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Table of Contents

1. EXECUTIVE SUMMARY................................................................................................................................2
2. INTRODUCTION..............................................................................................................................................2
3. SCOPE ................................................................................................................................................................3
4. EQUIPMENTS AND MATERIALS ................................................................................................................3
4.1. EQUIPMENTS ..............................................................................................................................................3
4.2. MATERIALS.................................................................................................................................................3
5. LOT SELECTION .............................................................................................................................................4
6. ABBREVIATIONS ............................................................................................................................................4
7. METHODS .........................................................................................................................................................4
7.1. TRYPIC DIGESTION ..................................................................................................................................4
7.2. HPLC SEPARATION AND MASS SPECTROMETRY ANALYSIS .....................................................4
8. RESULTS ...........................................................................................................................................................5
...
9. CONCLUSIONS ................................................................................................................................................8
............ ...
..............
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10. REFERENCES...................................................................................................................................................9
.................
..... .........
....
11. REVISION HISTORY.....................................................................................................................................10
.........................
.................
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Table of
o Figures

FIGURE 1. ALXN1820 AMINO ACID SEQUENCE

UENCE WITH DISULFIDE BONDS......................................................................2
QUENCE
FIGURE 2. SCHEMATIC REPRESENTATION OFF ALXN
ALXN1820 WITH DISULFIDE BONDS .......................................................3
FIGURE 3. TOTAL ION CURRENT CHROMATOGRAM RA A OF ALXN1820 RS REDUCED (TOP) AND NON-REDUCED
(BOTTOM) TRYPTIC PEPTIDE MAP ........................................................................................................................6

Table of Tables
TABLE 1. RP-HPLC-MS GRADIENT ...............................................................................................................................5
TABLE 2. CONFIRMATION OF CYSTEINE CONTAINING PEPTIDES OF ALXN1820 RS BY REDUCED TRYPTIC LC-MS/MS
PEPTIDE MAPPING ................................................................................................................................................7
TABLE 3. CONFIRMATION OF DISULFIDE LINKED PEPTIDES OF ALXN1820 RS BY NON-REDUCED TRYPTIC LC-
MS/MS PEPTIDE MAPPING ...................................................................................................................................7

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1. EXECUTIVE SUMMARY

Reduced and non-reduced tryptic peptide mapping with LC-MS/MS was performed on
ALXN1820 Reference Standard (RS18200420). This report summarizes the confirmation of 2
intra-chain disulfide linked peptides (Table 3) and peak identification of both reduced (peptides
containing cysteine residues) and non-reduced peptides (disulfide linkages) (Figure 3).

2. INTRODUCTION

ALXN1820 is a recombinant, humanized VHH bispecific antibody that binds to human

properdin and serum albumin. The VHH bispecific antibody consists of a single polypeptide
chain of 256 amino acids, which is comprised of an anti-albumin domain at the N-terminus that
is fused to a C-terminal anti-properdin domain via a 14 amino acid linker. The amino acid
sequence and a schematic representation of ALXN1820 is shown in Figure 1 and Figure 2
respectively. The variable domains that form the serum albumin and properdin binding sites
consist of llama complementarity determining regions
gio grafted to human germline frameworks.
There are 2 intra-chain disulfide bonds, one disulfide
ulfide bond
b localized in each VHH domain.
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Figure 1. ALXN1820 Amino Acid Sequence
quence Disulfide Bonds
ce with D
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Residues are numbered sequentially starting with the mature N-terminus. The putative complementarity-determining
regions are underlined. The peptide linker is italicized. The predicted intra-chain disulfide bonds are illustrated with
connecting lines. Arg and Lys tryptic sites are bold and blue.

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Figure 2. Schematic Representation of ALXN1820 with Disulfide Bonds

Red dashed bars represent intra-disulfide bonds. Anti-albumin domain at the N-terminus is fused to anti-properdin
domain via a 14 amino acid linker (blue line). CDR represents complementarity determining regions. FR represents
framework regions.

3. SCOPE

The scope of this work is to confirm the presence of 2 intra-chain disulfide linked peptides, e.g.,
disulfide linkage between C22 and C95 in the anti-albumin domain, and between C159 and C232
in the anti-properdin domain, shown in Figure 2. Reduced tryptic peptide mapping was
performed to confirm the masses and location of individual
ind peptides containing cysteine residues
whereas non-reduced tryptic peptide mapping was perf performed to confirm the identity of disulfide
pe
linked peptides. Results from the identificationn of
o both reduced
red (peptides containing cysteine
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residues) and non-reduced (disulfide linkedd peptides) tryptic peptides are detailed in this report.
ptides tryp

4. EQUIPMENTS AND MATERIALS

ERIALS
AL
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4.1. EQUIPMENTS

4.1.1 UltiMate UHPLC

PLC System
HPLC Sy (Thermo Scientific)
4.1.2 Thermo Orbitrap
ap
pFFusion Mass Spectrometer (Thermo Scientific)

4.2. MATERIALS

4.2.1 ALXN1820 (Reference Standard RS18200420)

4.2.2 AccuMAP™ Low pH Protein Digestion Kits (Promega, Cat # VA1040)
4.2.3 Tris(2-carboxyethyl)phosphine (Sigma, CAS# 51805-45-9)
4.2.4 N-Ethylmaleimide (Sigma-Aldrich, CAS#128-53-0)
4.2.5 Trypsin Platinum, Mass Spectrometry Grade, Frozen (Promega, Cat#
VA9000)
4.2.6 Waters Acquity UPLC Peptide CSH C18 column (130Å, 1.7 μm, 2.1 x
150 mm) (Waters, Cat# 186005298)
4.2.7 Acetonitrile, UHPLC-MS grade, 0.1 micron filtered (Thermo Scientific,
Cat # A956-1 1L)
4.2.8 Water, UHPLC-MS grade, 0.1 micron filtered (Thermo Scientific, Cat #
W8-1 1L)
4.2.9 Trifluoroacetic acid (TFA), Sequencing grade (Thermo Scientific, Cat #
28904, 10 x 1 mL)
4.2.10 Mobile phase A: 0.02% trifluoroacetic acid in water (200 μL of TFA was
added into 1 L of UHPLC-MS grade water, followed by mixing)

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4.2.11 Mobile phase B: 0.02% trifluoroacetic acid in acetonitrile (200 μL of TFA

was added into 1 L of UHPLC-MS grade acetonitrile, followed by mixing)
4.2.12 Purified Water (e.g. Milli-Q or Molecular Grade Water)
4.2.13 HPLC injection vials with caps (Waters)
4.2.14 General laboratory supplies

5. LOT SELECTION

ALXN1820 Reference Standard (RS18200420) was used for peptide mapping.

6. ABBREVIATIONS

6.1 UHPLC: Ultra High-Performance Liquid Chromatography

6.2 UPLC: Ultra Performance Liquid Chromatography
6.3 MS: Mass Spectrometry
6.4 LC-MS: Liquid Chromatography coupled w with Mass Spectrometry
6.5 LC-MS/MS: Liquid Chromatography coupled
oupled with
w Tandem Mass Spectrometry
6.6 TIC: Total Ion Current
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6.7 RS: Reference Standard
6.8 TCEP: Tris(2-carboxyethyl)phosphine
phinee
6.9 Room Temperature: 20 – 25°C
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7. METHODS

7.1. TRYPIC DIGESTION

N

Tryptic digestion of ALXN1820 RS was performed using the AccuMAPTH Low pH Protein
Digestion Kit. Briefly, 50 μg of protein samples were added into the mixture of 20 μL
AccuMAPTH denaturing solution and 6 μL of AccuMAPTH 10X low pH reaction buffer. Next,
samples were alkylated by incubating with 2 μL of 100 mM NEM at 37 °C for 30 minutes.
Samples were then pre-digested by adding 25 μL of AccuMAPTH Low pH Resistant rLys-C
solution and incubating at 37 °C for 4 hours, rLys-C to protein ratio was 1:10 (w:w) at this stage.
Finally, samples were diluted with water followed by the addition of rLys-C (final rLys-C to
protein ratio is 1:5 (w:w)) and trypsin platinum (trypsin to protein ratio is 1:5 (w:w)) for
overnight incubation at 37 °C. Protein digest was split into 2 x 150 μL aliquots and labelled as
reduced and non-reduced. In the reduced vial, 3 μL of 500 mM TCEP was added to reduce the
disulfide bonds (final concentration of TCEP is 10 mM), in the other aliquot 3 μL of water was
added as a control, both vials were kept at room temperature for 30 mins. Finally, the reaction
was quenched by adding 2 μL TFA.

7.2. HPLC SEPARATION AND MASS SPECTROMETRY ANALYSIS

The samples were injected on a Waters Acquity UPLC Peptide CSH C18 column (130Å, 1.7 μm,
2.1 x 150 mm) at 60 °C, followed by electrospray ionization and mass analysis. Peptides were
eluted with an acetonitrile gradient from 1 % to 60 % with 0.02% TFA over 60 minutes at a flow

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rate of 0.2 mL/min (Table 1). The Thermo Orbitrap Fusion was set up to collect one full-scan
spectrum at a resolution of 60,000, followed by 3 seconds of data-dependent MS/MS of the most
abundant ions. MS/MS spectra were collected with monoisotopic precursor selection and
dynamic exclusion, using higher-energy collisional dissociation at 28 %. LC-MS/MS data was
analyzed using Protein Metrics Byos software v4.4. Reduced and non-reduced peptides were
confirmed by both mass (MS) and fragmented ions (MS/MS).

Table 1. RP-HPLC-MS Gradient

Time (min) Flow (mL/min) %A %B
0.0 0.2 98.0 2.0
2.0 0.2 98.0 2.0
30.0 0.2 80.0 20.0
42.0 0.2 70.0 30.0
52.0 0.2 62.0 38.0
60.0 0.2 40.0 60.0
61.0 0.4 0.0 100.0
62.5 0.4 0.0 100.0
63.0 0.4 98.0 2.0
64.0 0.4 2.0 98.0
65.0 0.4 98.0
9 2.0
66.0 0.4 2.
2.0 98.0
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67.0 0.4
.4 98.0
9 2.0
69.0 0.2 98.0 2.0
72.0 0.22 98.0 2.0
75.0 0.2 98.0 2.0
80.0 0.2 98.0 2.0
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8. RESULTS

The TIC chromatograms showing reduced (top), and non-reduced (bottom) Lys-C/tryptic peptide
profiles are shown in Figure 3. As shown, peptides containing cysteine residues are labelled as
C22, C95, C159, and C232 to indicate the location of cysteines in ALXN1820 whereas non-
reduced tryptic peptides are labelled as C22-C95 and C159-C232 to represent the disulfide
linkage between C22 and C95, and between C159 and C232.

The peptide identity of each peak was determined by its percussor mass, within 10 ppm, and
fragment ions. The masses of individual peptides containing cysteine residues and disulfide
linked peptides were determined and were consistent with the theoretical masses of the expected
amino acid sequence of both individual reduced (Table 2) and disulfide linked peptides (Table
3). Both intra-chain disulfide bonds were confirmed and are reported in Table 3. The spectra
representing MS and MS/MS information used for the identification of the listed peptides are
reported in eLN 13450 while information on sample preparation is listed in eLN 12786.

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Figure 3. Total Ion Current Chromatogram of ALXN1820 RS Reduced (Top) and Non-Reduced (Bottom) Tryptic Peptide
Map

5.0e+08 Reduced C232

3.0e+08

C22 C95
C159
1.0e+08

5 10 15 20
W 25 30 35 40 45

Intensity
5.0e+08
Non-Reduced UD I C159 - C232

3.0e+08
'
C22 - C95

1.0e+08

5 10 15 20 25 30 35 40 45
Minutes

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Table 2. Confirmation of Cysteine Containing Peptides of ALXN1820 RS by Reduced

Tryptic LC-MS/MS Peptide Mapping
Theoretical Observed
CysLoc Residues Confirmed Sequence a RT (min)
mass (Da) mass (Da)

C22 20-38 LSCAASGRPVSNYAAAWFR 33.1 2025.9843 2025.9838

C95 87-100 AEDTAVYYCAAVFR 36.6 1577.7184 1577.7175

C159 157-164 LSCAASGR 9.8 763.3647 763.3641

C232 213-239 NTLYLQMNSLKPEDTAVYYCNALQYEK 39.1 3211.5206 3211.5205

Mass (Da) and RT (min) were obtained from Protein Metrics Inc. Byos® v4.4 software using Byologic workflow.
Peptide masses were confirmed within 10 ppm of theoretical mass, peptides containing cysteine residues listed here are
unmodified cysteine residues.
a. Sequence confirmed by mass and MS/MS.

Table 3. Confirmation of Disulfide Linked Peptides

ptid of ALXN1820 RS by Non-Reduced
Tryptic LC-MS/MS Peptide Mapping
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CysLoc a Residues es b
Disulfide Linked Peptides RT (min) Theoretical Observed
Mass (Da) Mass (Da)
20-38 LSCAASGRPVSNYAAAWFR
RPVSNYAAAWFR
SNYAAAWFR
C22 - C95 AEDTAVYYCAAVFR
VFR 35.7 3601.6871 3601.6848
87-100
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157-164 LSCAASGR
LS
SCAA
C159 - C232 NSLKPEDTAVYY
SLKPEDTAVY C
NTLYLQMNSLKPEDTAVYYCNALQYEK 34.7 3972.8696 3972.8675
213-239
Mass (Da) and RT (min) were obtained from m Protein Metr
Metri
Metrics Inc. Byos® v4.4 software using Byologic workflow.
Peptide masses were confirmed within 10 ppm off theore
theoretical mass.
ness (e.g.,
a. ‘-‘ stands for disulfide bond between two cysteines ( C22 and C95 and C159 and C232)
b. Sequence confirmed by mass and MS/MS. Neighboring bolded cysteines are linked by inter tryptic peptide disulfide

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9. CONCLUSIONS

ALXN1820 RS was analyzed by reduced and non-reduced Lys-C/tryptic LC-MS/MS

peptide mapping. The masses of individual peptides containing cysteine residues and
disulfide linked peptides were determined and were consistent with the theoretical masses of
the expected amino acid sequence of both individual reduced (Table 2) and disulfide linked
peptides (Table 3). Both intra-chain disulfide bonds were confirmed and are reported in
Table 3.

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10. REFERENCES

10.1 Non-Reduced Peptide Mapping for Disulfide Bond Analysis

(WPD-PC-140-00D)

10.2 eLNs 12786 and 13450

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11. REVISION HISTORY

Version Change Type

Revision Summary Justification
No. (New, Revise, or Admin)

To support the
1.0 New Document N/A
ALXN1820 Project

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