure of molecular disorder) and free energy (∆G).

� Free energy describes the overall stability of the system.

� Positive ∆G: the folded protein is more stable than the unfolded protein.


• Practical implications: protein with lower heat capacity is more rigid, more selective when binding a ligand. Protein with higher heat capacity is more flexible, less selective.

• Unfolding of a protein is dependent on the non-covalent intramolecular bonds such as hydrophobic interactions, hydrogen bonds, salt bridges, and conformational entropy. When the temperature is raised, as in a DSC scan, a protein unfolds and the sum of all of the enthalpy changes are measured and referred to as the ∆Hcalorimeter value (∆Hcal). Overall, the protein unfolding event is endothermic as the contribution from breaking hydrogen bonds out-weighs the contribution of the exothermic events such as the disruption of hydrophobic interactions. The unfolding transition for almost all protein domains occurs at a characteristic temperature called the transition midpoint, Tm, where the enthalpy changes and the entropy change, i.e. T∆S, are equal. Since ∆G =∆H-T∆S and ∆G = 0 at this temperature.

• The shape of the peak in the thermogram provides further insight into the properties of the sample. If denaturation occurs within a narrow temperature range, then the transition is considered highly cooperative (5).

• The number of peaks observed has been proposed to be related to the flexibility of the hinge region (6). Previous studies on multi-domain proteins suggest these complex thermograms and overlapping peaks arise from interactions between domains (7). Some DSC studies have succeeded in identifying the unfolding temperature of each separate domain within the heavy chain portion (8).


Experimental

� All samples were diluted to 0.5 mg/mL. Exact concentration were used in the analysis.

� 550 µL of the diluted samples was loaded into the 300 µL sample cell (with a total of 165 µg in cell).

� The dilution buffer, 10 mM PBS, pH 7.4 was loaded into the reference cell.

� Samples were scanned from either 25 to 95 ⁰ C or 4 to 95 ⁰ C at 1 ⁰C/min with a 600 s equilibration period.

� All samples were collected in duplicate.

� A background of 10 mM PBS buffer was subtracted from each set of data.

DSC Case Study: Pfizer ADC mimics...

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