Photoinduced Aggregation of a Model Antibody–Drug Conjugate

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PHOTO-INDUCED AGGREGATION OF A MODEL ANTIBODY-DRUG CONJUGATE Gregory M. Cockrell, Michael S. Wolfe, Janet L. Wolfe, and Christian Schoneich Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5006799 • Publication Date (Web): 16 Apr 2015 Downloaded from http://pubs.acs.org on April 21, 2015

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Molecular Pharmaceutics

1

PHOTO-INDUCED AGGREGATION OF A

2

MODEL ANTIBODY-DRUG CONJUGATE

3 4

Gregory M. Cockrell,1 Michael S. Wolfe,1 Janet L. Wolfe,1 and Christian

5

Schöneich*,2

6 7

1

8

States

9

2

10

Wolfe Laboratories Inc., 134 Coolidge Ave., Watertown, MA 02472, United

Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant

Avenue, Lawrence, Kansas 66047, United States

11 12 13

*Corresponding Author:

14

Christian Schöneich, Ph.D.

15

Department of Pharmaceutical Chemistry

16

The University of Kansas

17

2095 Constant Avenue

18

Lawrence, KS 66047

19

Phone: (785) 864-4880

20

Fax: (785) 864-5736

21

Email: [email protected]

22 23 ACS Paragon Plus Environment

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Abstract

2 3

During synthesis, purification, and especially storage, antibody-drug conjugates

4

(ADCs) may be exposed to various types of light. Several of the drugs commonly

5

conjugated to antibodies contain photo-sensitive functional groups. Exposure to

6

light could generate an excited state of the drug that subsequently triggers drug

7

and/or protein degradation. In order to mimic and study photo-induced ADC

8

degradation, we designed a model ADC in which the monoclonal antibody (mAb)

9

trastuzumab was treated with the amine-reactive probe eosin-5-isothiocyanate to

10

yield an antibody-eosin conjugate (T-EO). Photo-induced degradation was

11

monitored by size exclusion chromatography (SEC), dynamic light scattering

12

(DLS), SDS-PAGE under reducing and non-reducing conditions, and MS/MS

13

analysis. SEC analysis of the model ADC showed the formation of higher

14

molecular weight species directly following a 20 W-hr/m2 exposure of UVA light.

15

DLS analysis of these samples showed the formation of larger soluble particles,

16

and precipitate was observed 24 hours post light exposure. These results were

17

not seen in control samples of the model ADC that were shielded from light.

18

Furthermore, these results were not seen in control samples containing mAb

19

alone, suggesting that aggregation was the result of light exposure of the

20

conjugate. Importantly, when eosin-5-isothiocyanate was added separately to

21

solutions containing mAb, i.e. without conjugation, the extent of photo-induced

22

aggregation was substantially less, indicating that the conjugation of the photo-

23

sensitizer to the mAb specifically promoted photo-induced aggregation. Reducing

ACS Paragon Plus Environment

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1

and non-reducing SDS-PAGE suggested that photo-induced inter-chain covalent

2

cross-linking occurred through a mechanism other than disulfide formation. Using

3

peptide mapping and MS/MS analysis we identified key peptides in the T-EO

4

sequence which undergo photo-degradation. Finally, we also show that cross-

5

linking products formed in as little as 1 h exposure to ambient light. These

6

findings suggest that precautions should be taken to ensure minimal exposure to

7

light

8

photosensitive drugs.

during

synthesis,

purification,

and

storage

of

ADCs

containing

9 10 11

Keywords:

12

antibody-drug conjugate, monoclonal antibody, aggregation, photo-degradation,

13

dynamic light scattering, size exclusion chromatography

14


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Introduction

2 3

Antibody-drug

conjugates

(ADCs),

monoclonal

antibodies

(mAbs)

4

covalently linked with cytotoxic payloads, have become an interesting and

5

exciting breakthrough in pharmaceutical research.1-3 Taking advantage of the

6

high binding specificity of mAbs for cell-surface antigens is an important strategy

7

in developing therapeutics that selectively kill specific cell populations for

8

diseases such as cancer. ADCs can ideally reduce the nondiscriminatory

9

cytotoxicity exhibited by most chemotherapy drugs, leading to more efficacious

10

treatments with fewer side effects. However, the success of this strategy is highly

11

dependent on the physical and chemical stability of the ADC. For example,

12

aggregation of therapeutic proteins is a challenging problem that may cause

13

adverse clinical effects such as immunogenicity or decreased potency.4

14

Several factors define the structural stability of ADCs. Conjugation of a

15

drug to different sites on the mAb can have a direct effect on the charge,5

16

hydrophobicity,6 and thermostability7-9 of ADCs, all factors affecting the

17

propensity for protein aggregation. Native proteins aggregate through several

18

mechanisms that may be amplified by chemical conjugation of a drug, including

19

non-covalent self-association of intact proteins, unfolding, covalent linkage, and

20

chemical degradation.10 Aggregation of intact proteins can occur through

21

electrostatic and/or hydrophobic forces, in which protein-protein interactions are

22

favored over protein-solvent interactions.11 Drugs commonly conjugated to mAbs

23

include microtubule/tubulin polymerization inhibitors (auristatins), 12, 13 DNA minor


4

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1

groove binders (duocarmycins),14 and topoisomerase II inhibitors (doxorubicins),

2

15-19

3

solubility.20 Chemical conjugation of a drug to a mAb can also directly affect the

4

electrostatic surface charge of the protein.5 Thus, ADCs can be highly

5

susceptible to aggregation through self-association of native conjugate.

6

Aggregation through unfolding is similar to the pathway of aggregation through

7

self-association of native protein, except that aggregation proceeds through

8

intermediates unfolded to varying degrees that contain exposed hydrophobic

9

residues. These unfolded intermediates, in equilibrium with the native folded

10

all of which are large, hydrophobic molecules that have limited aqueous

protein, are key precursors for irreversible aggregate formation.21

11

Alternative aggregation pathways proceed through chemical reactions.

12

Aggregation through covalent linkages occurs through cross-linking reactions

13

between protein chains. The most common reaction is the formation of disulfide

14

linkages either via oxidation of free cysteine residues located on the surface of

15

the protein or through disulfide exchange. Chemical cross-linking can also occur

16

through the formation of dityrosine. While this reaction occurs less frequently, the

17

formation of dityrosine is a known product of tyrosine photon absorption.22,

18

Oxidation24 or deamidation25,

19

Furthermore, aggregation can proceed through subsequent cross-linking

20

reactions to oxidation products. Torosantucci et al.27 demonstrated the potential

21

for 1,4- and 1,6-additions of nucleophiles to Tyr oxidation products, which lead to

22

covalently cross-linked products resulting in aggregation. 27, 28

26

23

can induce aggregation by triggering unfolding.


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1

While these main pathways of aggregation can often be minimized

2

through solution conditions such as reaction additives,29 pH,30-32 ionic strength,30-

3

33

4

alluded to above, light exposure can facilitate the formation of aggregates.

5

Extensive work has been done on the mechanisms of photo-induced damage to

6

proteins.23, 35 Light exposure can induce unfolding due to photo-oxidation of the

7

protein, leading to aggregation.36 Proteins also have the potential to aggregate

8

through photo-induced cross-linking reactions involving the formation of

9

dityrosine,35, 37 cross-linking between Cys and Trp residues,38 Cys and Tyr,39 and

10

between oxidized His residues.40-42 Other photo-degradation studies have

11

observed the pH-dependent aggregation of IgG1 after as little as 1 minute of UV

12

light exposure.43 The UV exposure of IgG1 can result in the cleavage of Trp side

13

chains through Trp radical cation formation,44 in which the ensuing radicals can

14

react with oxygen and/or thiols.44, 45

and excipient selection,34 environmental factors such as temperature32 and, as

15

In some types of ADCs, light-sensitive functional groups that are not

16

normally found in native proteins are introduced via drug conjugation. Examples

17

from developmental ADCs include anthracylines,18 duocarmycins,2 porphyrins,

18

chlorines, and bacteriochlorins.46 Furthermore, conjugation of any drug to an

19

antibody may change the intrinsic photo-sensitivity of the construct due to

20

changes in surface hydrophobicity. However, to date there is no published

21

information on photo-induced degradation of ADCs. Here, we investigated the

22

effects of light exposure on the physical stability of a model ADC with an average

23

DAR of 1.2, conjugated through Lys residues. As the antibody portion of this


6

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1

model ADC, we selected trastuzumab (Herceptin®), which has been shown to

2

have

3

trastuzumab conjugated with the drug DM1 (trastuzumab emtansine; Kadcyla®)

4

is one of only two currently marketed ADCs.48-50 Eosin was selected as the small

5

molecule surrogate for our model ADC system. Eosin contains a network of

6

aromatic rings and bears a structural resemblance to classes of anti-cancer

7

drugs such as anthracyclines. Importantly, the use of eosin as a model allowed

8

us to evaluate the general photosensitivity of an antibody-small molecule

9

conjugate without the risk of drug exposure of laboratory personnel.

excellent

long-term

physical

and

chemical

stability.47

Moreover,

10

Eosin is a known photosensitizer for singlet oxygen formation51 and has

11

been used to study the intermolecular photo-oxidation of proteins.52 The data

12

presented demonstrate the direct correlation between a covalently linked model

13

drug and protein aggregation upon light exposure. Understanding these

14

photochemical effects on ADCs is essential and will have a future impact on

15

processing conditions, formulation strategies, and storage conditions of ADCs as

16

they become marketable therapeutics.

17


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Materials and Methods

2 3

Materials. Trastuzumab was purchased from Myoderm (Norristown, PA) and

4

provided as a lyophilized powder containing trehalose dihydrate, L-histidine, and

5

polysorbate 20. The lyophilized powder was reconstituted as a 21 mg/mL stock

6

solution of trastuzumab containing final concentrations of 20 mg/mL trehalose

7

dihydrate, 0.5 mg/mL L-histidine, pH=6, and 0.09 mg/mL polysorbate 20. Eosin-

8

5-isothiocyanate was purchased from Invitrogen (Carlsbad, CA) and used as

9

received.

OmniPur 10X phosphate buffered saline (PBS) liquid concentrate

10

(Millipore, Darmstadt, Germany) was diluted with deionized water to a final 1X

11

concentration (50 mM sodium phosphate, 137 mM NaCl at pH 7.4). Trypsin

12

digestion kits were purchased from Protea Biosciences (Morgantown, WV).

13

Materials used for SDS-PAGE analysis were purchased from Invitrogen.

14

NuPAGE® Novex® 1.0 mm, 10 well 12% Bis-Tris gels, the Mark 12TM unstained

15

standard, and NuPAGE® LDS sample buffer (4X) were used as received.

16

NuPAGE® MOPS SDS running buffer (20X) was used following a 20-fold dilution

17

with deionized water for a final concentration of 50 mM MOPS, 50 mM Tris Base,

18

0.1% SDS, and 1 mM EDTA at pH=7.7. Novex® colloidal blue staining kits were

19

used following manufacturer guidelines. All deionized water (18 MΩ cm) used in

20

buffer and sample preparation was obtained from a Barnstead NANOpure Infinity

21

water purification system.

22


8

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The 21 mg/mL stock

1

Synthesis of trastuzumab-eosin (T-EO) conjugate.

2

solution of trastuzumab described above was buffer exchanged into 0.1 M

3

bicarbonate, pH= 9.1, using microcentrifuge tubes containing a 3 kDa molecular

4

weight cut-off (MWCO) filter (Millipore). Separately, a 10 mg/mL stock solution of

5

eosin-5-isothiocyanate (dye) was prepared in DMSO. To a microcentrifuge tube,

6

143 µL of the trastuzumab stock solution, 837 µL of 0.1 M bicarbonate buffer

7

(pH=9.1), 9 µL of the 10 mg/mL eosin-5-isothiocyanate stock solution (6

8

equivalents), and 11 µL of DMSO were added, such that the final protein

9

concentration was 3 mg/mL in bicarbonate buffer with a solvent content of 2%

10

DMSO. The vial containing the reaction mixture was covered in aluminum foil

11

and gently agitated at room temperature for approximately 1 h. Excess dye was

12

subsequently removed by dialysis against PBS, pH=7.4, at 4 °C for 24 h using 10

13

kDa MWCO Slide-a-lyzer dialysis cassettes (Pierce, Rockford, IL).

14 The dialyzed protein conjugate solution was

15

Sample Characterization.

16

analyzed by size exclusion chromatography (SEC, method outlined below) to

17

confirm the removal of free dye. Direct measurement of the protein at 280 nm

18

and eosin absorbance at 530 nm was performed on a Spectramax M5 UV/Vis

19

spectrophotometer (Molecular Devices, Sunnyvale, CA). These absorbance

20

values were used to calculate the average dye-to-antibody ratio (DAR) by using

21

the following protocol for amine-reactive probes obtained from Invitrogen. First,

22

280 the absorbance of the trastuzumab at 280 nm ( Atrastuzuma b ) was corrected for the

23

contribution of eosin to the total absorbance at 280 nm.


9


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1

2

Eqn. 1:

280 trastuzumab

A

=A

280 sample

−A

530 sample

 Aeo280sin  530  Aeo sin

  

3

280 530 Asample and Asample are direct absorbance measurements of the T-EO conjugate

4

sample at 280 nm and 530 nm, respectively. Aeo280sin and Aeo530sin are absorbance

5

values obtained from free eosin dye at 280 nm and 530 nm, respectively. This

6

correction factor was determined to be 0.25. The corrected Atrastuzumab was then

7

used to calculate the protein concentration using Beer’s Law, where ε trastuzumab =

8

225,000 M-1 cm-1. The average DAR was then calculated using the following

9

equation:

280

530 ASample × MWtrastuzumab DAR = 530 [trastuzumab]× ε eosin

10

Eqn. 2:

11

530 where ASample =

12

MW trastzumab = molecular weight of trastuzumab (145,531.5 Da), [trastuzumab] =

13

concentration of trastuzumab in units of mg/mL, and ε eo sin = extinction coefficient

14

of eosin at 530 nm (90,000 M-1 cm-1). This wavelength is the absorption

15

maximum of eosin in the presence of protein.53

direct absorbance measurement of T-EO sample at 530 nm,

530

16

For comparison with the T-EO conjugate, a 1 mg/mL sample of

17

trastuzumab was spiked with the free dye, eosin-5-isothiocyanate in PBS,

18

pH=7.4. The eosin-spiked trastuzumab (T+EO) sample had a final concentration

19

of 3.4 µg/mL eosin-5-isothiocyanate, representing 1 eq of dye to antibody.

20


10

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1

Size Exclusion Chromatography. Size exclusion chromatography (SEC) was

2

performed on a Shimadzu Prominance LC-20 AT HPLC system. Approximately

3

10 µg of protein was loaded onto a TSKgel G3000SW xL (7.8 mm x 30 cm, 5 µm)

4

column (Tosoh Haas Bioscience, San Francisco, CA), and eluted using an

5

isocratic flow (20 min, 1.0 mL/min) of 50 mM sodium phosphate, 50 mM sodium

6

chloride at pH = 6.9. The eluate was monitored by UV detection at a wavelength

7

of 280 nm. Integration of the peak areas was performed to calculate the percent

8

peak area of each peak relative to the total area of all peaks. Peaks eluting

9

before the main peak are reported as higher molecular weight species (HMWS).

10 11

Dynamic Light Scattering. The size distribution of soluble particles in each

12

sample was measured on a Malvern Zetasizer Nano ZS90 (Malvern Instruments,

13

Worcestershire, UK). Samples were equilibrated at 25 ± 0.1 °C for 600 s prior to

14

each measurement and this temperature was held constant throughout the

15

experiment. Results are reported as averaged particle sizes (Z-average) in each

16

sample, which were calculated based on autocorrelation functions of the

17

scattered light using the Malvern Zetasizer software. For a single sample, the

18

result is the Z-average given as a repetition of three measurements. Values are

19

reported with standard deviations as the Z-average of three independent

20

samples.

21 22


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1

Light Exposure. Samples were exposed to UVA (315-400 nm) light in a Caron

2

6540-1 photostability chamber with light uniformity of ± 10%. The temperature

3

was controlled at 25 ± 0.2 °C. Initial 1 mg/mL solutions of trastuzumab / T-EO

4

were equally split into two vials. One of the vials was wrapped in aluminum foil,

5

protecting it from any light exposure (dark control). The second vial was left

6

unaltered. Both the dark control vial and sample vial were placed in the

7

photostability chamber under the same conditions. Samples underwent an

8

accumulated exposure of 20 W-hr/m2 when the UVA lamp was turned off. All

9

samples were covered in foil prior to removal from the photostability chamber.

10

Dark control samples and light exposed samples were analyzed by SEC and

11

DLS directly after exposure. Dark control samples and light exposed samples

12

were analyzed by DLS and reducing/non-reducing SDS-PAGE 24 h after

13

exposure.

14

A single control sample of 1 mg/mL T-EO in PBS, pH=7.4 was stored in a

15

clear glass vial on a shelf with exposure to ambient light. Ambient light conditions

16

included indoor fluorescent lighting with window-filtered daylight (assuming 12 h

17

of daylight for a 24 h cycle). The typical UVA light intensity for this type of

18

ambient light source is estimated to be 17 W/m2.54 The temperature of this

19

sample was not regulated. DLS measurements of the sample were taken prior to

20

storage and 24 h after storage. Aliquots at a volume of 2 µL were removed every

21

hour for 7 h and prepared for SDS-PAGE analysis. The stored T-EO sample was

22

allowed to sit on the shelf for 24 h (12 h, lights on, 12 h, lights off). After 24 h, a


12

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1

final aliquot was removed for gel electrophoresis and a final DLS measurement

2

was performed.

3 4

Gel Electrophoresis. SDS-PAGE analysis was performed in NuPAGE® 12%

5

Bis-Tris precast gels with NuPAGE® MOPS running buffer (Invitrogen). Samples

6

were prepared with a target of 2 µg of protein loaded into each well. For samples

7

analyzed under reducing conditions, the protein samples were prepared in

8

NuPAGE® LDS sample buffer containing a final concentration of 50 mM

9

dithiothreitol (DTT). These samples were heated to 95 °C for approximately 1 min

10

prior to loading in the sample wells. For samples analyzed under non-reducing

11

conditions, the protein samples were prepared in NuPAGE® LDS buffer

12

containing no DTT. A volume of 5 µL of Mark12TM molecular weight marker was

13

loaded into the first well of each gel. Gels were developed using a constant

14

voltage of 150 V for approximately 1.5 h. After developing, the gels were stained

15

with Novex® Colloidal blue staining kit. The stained gels were imaged using a

16

GS-800 calibrated densitometer and analyzed with Quantity One Software (Bio-

17

Rad Laboratories, Hercules, CA).

18 19

Peptide Mapping and MS/MS Analysis. Samples of trastuzumab or T-EO at

20

concentrations of 1.7 mg/mL were exposed to 20 W-hr/m2 UVA light following the

21

procedure above. Both the control samples and light-exposed samples of each

22

were prepared for tryptic digests in the following way (under exclusion of light). A

23

volume of 200 µL of the protein sample was added to 750 µL of denaturing buffer


13


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Page 14 of 61

1

consisting of 6 M guanidine hydrochloride,1 mM EDTA, and 0.25 M Tris, pH =

2

7.5. To this solution, 25 µL of 0.5 M DTT was added. The solution was incubated

3

at 37 °C for 30 min. After incubation, 50 µL of 0.5 M iodoacetamide was added,

4

and the solution was incubated at room temperature for an additional 30 min. A

5

volume of 25 µL of 0.5 M DTT was added after 30 min. A Sephadex G-25 column

6

was pre-equilibrated with a 0.1 M Tris buffer, pH=7.8 containing 2 M urea. The

7

denatured protein solution was loaded on the column and eluted with 0.1 M Tris

8

buffer, pH=7.8, containing 2 M urea in 750 µL increments. A total of 7 fractions

9

were collected and monitored for absorbance at 280 nm. Trypsin (5 µL of a 1

10

mg/mL solution) was added directly to the fraction with the highest absorbance at

11

280 nm. The digest solution was covered in foil and incubated at 37 °C for

12

approximately 14 h. The digest was then quenched by the addition of 1 µL

13

trifluoroacetic acid (TFA), 99%.

14

Analysis of the protein digests was performed using ultra performance

15

liquid chromatography (UPLC) on a Waters ACQUITYTM H-Class BIO system

16

(Milford, MA) equipped with a tunable ultraviolet detector. A volume of 100 µL

17

was loaded onto a Waters ACQUITYTM UPLC BEH C18 (2.1 mm x 50 mm, 1.7

18

µm) column. Peptides resulting from the tryptic digests were eluted off of the

19

column using a linear gradient starting with 98% of mobile phase A (0.1% TFA in

20

water) and 2% of mobile phase B (0.1% TFA in acetonitrile) to 55% mobile phase

21

A and 45% mobile phase B within 120 min at a flow rate of 0.3 mL/min. Peptides

22

eluting from the column were monitored at 280 nm and 530 nm.


14

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1

Individual peaks of the tryptic digest of the T-EO control were selected for

2

analysis by mass spectrometry. The peptides eluting at 50.5 min, 54.5 min, and

3

77.5 min were collected in three separate fractions as they were detected by UV

4

light (refer to peptide mapping results). The peaks at 50.5 min and 54.5 min were

5

of interest because they appeared to be degraded between the dark control and

6

light exposed T-EO samples. The peak at 77.5 min was of interest because this

7

peak was not present in the peptide map of trastuzumab alone, indicating a

8

major site of eosin labeling. This was also confirmed by significant absorbance at

9

530 nm. The fractions were covered in foil and analyzed by tandem mass

10

spectrometry (MS/MS). The fractions were infused directly into an AB SCIEX API

11

4000 QTRAP mass spectrometer using electrospray ionization in the positive

12

mode. Parent masses were identified following a Q1 scan between 200 and 2400

13

amu. Identified parent masses were subsequently input for further analysis using

14

enhanced product ion scans. Operating conditions used to obtain the mass

15

spectra were: declustering potential = 75 V, temperature = 600 °C, and collision

16

energy= 55 eV.

17

Expected monoisotopic masses for peptides resulting from the tryptic digest of

18

trastuzumab were calculated using the PeptideMass55 tool on the ExPASy web

19

server56,

20

light chains were input with the option for cysteine derivatization by

21

iodoacetamide. Once potential peptides were identified by parent masses, the

22

expected b and y fragment ions were calculated using the MS-Product tool of

23

Protein Prospector v 5.12.1 developed by University of California, San Francisco

57

(http://web.expasy.org/peptide_mass). Sequences for the heavy and


15


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Page 16 of 61

1

(http://prospector.ucsf.edu). The calculated fragments were compared to the

2

experimental product ion scans to verify the identified peptide sequences.


16

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1


Results

2 3

Pre-Exposure Measurements. Samples of trastuzumab, trastuzumab-eosin

4

conjugate (T-EO), and eosin spiked trastuzumab (T+EO) at 1 mg/mL in PBS,

5

pH=7.4, were analyzed by size exclusion chromatography (SEC) and dynamic

6

light scattering (DLS) in order to establish baseline levels of higher molecular

7

weight species (HMWS) and larger particle sizes, respectively, prior to light

8

exposure testing. PBS was chosen as the buffer in this study because it is not

9

affected by light, allowing for the effects of light exposure on the mAb to be

10

studied independently from the effects light may have on other buffers (e.g., His).

11

SEC analysis of trastuzumab showed a single peak eluting at 8.5 min. Particle

12

size distribution by DLS also showed a single peak with an averaged particle

13

radius (Z-average) of 5.2 ± 0.01 nm (Table 1). For the T-EO conjugate, SEC

14

showed a single peak eluting at 8.5 min. DLS measurements determined the Z-

15

average of T-EO as 5.8 ± 0.09 nm (Table 1), with no peaks corresponding to

16

larger sized particles. These parameters were used to confirm that no

17

aggregates were present prior to light exposure. These initial values were also

18

used to confirm that no aggregates formed in the dark controls due to

19

temperature or buffer conditions during placement in the photostability chamber.

20 21

Light exposure does not cause trastuzumab to aggregate. Dark controls and

22

trastuzumab samples, each at a concentration of 1 mg/mL in PBS, pH=7.4, were

23

placed in the photostability chamber ensuring that each experienced identical


17


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Page 18 of 61

1

temperature. For samples that were exposed to light, DLS and SEC

2

measurements were performed directly after an exposure of 20 W-hr/m2 UVA

3

light was reached. Figure 1A displays the particle size distribution of a dark

4

control of trastuzumab.

5

average of 5.2 ± 0.04 nm. Figure 1B shows a particle size distribution with a Z-

6

average of 5.2 ± 0.04 nm for trastuzumab exposed to 20 W-hr/m2 UVA light,

7

revealing no difference in particle size between the control and light-exposed

8

trastuzumab samples. All DLS measurements are summarized in Table 1.

The graph depicts one peak corresponding to a Z-

9

SEC chromatograms of dark control and light-exposed trastuzumab are

10

shown in Figure 2A, exhibiting single peaks eluting at 8.5 min. No higher

11

molecular weight peaks appeared in the light-exposed sample, indicating no

12

photo-induced aggregation. These findings correlated with the particle size

13

measurements shown in Figures 1A and 1B. The DLS and SEC data together

14

showed that light exposure, temperature, or buffer used in this study did not lead

15

to aggregation of trastuzumab. After initial DLS and SEC measurements, the

16

samples were stored at 4 °C without further light exposure, and the DLS

17

measurements were repeated after 24 h. In the case of trastuzumab, DLS

18

showed a single peak in the size distribution graph which correlated to a Z-

19

average of 5.2 ± 0.02 nm. Accordingly, 24 h storage of dark control and light-

20

exposed trastuzumab at 4 oC did not result in any measurable aggregation.

21 22

Light exposure causes significant aggregation of T-EO. The T-EO

23

conjugates used in this study had a low average dye-to-antibody ratio (DAR). By

24

direct absorbance of the conjugate at 280 nm and 530 nm, the DAR was


18

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1

calculated to be 1.2. The light exposure of T-EO at 1 mg/mL in PBS, pH=7.4, was

2

performed as in the trastuzumab experiments described above. Figure 1C

3

depicts the size distribution of a dark control of T-EO. The graph shows a single

4

peak that corresponds to a Z-average of 5.8 ± 0.01 nm. Dark control samples

5

remained constant with a Z-average of 5.8 nm from pre-exposure through post-

6

exposure, including 24 h storage (see below), which indicated that temperature

7

and buffer conditions were not causing aggregation (Table 1). The size

8

distribution by DLS of the T-EO sample that was exposed to 20 W-hr/m2 UVA

9

light is shown in Figure 1D. Directly after light exposure the Z-average increased

10

to 133.2 ± 58.13 nm, as compared to 5.8 nm of the pre-exposed and dark control

11

samples. The representative size distribution versus intensity graph in Figure 1D

12

shows two peaks with average hydrodynamic radii of 6.5 nm and 1673 nm.

13

Visual inspections of both the sample and dark control vials were conducted; no

14

precipitate had formed directly after accumulated exposure of 20 W-hr/m2.

15

SEC chromatograms of the control and light-exposed T-EO are shown in

16

Figure 2B. The dark control sample displayed a single peak eluting at 8.5 min.

17

The light-exposed sample of T-EO showed a total of two peaks. The main peak

18

was approximately half the intensity of the control sample. The main peak was

19

also slightly shifted to the left in the light-exposed sample, indicating that the

20

light-exposed sample is hydrodynamically larger than the control sample. A

21

possible explanation for this phenomenon is that the exposure to light could

22

cause some of the disulfide bonds to break, which may increase the

23

hydrodynamic radius of the protein. Similar observations had been reported


19


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Page 20 of 61

1

earlier when bovine somatotropin was exposed to light.58 However, we also have

2

to consider the possibility of higher molecular weight species generated by

3

covalent cross-linking of antibody fragments to the antibody, i.e. the generation of

4

species with hydrodynamic radii between those of antibody monomer and dimer

5

(see below). An earlier eluting peak at 7.2 min represented 14% of the total peak

6

area. Hence, both SEC and DLS indicated the formation of aggregates directly

7

after the exposure to 20 W-hr/m2 UVA light, detecting both a higher molecular

8

weight species and larger particles not seen in control samples.

9

Dark control and light-exposed T-EO were stored at 4 °C after DLS and

10

SEC measurements. After 24 h, visual inspection of the light-exposed T-EO

11

revealed a visible precipitate that settled on the bottom of the vial. This

12

precipitate was not visible in the dark control. DLS measurements were repeated

13

after 24 h storage. The Z-average of the T-EO dark control remained at 5.8 nm.

14

DLS measurements of the light-exposed sample were of poor quality due to

15

precipitation and are, therefore, not reported.

16 17

Light exposure does not cause the aggregation of trastuzumab in the

18

presence of free dye. For comparison with the T-EO conjugate, a 1 mg/mL

19

sample of trastuzumab was spiked with the free dye, eosin-5-isothiocyanate in

20

PBS, pH=7.4. The eosin-spiked trastuzumab (T+EO) sample had a final

21

concentration of 3.4 µg/mL eosin-5-isothiocyanate, representing 1 eq of dye to

22

antibody. Pre-light exposure DLS measurements revealed a Z-average of 5.4 ±

23

0.004 nm. Figure 1E shows the size distribution versus intensity for a T+EO dark


20

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1

control. The graph shows a single peak representing a Z-average radius of 5.3 ±

2

0.03 nm. Figure 1F shows the size distribution versus intensity for the T+EO

3

sample after 20 W-hr/m2 UVA light exposure. The Z-average of the T+EO light

4

exposed sample was 5.4 ± 0.10 nm, indicating that the free eosin dye in solution

5

was not causing aggregation of trastuzumab during light exposure.

6

SEC chromatograms of dark control and light-exposed T+EO are shown in

7

Figure 2C. SEC analysis of T+EO dark control displayed a main peak eluting at

8

8.5 min and a smaller molecular weight peak eluting at 12.5 min. The later eluting

9

peak was identified as free dye following an injection of only eosin-5-

10

isothiocyanate onto the column. The chromatogram of T+EO after exposure to 20

11

W-hr/m2 UVA light was identical to that of the T+EO dark control, i.e. showing a

12

major peak eluting at 8.5 min. By comparison of dark control and light-exposed

13

T+EO, there did not appear to be any change in the peak corresponding to the

14

free dye. The SEC chomatograms correlated with DLS measurements in that no

15

photo-induced aggregation was detected for the T+EO immediately after light

16

exposure.

17

significant changes in the Z-average values for the dark control and light exposed

18

sample (Table 1).

After 24 h of storage at 4 °C, DLS measurements revealed no

19 20

SDS-PAGE

analysis

reveals

non-reducible

cross-linking

21

degradation product of T-EO. SDS-PAGE analysis of dark control and light-

22

exposed (20 W-hr/m2 UVA light) samples was performed under reducing and

23

non-reducing conditions in order to further identify HMWS that were not resolved


as

photo-

21


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Page 22 of 61

1

by SEC analysis (Figure 3). HMWS under reducing conditions are defined as

2

those above the expected molecular weight of the heavy chain (50 kDa). HMWS

3

under non-reducing conditions are defined as those above the expected

4

molecular weight of trastuzumab (150 kDa) and are directly comparable to the

5

SEC data. Under reducing conditions, the trastuzumab control sample displayed

6

two bands as expected for the light (25 kDa) and heavy (50 kDa) chains (Figure

7

3A, Lane 3), both at relative quantities of 50% (Table 2). These quantities were

8

obtained by densitometry. However, the degree of staining depends on the size

9

of the polypeptide chain. Thus, the values reported in Table 2 were first corrected

10

for the molecular weight assuming a linear correlation between molecular weight

11

and degree of staining. Values were then normalized such that the heavy and

12

light chains represent equal relative amounts. The values reported for the light-

13

exposed samples were calculated using the same normalization factors as the

14

dark controls. The relative quantities of the HMWS were reported as the

15

remainder of the total relative quantity of all detected bands. The light-exposed

16

sample of trastuzumab alone (Figure 3A, Lane 4) exhibited bands for the heavy

17

and light chains; however there were also two weaker bands that appeared at ca.

18

61 kDa and 55 kDa. These two HMWS had a total relative quantity of 18%, while

19

both the heavy chain and light chain equally decreased from 50% to 41% (Table

20

2). Under non-reducing conditions, both the control trastuzumab sample and the

21

light-exposed trastuzumab exhibited major bands at the expected molecular

22

weight of 150 kDa (Figure 3B, Lanes 3 and 4). There were no higher molecular

23

weight bands present to suggest the formation of aggregates. Although the light-


22

Page 23 of 61

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1

exposed trastuzmab sample exhibited bands slightly higher in molecular weight

2

than the heavy chain (Figure 3A, lane 4), these HMWS were not indicative of

3

cross-linking products that lead to aggregation, which is noted in Table 2. This is

4

based on data obtained under non-reducing conditions. It appears that these

5

species are not contributing to aggregate formation.

6

Two independent sets of T-EO samples were run on SDS-PAGE under

7

reducing and non-reducing conditions (Figure 3A and B, Lanes 5-8). The T-EO

8

dark control exhibited the expected major bands at 50 kDa and 25 kDa under

9

reducing conditions (Figure 3A, Lanes 5 and 7) and the expected major band of

10

150 kDa under non-reducing conditions (Figure 3B, Lanes 5 and 7). The light-

11

exposed samples of T-EO are shown in Figures 3A and 3B, Lanes 6 and 8.

12

Under reducing conditions, the intensities of the heavy and light chain bands

13

decreased, and several bands of HMWS appeared. These bands correspond to

14

molecular weights of ca. 150 kDa, 120 kDa, and 75 kDa. These HMWS totaled to

15

quantities of 41% (T-EO sample #1) and 49% (T-EO sample #2) relative to the

16

heavy and light chains in the two T-EO samples (Table 2). Two of these bands

17

were faintly visible in the T-EO control samples (Figure 3A, Lanes 5 and 7),

18

however the intensities were significantly lower than in the light-exposed samples

19

in Lanes 6 and 8, and they were not registered by the Quantity One analysis

20

software. Under non-reducing conditions significant bands appeared at molecular

21

weights higher than the expected 150 kDa band. These bands were above the

22

200 kDa upper limit of the MW ladder, however molecular weight values were

23

extrapolated from the standard curve. These bands corresponded to species with


23


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Page 24 of 61

1

molecular weights of ca. 300 kDa and 215 kDa (Figure 3B, Lanes 6 and 8). It is

2

expected that these values have significant error associated as they are outside

3

the linear range of the gel. Therefore, the band extrapolated to 215 kDa may

4

actually reflect the addition of multiple species of 25 kDa, i.e. the light chain, to

5

the original protein with 150 kDa (i.e., generating products with apparent

6

molecular weights of 200 kDa or 225 kDa). In either case, this result is significant

7

as it is between the expected values of 150 kDa and 300 kDa (monomeric and

8

dimeric species, respectively) under non-reducing conditions.

9

This set of results indicated that covalent inter-chain cross-linking may be

10

a pathway towards aggregation of T-EO. This cross-linking did not occur through

11

disulfide bond formation, as the cross-linked products were non-reducible. The

12

HMWS bands observed in the two T-EO samples during SDS-PAGE under

13

reducing conditions (Figure 3A, Lanes 6 and 8) corresponded to molecular

14

weights of two heavy chains and two light chains (150 kDa), two heavy chains

15

and one light chain (125 kDa), and one heavy chain and one light chain (75 kDa).

16

Under non-reducing conditions (Figure 3B, Lanes 6 and 8) a cross-linked product

17

was observed at ca. 300 kDa, which corresponded to two cross-linked

18

monomers. Lanes 6 and 8 also showed dark bands located in the sample wells

19

suggesting even higher molecular weight species that did not load onto the gel.

20

Finally, the non-reducing SDS-PAGE exhibited a cross-linked species between

21

the expected 150 kDa and 300 kDa bands. This molecular weight did not

22

correspond to any combination of monomeric species that were expected on

23

non-reduced SDS-PAGE. This result suggested that some portion of the T-EO


24

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1

sample underwent fragmentation as well as cross-linking. There were very faint

2

lower molecular weight bands observed in the T-EO samples at 50 kDa and 25

3

kDa (Figure 3B, Lanes 6 and 8) that would also indicate that fragmentation had

4

occurred after light exposure. These species were very low in concentration, as

5

indicated in the gel, and were likely under the detection limit of the SEC method.

6

Finally, a set of the T+EO samples under reducing conditions is shown in

7

Figure 3C. The dark control sample under reducing conditions (Figure 3C, Lane

8

3) showed only the two expected bands at 50 kDa and 25 kDa with relative

9

quantities of 50% for both the heavy and light chains. The light-exposed T+EO

10

sample (Figure 3C, Lane 4) exhibited the heavy and light chain bands as well as

11

one higher molecular weight band at ca. 120 kDa. It is possible that a small

12

amount of the reactive eosin-5-isothiocyanate could have conjugated to the mAb

13

leading to the small amount of cross-linking observed. The T+EO solution was

14

prepared at pH=7.4, which is well below the pKa of the Lys side chain, and

15

therefore any conjugation should be minimal. The relative quantity of the 120 kDa

16

band was 7%. Interestingly, the relative quantity of the light chain remained the

17

same while the relative quantity of the heavy chain was reduced to 43% upon

18

light exposure. The amount of cross-linking products was significantly less in the

19

T+EO sample relative to the T-EO samples (7% vs. ~50%; Table 2). This result

20

suggested that the covalent linkage of the dye to the mAb significantly amplifies

21

the photo-induced cross-linking products, possibly leading to the aggregation

22

observed by other analytical methods.

23


25


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Page 26 of 61

1

T-EO aggregates upon ambient light exposure. A sample of T-EO was stored

2

at ambient temperature on a shelf in a clear glass vial, allowing exposure to

3

ambient light. The sample was not shielded from the indoor fluorescent lighting or

4

from the window-filtered sunlight. Baertschi et al.54 have estimated that the

5

typical light intensity for this type of exposure is 17 W/m2 UVA light. Using this

6

estimate, 1 hr under ambient light conditions is comparable to the 20 W-hr/m2

7

exposure in the photostability chamber. However, this is an estimate and the

8

actual exposure may be less due to factors such as light uniformity. Prior to this

9

exposure, an initial DLS measurement revealed a single peak corresponding to a

10

Z-average radius of 5.8 nm (Figure 4A), consistent with previous measurements

11

(see Table 1). After a 24 h cycle of storage at ambient light (i.e., a period with 12

12

h light on and 12 h light off) and ambient temperature, DLS analysis showed

13

three peaks, with a Z-average of 14.5 nm (Figure 4B).

14

observed in the sample vial based on visual inspection. Particle size distribution

15

appeared different than was observed for the samples exposed in the

16

photostability chamber (3 peaks vs. 2 peaks, respectively). It is expected that

17

DLS measurements will exhibit some variability between samples containing

18

aggregates. This is because aggregation mechanisms are not homogeneous

19

processes and that aggregates of different particle sizes are present at any given

20

time. It is also possible that the actual exposure of light was less than that of the

21

photostability chamber as light uniformity was uncontrollable (e.g., cloud

22

coverage, time of day, etc.). It is expected that lower levels of light exposure

23

would result in slower aggregate formation.


No precipitate was

26

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1

Aliquots of the T-EO sample exposed to ambient light conditions were

2

removed every hour and immediately prepared for SDS-PAGE under reducing

3

conditions (Figure 4C). After the first hour of exposure, the T-EO sample

4

displayed several HMWS bands corresponding to molecular weights of ca. 175

5

kDa, 150 kDa, 120 kDa, and 80 kDa (Figure 4C, Lane 3). Lanes 3-10

6

(corresponding to 1-7 h and 24 h exposure) all contained these HMWS bands;

7

however the relative quantities of all bands, including the heavy and light chain

8

bands, seemed to decrease over time suggesting the formation of potentially

9

insoluble aggregates. Small levels of HMWS are seen in the control sample

10

(Figure 4C, Lane 2). This was likely due to brief uncontrollable light exposure

11

during SDS-PAGE sample preparation as initial DLS results indicated no species

12

of higher particle sizes.

13 14

Peptide Mapping and MS/MS Results. Peptide mapping of trastuzumab and T-

15

EO samples was conducted in order to identify differences between control and

16

light-exposed samples on a closer level than was allowed by SDS-PAGE. Dark

17

control samples of trastuzumab and T-EO were reduced, and free cysteines were

18

alkylated with iodoacetamide. Samples were then digested with trypsin, and a

19

peptide map was generated by RP-UPLC (Figure 5). The T-EO peptide map

20

monitored at 280 nm (Figure 5A, blue trace) displayed several small peaks with

21

retention times after 60 min not observed in the trastuzumab map (Figure 5A, red

22

trace). These peaks were confirmed to be eosin conjugation sites by

23

chromatograms of each monitored at 530 nm, (Figure 5B) the λmax of eosin.


27


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Page 28 of 61

1

Trastuzumab and T-EO had identical peptide maps at 530 nm for retention times

2

< 60 min, which was likely background noise from simultaneous monitoring at

3

280 nm. Between 60 and 100 min the T-EO peptide map exhibited several peaks

4

at 530 nm that were not visible in the trastuzumab sample, suggesting eosin

5

conjugation to these peptides. The most notable difference between the peptide

6

maps was the peak at 77.5 min in the T-EO sample. This peak was not observed

7

in the trastuzumab peptide map at 280 nm (Figure 5A, blue trace) and was

8

significantly larger than any other 530 nm peak (Figure 5B, blue trace), indicating

9

a site of eosin conjugation. However, the mass spectrum of the isolated peak

10

suggested the co-elution of several peptides that did not absorb at 530 nm. The

11

concentrations of these peptides, including the peptide of suspected eosin

12

conjugation, were low, and thus MS/MS data were unobtainable due to

13

instrument limitations.

14

Figure 6A shows the RP-UPLC trace of the peptides resulting from the

15

tryptic digest of the trastuzumab dark control sample and the trastuzumab

16

sample exposed to 20 W-hr/m2 UVA light. These two traces were identical, and

17

all of the peaks in the light-exposed sample were accounted for in the control

18

sample. Figure 6B shows the peptides resulting from the tryptic digest of the T-

19

EO dark control sample and the T-EO sample exposed to 20 W-hr/m2 UVA light.

20

The peptide map of light exposed T-EO did not show the appearance of any new

21

peak when compared to the dark control sample, but clearly shows the

22

disappearance or reduction in peak area of two key peaks (Figure 6B). The first

23

peptide of interest eluted from the column at 50.5 min (red trace) and was greatly


28

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1

diminished in the light-exposed sample (blue trace). The second peptide of

2

interest, eluting at 54.5 min, had completely disappeared in the light-exposed

3

sample. These peptides from the T-EO dark control sample were collected in

4

separate fractions from the column and further analyzed by mass spectrometry.

5

The peptide eluting at 50.5 minutes was determined to have a parent

6

mass (m/z) of 840.2 Da, which matched trastuzumab heavy chain residues 279-

7

292 (FNWYVDGVEVHNAK) with a charge of +2. The MS/MS data of this peptide

8

are shown in Figure 7A with the matched b and y fragment ions calculated using

9

Protein Prospector. This sequence contained the residues Phe, Tyr, Trp, and His

10

that are susceptible to photo-degradation. The peptide sequence of T-EO that

11

disappeared (Figure 6B, red trace at 54.5 minutes) upon light exposure had a

12

parent mass (m/z) of 934.8 Da. This corresponds to residues 421-443 in the CH2

13

domain

14

WQQGNVFSCSVMHEALHNHYTQK was confirmed by MS/MS. Figure 7B

15

displays the MS/MS spectra of the peptide and the identity of the sequencing ion

16

fragments as calculated by Protein Prospector. There are several residues in this

17

sequence that are highly susceptible to photo-degradation including Trp, Phe,

18

Cys, Met, His, and Tyr.

of

the

Fc

region

with

a

charge

of

+3.

The

sequence

19

The peptide maps and MS/MS data presented only focus on the most

20

significant differences between the control and light-exposed T-EO samples

21

(Figure 6). The two major differences were the peptides described above, both of

22

which are rich in amino acids highly susceptible to photo-degradation

23

example, non-reducible cross-links can form between two Tyr residues,35,


23, 35

. For 37

or

29


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Page 30 of 61

1

between two oxidized His residues.40-42 In addition, Cys and Trp38 or Cys and

2

Tyr39 residues can form non-reducible cross-links. The presence of these amino

3

acids is a likely reason for the photo-induced disappearance of the parent

4

sequences. It is important to note that no major peak in the trace could be

5

attributed to a cross-linked peptide; however, this is to be expected: it is very

6

likely that the cross-linking observed by SDS-PAGE was due to an array of

7

different cross-linking reactions. The total sum of all cross-linking products would

8

result in a measurable change to the overall protein (i.e., inter-chain cross-

9

linking) that is observable by SDS-PAGE. However, on a more detailed

10

molecular level the types of cross-linking reactions and the areas on the protein

11

where the cross-linking occurs are very heterogeneous and likely the reason that

12

no one cross-linking product could be attributed to a major single peak in the

13

peptide map. It is more likely that some of the smaller peaks closer to the

14

baseline that are present in the light-exposed sample (Figure 6B, blue trace) and

15

not in the control sample (Figure 6B, red trace) represent cross-linked peptides.

16

The peptides that correspond to these peaks are very low in concentration and

17

thus MS/MS data were not obtained. Identifying cross-linked species in the T-EO

18

peptide map is a subject for further study.

19


30

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1


Discussion

2

The data presented in this study provide evidence that covalent linkage of

3

a photosensitive small molecule to a mAb can facilitate aggregation upon

4

exposure to light. We also provide evidence that non-reducible cross-linking is a

5

possible pathway towards the observed aggregation. Three independent

6

techniques, including DLS, SEC, and SDS-PAGE were applied to assess the

7

formation of trastuzumab and T-EO aggregates or cross-linked species upon

8

exposure to 20 W-hr/m2 UVA light. The cumulative results were that 1)

9

trastuzumab does not aggregate upon exposure to 20 W-hr/m2, 2) the addition of

10

the photosensitive group to the mAb causes significant aggregation and/or cross-

11

linking upon light exposure, and 3) the cross-linking reactions that likely lead to

12

aggregation are enhanced by the covalent linkage and not just the presence of

13

the photosensitive dye in solution. Peptide mapping and MS/MS revealed two

14

peptides in the trastuzumab sequence that were significantly affected by light

15

exposure. These peptides were rich in residues that are susceptible to photo-

16

degradation, including cross-linking reactions. Further MS studies will need to

17

address the nature of the photoproducts, including the various potential covalent

18

cross-links.

19

We also show that aggregation occurs in as little as 1 hour (lights on)

20

under ambient light conditions. These conditions included artificial lighting lamps

21

with a typical UV intensity of 0.1- 0.3 W/m2 and window-filtered sunlight with an

22

estimated intensity of 17 W/m2.54 While this is an estimated light intensity, the

23

total exposure is likely less than that due to the lack of light uniformity. Therefore,


31


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Page 32 of 61

1

aggregation was observed in a sample in the first hour at a light intensity of < 17

2

W/m2. Collectively, these results show that significant caution should be applied

3

in developing ADCs, especially those with photo-sensitive payloads.

4

There are certain factors, including DAR, that vary between ADCs. The

5

model ADC system used in this study was a trastuzumab-eosin conjugate with

6

an average DAR of 1.2. This is a relatively low DAR compared to the two ADCs

7

currently on the market. Both Kadcyla® (trastuzumab entansine)48 and Adcetris®

8

(brentuximab vedotin)59 have average DARs of 4, and most ADCs in

9

development have an average of 3-4 drugs per antibody.3 The aggregation

10

observed in this study with just one dye molecule per antibody is likely to occur

11

faster, and with less light exposure, as the DAR increases. Therefore,

12

marketed/developmental ADCs with average DARs of 3-4 are potentially more

13

susceptible to photo-induced aggregation, depending on the photochemical

14

nature of the drug, than the model system presented here. We note that the

15

process of drug conjugation itself may contribute to the ultimate photosensitivity

16

of an ADC, especially when the reaction conditions applied for conjugation (i.e.,

17

the presence of organic co-solvent, alkaline pH etc.) impact the conformation,

18

physical and/or chemical stability of the antibody.

19

Photo-induced aggregation of ADCs is highly dependent on the

20

photosensitivity of the payload attached to the mAb. Here we demonstrate that

21

our model antibody trastuzumab exhibited no aggregation induced by the

22

exposure to 20 W-hr/m2 until the covalent addition of our model drug eosin. Eosin

23

is a good photo-sensitizer51 and not all drugs currently used in the development


32

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1

of ADCs are as photosensitive. An example of a common ADC payload that is

2

likely more stable to light exposure is monomethyl auristatin E (MMAE).

3

However, there are photosensitive drugs used in the development of ADCs that

4

likely will exhibit photo-degradation, and possibly photo-induced aggregation

5

such as observed for T-EO. Eosin was used in this study to mimic drugs such as

6

anthracyclines. Examples of anthracyclines that have been used in ADC

7

development are doxorubicin and its derivatives.15-19 Doxorubicin is a very

8

photoreactive compound, where absorption of a photon results in either photo-

9

degradation of the drug itself, or the generation of hydrogen peroxide.60

This

10

ROS could potentially damage the mAb portion of the ADC and lead to the

11

formation of aggregates. Other drugs with photosensitive moieties used in ADC

12

development include cyclopropapyrroloind-4-one (CPI), cyclopropabenzindol-4-

13

one (CBI), duocarmycins,2 and CC-1065.61

14

A specific treatment method where the results of this study have direct

15

implications is the use of ADCs in antibody-directed phototherapy (ADP). ADP

16

specifically refers to delivering photosensitizers to tumor cells through antibody

17

conjugation. There are currently many photosensitive drugs approved for clinical

18

use,46

19

selectivity for tumor cells. Photosensitizer drugs are mostly porphyrins, chlorines,

20

or bacteriochlorins, all of which have a strong absorption in the visible region.46

21

Based on the results of this study with the comparable small molecule eosin, it is

22

not unreasonable to suggest that ADCs developed for ADP will undergo

23

significant photo-degradation, and possibly aggregate, upon light exposure. With

which by using a mAb delivery system could potentially increase


33


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Page 34 of 61

1

the many drugs used in the development of ADCs and the concern for

2

photochemical degradation events, it may be useful to study the quantum yields

3

for photoreactions of these common photosensitizing groups when they are

4

conjugated to the protein.

5

The major result observed for the model ADC system T-EO was

6

significant aggregation upon light exposure. By SDS-PAGE, we show that major

7

photo-degradation products are generated through non-reducible cross-linking.

8

Though cross-linking was observed for this system, an array of other photo-

9

degradation reactions can occur. One such reaction is photo-induced

10

fragmentation. This reaction occurs through cleavage of the backbone, with α-

11

carbon radicals known as key intermediates in the presence of O2.62

12

Fragmentation can also occur through disulfide bond breakage. There is some

13

evidence of photo-induced fragmentation of T-EO by SDS-PAGE analysis,

14

though not to the extent of aggregate formation. Under non-reducing conditions,

15

T-EO exhibited a band between the expected bands of 150 kDa and 300 kDa,

16

trastuzumab monomer and dimer, respectively (Figure 3B, Lanes 6 and 8). There

17

were also two lower molecular weight bands slightly visible at 25 kD and 50 kDa

18

for the two light-exposed samples. These data suggest that fragmentation is

19

occurring upon light exposure which is consistent with the SEC data. As

20

mentioned earlier, one possible photo-degradation reaction that could occur is

21

breakage of disulfide linkages as well as cross-linking through bonds other than

22

disulfides. Breakage of disulfide linkages could make the protein appear

23

hydrodynamically larger as seen in the SEC chromatogram for T-EO (Figure 2B).


34

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1

These fragmented species may dissociate from the rest of the protein if not

2

bound through other covalent means, which would explain the band between the

3

expected 300 kDa and 150 kDa bands, as well as the faint 25 kDa and 50 kDa

4

bands observed during non-reducing SDS-PAGE of the light-exposed T-EO

5

sample. These species could also stay associated through covalent linkages

6

other than disulfides that form as a result of light exposure, which may explain

7

the cross-linked species observed during reducing SDS-PAGE, as well as the

8

300 kDa band and other HMWS that did not load onto the gel under non-

9

reducing conditions.

10

Fragmentation can also explain the HMWS bands present in the light-

11

exposed trastuzumab sample (Figure 3A, Lane 4). The presence of these two

12

species under reducing conditions indicated that some photo-degradation

13

occurred in the trastuzumab sample that did not lead to aggregation.

14

Aggregation was not observed by SEC and DLS measurements of light-exposed

15

trastuzumab samples, i.e. no formation of HMWS or larger particle sizes,

16

respectively. Also, the only band exhibited by this sample under non-reducing

17

conditions was the expected 150 kDa band (Figure 3B, Lane 4). It is possible that

18

when exposed to light a small amount of the trastuzumab sample underwent

19

inter-chain cross-linking through any bond other than disulfide as well as

20

fragmentation.

21

fragmentation is not observed because these parts stay bound through disulfide

22

bonds. Under reducing conditions, these disulfide bonds are broken and the

23

small amount of one short heavy chain or one short light chain fragment will stay

Under

non-reducing

conditions

during


SDS-PAGE,

the

35


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Page 36 of 61

1

bound to another heavy chain through a covalent bond other than a disulfide

2

bridge (photo-induced cross-linking); these would explain the two bands

3

observed by reducing SDS-Page in Figure 3A, lane 4. Photo-degradation of

4

trastuzumab occurs to a much lower extent and does not result in aggregation as

5

compared to T-EO. Photo-induced

6

aggregation

is

particularly

concerning

for

ADC

7

development as exposure to light is difficult to control from manufacturing to

8

patient administration. In fact, great care was taken to ensure that all samples

9

were protected from light in this study prior to the light exposure tests. However,

10

the dark control samples of T-EO exhibit very faint bands of HMWS by SDS-

11

PAGE analysis (Figure 3A and 3B, Lanes 5 and 7), likely due to brief,

12

uncontrollable ambient light exposure during preparation of the samples. While

13

these bands are negligible based on densitometry at the time of analysis, these

14

HMWS could initiate nucleation events, driving the ADC towards aggregation.

15

Figure 4 shows that this model ADC is very sensitive to ambient light, with

16

several HMWS forming in as little as 1 hour at a light intensity < 17 W/m2. These

17

aggregation products would represent impurities in the active pharmaceutical

18

ingredient and require additional purification steps for removal. However, these

19

aggregates are of greater concern because of potential safety risks to patients.4 Exposures of 20 W-hr/m2 UVA light were consistently used throughout this

20 21

study.

Current

guidelines

outlined

by

the International

22

Harmonisation of Technical Requirements for Registration of Pharmaceuticals for

23

Human Use (ICH) state that samples should be exposed to light near UV energy


Conference

on

36

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1

for no less than 200 W-hr/m2 during photostability testing of new drug substances

2

and products.63 The model ADC used in this study, and presumably ADCs with

3

photosensitive groups attached, would not pass this criteria.

4

Formulation development of the ADC could potentially reduce the photo-

5

degradation reactions leading to aggregation. The work conducted in this study

6

was performed in a simple PBS, pH 7.4, system. We chose PBS as a buffer in

7

our system because it is not photo-degradable, allowing the study of protein

8

degradation independent of buffer effects. Although PBS, pH 7.4, may not be a

9

relevant formulation for therapeutics, we demonstrate that this model buffer

10

system alone does not cause the formation of trastuzumab aggregates or T-EO

11

aggregates and that the observed aggregation of T-EO can be attributed directly

12

to light exposure. Thus, special consideration when developing formulations must

13

be given not only to aggregation, but also to photo-induced aggregation.

14

It is likely that residues susceptible to photo-degradation located near

15

conjugation sites of the dye in 3D space were the areas affected the most by light

16

exposure. This means that predicting these photo-degradation events may be

17

particularly challenging in an ADC system with drug loading heterogeneity as a

18

factor. Therefore, another method to reduce photo-degradation of ADCs could

19

be engineering certain residues for sites of specific drug loading. This has been

20

employed in many studies in an effort to reduce the heterogeneity of drug loading

21

in ADC development.64, 65 These sites could potentially be engineered to be away

22

from residues susceptible to photo-degradation. A pre-requisite for this would be

23

access to a 3D crystal or solution structure of the antibody. Linker length or


37


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Page 38 of 61

1

chemistry may also be variables that could potentially reduce photo-induced

2

degradation of the ADC, and could be used as a tandem strategy with

3

engineered drug loading sites. In our model, the drug was covalently attached

4

through a thiourea bond. Extending the distance between the photosensitizer and

5

the mAb may reduce the extent of photo-degradation. Though there have been

6

no studies to show an inverse correlation between linker length and the amount

7

of photo-degradation, we showed that unconjugated dye does not cause

8

appreciable amounts of photo-induced aggregation.

9

Packaging materials should also be considered in the manufacturing of

10

ADCs. In this study eosin was used to model the drug payload, which has an

11

absorption maximum at 530 nm.53 Eosin also strongly absorbs around 280 nm.

12

Limiting the light exposure in the absorption range of the payload would likely

13

reduce the impact of photo-degradation products.

14

Future studies are of course required to more fully understand the photo-

15

induced aggregation in ADCs. Here we present a case where a model ADC

16

exhibited aggregation as a direct result of light exposure. Further work should be

17

directed to understand the positive or negative effects of changing variables such

18

as concentration, types of model drugs, linker chemistry, and buffer

19

systems/formulations. In any event, the results of the present work should be a

20

cause of concern for the light exposure of ADCs.

21


38

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1


Conclusion

2

Here we present a case where covalently attaching a photosensitive dye

3

to a mAb exhibited aggregation products as a direct result of light exposure.

4

Based on this work, it is not unreasonable to suggest that any photosensitive

5

drug covalently attached to a mAb could cause significant, irreversible

6

aggregation upon the exposure to light. The extent of the photosensitivity will be

7

dependent on the nature of the drug conjugated to the mAb. Many anti-

8

proliferation drugs contain fused ring systems and other moieties that are highly

9

photosensitive, and ADP requires a photosensitive drug to be conjugated. The

10

results of this study should yield a concern for photo-induced aggregation of

11

ADCs with photosensitive drugs. Great care should be taken to minimize the

12

effects of light on such ADCs through formulation strategies and packaging, thus

13

helping to minimize risks associated with the safety of the patients in need of

14

these drugs.

15 16

Disclosure

17

The authors declare no competing financial interest.

18 19

Acknowledgments

20

Funding for this project was provided by and research was performed at Wolfe

21

Laboratories.

22 23


39


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Page 40 of 61

1 2

Abbreviations

3

ADC: antibody-drug conjugate

4

ADP: antibody-directed phototherapy

5

DAR: dye-to-antibody ratio

6

DLS: dynamic light scattering

7

HMWS: higher molecular weight species

8

HPLC: high performance liquid chromatography

9

MS/MS: tandem mass spectrometry

10

mAb: monoclonal antibody

11

ROS: reactive oxygen species

12

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

13

SEC: size-exclusion chromatography

14

T-EO : trastuzumab-eosin conjugate

15

T+EO: trastuzumab in the presence of free eosin-5-isothiocyanate

16

UPLC: ultra performance liquid chromatography

17

UVA: near ultraviolet

18


40

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1

Page 52 of 61

Figures

2 3 4 5 6

7 8 9 10 11 12 13 14

Figure 1. Representative particle size distribution vs. intensity DLS measurements of A) 2 trastuzumab (dark control), B) trastuzumab directly after accumulated exposure of 20 W-hr/m , C) 2 T-EO (dark control), D) T-EO directly after accumulated exposure of 20 W-hr/m , E) T+EO (dark 2 control), and F) T+EO directly after accumulated exposure of 20 W-hr/m .


52

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1

2 3 4 5 6 7 8

Figure 2. SEC chromatograms of A) trastuzumab dark control sample (green) and trastuzumab light exposed sample (red), B) T-EO dark control sample (green) and T-EO light exposed sample (red) and C) T+EO dark control sample (green) and T+EO light exposed sample (red). Y-axis units are mAU at 280 nm.


53


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Page 54 of 61

1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Figure 3. (A) Reducing and (B) non-reducing SDS-PAGE of trastuzumab and T-EO samples. Both reducing and non-reducing gels: Lane 1, molecular weight ladder; Lane 2, blank; Lane 3, 2 trastuzumab (control); Lane 4, trastuzumab (20 W-hr/m exposure); Lane 5, T-EO sample #1 2 (control); Lane 6, T-EO sample #1 (20 W-hr/m exposure); Lane 7, T-EO sample #2 (control); 2 Lane 8, T-EO sample #2 (20 W-hr/m exposure); (C) Reducing SDS-PAGE of trastuzumab spiked with Eosin dye at 1 equivalent: Lane 1, molecular weight ladder; Lane 2, blank; Lane 3, T 2 + EO (control); Lane 4, T + EO (20 W-hr/m exposure).


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1 2 3 4

5 6 7 8 9 10 11 12 13

Figure 4. Dark control sample of T-EO exposed to ambient light. A) DLS measurement of sample prior to storage. B) DLS measurement of sample after 24 hours of storage and C) Reducing SDSPAGE of T-EO sample during storage. Aliquots pulled every hour during storage for 7 h and after 24 h. Lane 1, molecular weight ladder; Lane 2, t=0; Lanes 3 - 9; t= 1 – 7 hr; Lane 10, t=24 hr.


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1

2 3 4 5 6 7 8 9

Figure 5. RP-UPLC traces of the tryptic digests of trastuzumab (Red) and T-EO (Blue) control samples monitored at A) 280 nm and B) 530 nm.


56

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1 2

3 4 5 6 7 8 9 10 11

Figure 6. A) RP-UPLC traces of the tryptic digest of trastuzumab control sample (Red) and the 2 tryptic digest of trastuzumab sample after 20 W-hr/m exposure (Blue) monitored at 280 nm. B) RP-UPLC traces of the tryptic digest of T-EO control sample (Red) and the tryptic digest of 2 trastuzumab sample after 20 W-hr/m exposure (Blue) monitored at 280 nm.


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1 2 3 4 5 6

Page 58 of 61

Figure 7. MS/MS spectra of A) peptide eluting at 50.5 min from the tryptic digest of T-EO, which was significantly reduced in intensity upon light exposure, and B) peptide eluting at 54.5 min from the tryptic digest of T-EO, which disappeared upon light exposure.


58

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1


Tables

2 3 4 Z-Average (r.nm) Pre-Exposure

5 6 7 8 9 10 11 12 13 14 15

Dark Control

Sample, 20 W-hr/m

2

t=0

t = 24

t=0

t = 24

Trastuzumab

5.23 ± 0.01

5.25 ± 0.04

5.25 ± 0.04

5.22 ± 0.01

5.23 ± 0.02

T-EO

5.84 ± 0.09

5.78 ± 0.01

5.82 ± 0.13

133.20 ± 58.13

Precipitate

T + EO

5.38 ± 0.004

5.34 ± 0.03

5.35 ± 0.05

5.41 ± 0.10

5.37 ± 0.09

Table 1. Summary of DLS measurements for trastuzumab, T-EO, and T + EO. Dark controls and 2 samples exposed to 20 W-hr/m UVA light were analyzed directly after exposure (t=0) and 24 h after exposure (t=24). Reported values are the averaged particle radii in each sample (Zaverage). Standard deviations were calculated from Z-average measurements of three independent samples.


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Page 60 of 61

1 2

50 kDa 25 kDa HMWS

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Trastuzumab Control Sample 50% 41% 50% 41% 0% 18%*

T-EO #1 Control Sample 50% 26% 50% 33% 0% 41%

T-EO #2 Control Sample 50% 20% 50% 31% 0% 49%

T+EO Control Sample 50% 43% 50% 50% 0% 7%

Table 2. Relative quantities for bands shown on SDS-PAGE under reducing conditions as calculated by densitometry. Values were corrected for molecular weight assuming a linear correlation between MW and degree of staining. Values for control samples were then normalized such that the heavy and light chains represent equal relative amounts. Trastuzumab sample corresponds with Figure 4A, Lanes 3 and 4. T-EO #1 and T-EO #2 samples correspond with Figure 4A Lanes 5 – 8. T+EO sample corresponds with Figure 4C, Lanes 3 and 4. The heavy and light chains of trastuzumab are 50 kDa and 25 kDa, respectively. All observed higher molecular weight bands were summed and reported as higher molecular weight species (HMWS). *The observed HMWS in the trastuzumab samples did not lead to aggregation.


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1


TOC Figure

2 3 4 5


61

Photoinduced Aggregation of a Model Antibody–Drug Conjugate

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