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CHARACTERIZATION OF ISOFORMS OF CYSTEINE-CONJUGATED ANTIBODY DRUG CONJUGATES (ADCs) USING ON-LINE 2D-LC/MS

Robert Birdsall,1 Henry Shion,1 Frank W. Kotch,2 April Xu,3 Thomas J. Porter,4 Weibin Chen1

1. Pharmaceutical Business Operations, Waters Corporation, 34 Maple Street, Milford MA 01757

METHODS RESULTS AND DISCUSSION

CONCLUSION 2D-LC/MS provides unambiguous identification of positional isomers in cysteine-conjugated ADCs

Middle down MSE analysis provides a method for quick drug conjugation site confirmation

Efficient on-line methodology bypasses time consuming offline fractionation and sample preparation

2. Pfizer Bioprocess Research & Development, 401 N. Middletown Road, Pearl River, NY 10965

3. Pfizer Analytical Research & Development, 401 N. Middletown Road, Pearl River, NY 10965

4. Pfizer Analytical Research & Development, 1 Burtt Road, Andover, MA 01810

OVERVIEW

2D-LC/MS based methods enable

rapid verification of positional

isomers for accurate determination of

drug to antibody ratio (DAR) values

Conjugation sites were identified

using a middle down approach with

analysis of ADC fractions using MS

techniques

Figure 1. Positional isoforms of cysteine conjugated

ADCs.

8 # of drugs

0

positional isomers

= drug

HIC (1st dimension)

Mobile Phase:

A: 125 mM Sodium Phos-

phate Buffer, pH 6.7, 2.5M

ammonium sulfate

B: 125 mM Sodium Phos-

phate Buffer, pH 6.7

C: Isopropanol

D: 18 MΩ water

RPLC (2nd dimension)

Mobile Phase:

A: 18 MΩ water, 0.1% FA,

0.01% TFA (v/v)

B: Acetonitrile, 0.1% FA,

0.01% TFA (v/v)

1st Dimension (HIC)

Heart-cut

Retention time [min]

2nd Dimension TIC (RPLC)


TIC

[C

ou

nts

]

peak 1

peak 2

ADC

sub-unit 2x

2x

Expected DAR

Retention time (min)

Area % Area

(%)

Individual DAR

(exp. DAR x area)

0 4.66 3943 0.04 0.00

0 4.8 11615 0.11 0.00

0 5.11 22377 0.22 0.00

2 5.62 288610 2.84 0.06

2 6.06 58009 0.57 0.01

4 6.61 2757313 27.17 1.09

4 7.19 288157 2.84 0.11

4 7.72 63524 0.63 0.03

6 8.4 1420982 14 0.84

6 8.89 469456 4.63 0.28

6 9.89 1015204 10.01 0.60

8 10.6 3747624 36.93 2.95

Total DAR 5.97

= 0 Expected

drug load 2 4 6 8

6a

6b 6c

Table 1: Calculated DAR from HIC Analysis

peak 1 peak 2 23,575.0 Da 51,620.0 Da

HIC Analysis for Determination of DAR Value (1D mode)

HIC to RPLC-MS of Individual Drug Conjugate Using Heart-cut Fractionation (2D mode)

Figure 3. HIC analysis of cysteine-conjugated ADCs using a UV detector. Determination of drug distribution is readily achieved using

a HIC-based technique. DAR values of each conjugate can be calculated from the peak area and summed for the total DAR value.

Figure 4. Fractionation of individual drug conjugates using an H-Class Bio with 2D Technology. A) Using a heart-cut, DAR 8 was frac-

tionated onto the reversed phase column. B) The lack of inter-chain disulfide bonds results in two peaks when using a reversed phase gradient.

MS Analysis for the Determination of Sub-unit Mass from Heart-cut Fraction (2D mode)

Figure 5. MS analysis of fractionated drug conjugates. A) Peak 1 and B) peak 2 eluted from the reversed phase separation shown in

Figure 4B were analyzed with a Xevo G2 QTOF. Peak 1 and peak 2 were identified as the light chain and heavy chain with 1 and 3 drugs, respectively.

A) B)

A) B)

A) DAR 4

Positional Isomers Determination Using 2D-LC/MS

2nd Dimension Deconvoluted mass spectrum


TIC

[C

ou

nts

]


TIC

[C

ou

nts

]


TIC

[C

ou

nts

]

Possible sub-units using RPLC

74,439.0 Da 125,302.0 Da

125,302.0 Da 101,727.0 Da

23,575.0 Da

51,620.0 Da

102,484.0 Da

74,439.0 Da

23,575.0 Da

51,620.0 Da

102,484.0 Da

74,439.0 Da

23,575.0 Da

=

Isoform

=

=

C) DAR 6c

peak 1

peak 2

peak 1 peak 2

Mass [Da] Mass [Da]

102,484.0 Da 23,575.0 Da

peak 1 peak 2

peak 1

peak 2

peak 3

Figure 6. Positional isomer determination using 2D-LC/MS. Heart-cut fractions of A) DAR 4, B) DAR 6a, and C) DAR 6c were performed from in-

dividual HIC separations of cysteine conjugated ADCs. A reversed phase gradient of each cut produced up to 3 peaks representing sub-units of the positional isomers. Deconvolution of each peak resulted in unambiguous identification of the isoform for each fraction.

578.7 725.8 812.8 940.9 1041.9 1141.0 1238.1 Pro Val Thr Lys Ser Phe

y”

840.8 954.9 1101.9 1189.0 1317.1 1418.1 Thr Lys Ser Phe Asn y”

Observed m/z

Hydrophobic interaction chromatography

(HIC) is a leading technique for the

determination of average DAR and drug

loading distribution for cysteine

conjugated ADCs. However,

chromatographic peaks are not always

readily interpreted and may be

associated with conjugate isoforms

(Figure 1).

An on-line 2D-LC/MS approach was

applied to elucidate the structures of

positional isomers in cysteine ADCs.


1st Dimension

Heart-cut

B) DAR 6a


Heart-cut


Heart-cut

INTRODUCTION

Valve

Position 1: Sample is injected onto the 1st dimension

column and a HIC separation is carried out with a salt gradient using

Position 1: The binary pump is used to deliver a reversed phase gra-

dient to the 2nd dimension column and elute the fractionated peak of

interest

Position 2: The regenerative pump is used to elute

(heart-cut) a peak of interest to the 2nd dimension column where it

is trapped for desalting

Figure 2. Schematic of valve configuration for 2D-LC /MS

analysis of cysteine conjugated ADCs.

MS:

Capillary: 3kV

Sample cone: 45 V

Source temp: 120 °C

Desolvation temp: 350 °C

ADC Conjugation Site Determination Using 2D-LC/MSE A) mAb Control

Figure 7. Conjugation site determination using MSE. A) MSE analysis of the partially reduced light chain (LC) from the unconjugated mAb

(control) identified a C-terminus peptide fragment from the predicted fragments. B) MSE analysis of the LC fragment from DAR4 (Figure 6A), in-dicates a mass shift equal to the drug mimic in the same peptide fragment, confirming drug attachment in the DAR4 LC fraction.

Predicted fragments

O

HO S

H

C-terminal

Light chain

MSE

Predicted fragments

O

HO S

drug

C-terminal

Light chain

b” 1037.92 1136.99 1238.04 1366.13 1453.17 1600.23 1714.28 1871.49

Pro Val Thr Lys Ser Phe Asn Arg

y” 1614.19 1517.13 1418.07 1317.02 1188.92 1101.89 954.82 840.78

b” 1039.02 1138.09 1239.14 1367.23 1454.27 1601.33 1715.38 1872.59

Pro Val Thr Lys Ser Phe Asn Arg

y” 1238.10 1141.05 1041.98 940.93 812.84 725. 80 578.74 464.69

mAb control: C-terminal MSe peptide fragments from non-conjugated light chain

ADC: C-terminal MSe peptide fragments from conjugated light chain

Observed m/z

MSE

B) ADC

G0F

G1F

G0F

G1F

G2F

Mass [Da] Mass [Da] Mass [Da]

23,575.0 Da 74,439.0 Da 51,620.0 Da

peak 1 G0F G0F

G1F

G1F

peak 2 peak 3

Mass [Da] Mass [Da]

23,575.0 Da 101,727.0 Da

peak 1 G0F

G1F

G2F

peak 2

TUV/PDA

quater-

nary

pump

auto

sampler

Column 2 (RPLC)

Column 1 (HIC)

TUV/PDA

binary

pump

regenera-

tive pump

mass spec-

trometer

waste waste

TUV/PDA

quater-

nary

pump

auto

sampler

Column 2 (RPLC)

Column 1 (HIC)

TUV/PDA

binary

pump

regenera-

tive pump

mass spec-

trometer

waste waste

TUV/PDA

quater-

nary

pump

auto

sampler

Column 2 (RPLC)

Column 1 (HIC)

TUV/PDA

binary

pump

regenera-

tive pump

mass spec-

trometer

waste waste

Instrumentation

Waters ACQUITY H-Class Bio with 2D Technology

Waters ACQUITY® Xevo G2 QTof

Columns

Waters Protein Pak Hi Res HIC (available 3Q 2014)

ACQUITY UPLC Protein BEH C4, 300Å, 1.7 µm, 2.1 mm X 50 mm

characterization of isoforms of cysteine-conjugated ... · 2d-lc/ms provides unambiguous...

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