Supplementary Online Material
Table S1. Supplier, specificity and levels of the inhibitors used in protease activity diagnosis screening of
Lot B. Protease activity results presented relative to Lot B activity in the absence of inhibitors at both pH
6.0 and at pH 7.0. Notable reductions in protease activity are highlighted in bold red text. EDTA caused a
slight increase in fluorescence signal at pH 6.0, possibly due to its impact/interference on the fluorescence
of the BODIPY detection dye. Abbreviations used in table: AEBSF-HCl, 4-(2-Aminoethyl)
benzenesulfonyl fluoride hydrochloride; E-64, N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]agmatine; [1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-guanidinobutane]; EDTA,
ethylenediaminetetraacetic acid.
Sample Description
Inhibitor Source
Inhibitor Specificity
Inhibitor
Level (μM)
Relative
Activity at
pH 6.0
Relative
Activity at pH
7.0
Lot B control
NA
0
1.0 ± 0.2
1.0 ± 0.2
Protease inhibitor
cocktail (without
pepstatin A or E-64)
Sigma-Aldrich,
P2714
NA
Serine proteases,
cysteine proteases,
aminopeptidases,
metalloproteases
Used at
“1×”
1.00 ± 0.03
0.40 ± 0.07
Serine proteases
1000
0.9 ± 0.1
0.40 ± 0.07
Serine proteases
1
1.2 ± 0.2
0.7 ± 0.1
Serine and cysteine
proteases
20
0.9 ± 0.1
0.90 ± 0.08
Amino-peptidases
50
1.1 ± 0.1
1.00 ± 0.06
Cysteine proteases
15
0.8 ± 0.2
1.00 ± 0.06
metalloproteases
4000
1.6 ± 0.1
0.9 ± 0.1
Aspartyl proteases
10
0.11 ± 0.03
0.00 ± 0.04
AEBSF-HCl
Aprotinin
Leupeptin
Bestatin
E-64
EDTA
Pepstatin A
ThermoScientific,
78431
ThermoScientific,
78432
ThermoScientific,
78435
ThermoScientific,
78433
ThermoScientific,
78434
Avantor, 4040
ThermoScientific,
78436
Figure S1. Selected analytical and stability results for process 1a lots (black circles) and process 1b lots
(open triangles). The data corresponding to the lots are also labelled A through G, as identified in Table 1.
Panel A: correlation between protease activity assay counts and HCP levels. Panel B: correlation between
soluble fragmentation rates at 40°C and HCP levels.
2
10
A
Soluble aggregates
by HP-SEC (%)
Purity by HP-SEC (%)
100
95
90
B
8
6
4
2
0
0
1
2
3
0
1
Time (months)
5
3
15
C
4
*with aspartyl
Soluble fragments
by RP-HPLC (%)
Soluble fragments
by HP-SEC (%)
2
Time (months)
protease inhibitor
3
*
2
1
protease-free lot
0
D
10
*
*
5
*with aspartyl
protease inhibitor
protease-free lot
0
0
1
2
3
0
Time (months)
1
2
3
Time (months)
Figure S2. Stress stability results for mAb-1. Lot B produced using process 1a (black squares), Lot B
with added serine protease inhibitor (blue diamonds) and with added aspartyl protease inhibitor (orange
circles). The protease-free Lot G produced using process 1b is shown for comparison (open triangles).
Panel A: purity by HP-SEC. Panel B: soluble aggregates by HP-SEC. Panel C: soluble fragments by HPSEC. Panel D: soluble fragments by RP-HPLC.
Gel Electrophoresis
Gel electrophoresis of CHO cathepsin D samples were run on a 4-12% Bis-Tris SDS-PAGE gel
(NP0322BOX, Life Technologies, Grand Island, NY) under reducing conditions. Gels were developed
with silver stain (LC6070, Life Technologies) and Western blot at a 300 ng load. For Western blot, the gel
was transferred to a PVDF membrane (IB401002, Life Technologies), washed with 1X TBS-Tween and
incubated with 1 g/mL of AF4G5 murine anti-cathepsin D antibody (MA5-17236, ThermoFisher,
Waltham, MA). The blot was washed, incubated with a goat-anti mouse (HC+LC) secondary antibody
(0751-1809, KPL, Gaithersburg, MD) conjugated to alkaline phosphatase, and developed with BCIP/NBT
(5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) phosphatase substrate. The Western blot
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results confirmed that the major 48 kDa band in the silver stain gel of samples captured from both the
mAb-1 product and CCCS samples is CHO cathepsin D (lanes 4 and 5).
Figure S3. Reduced silver stained electropherogram and Western blot of affinity purification enriched
CHO cathepsin D samples.
Lane 1: Molecular weight marker.
Lane 2: Control sample of human liver cathepsin D (Enzo Life Sciences, SE199) showing bands at ~14
kDa and ~34 kDa on the silver stain and at ~34 kDa on the Western blot.
Lane 3: Control sample of mAb-1 product showing heavy (~50 kDa) and light chain bands (~25 kDa) on
the silver stain. Some Western blot signal was seen for mAb-1 HC and LC because the goat-anti mouse
secondary antibody was slightly cross-reactive with the human mAb-1 HC and LC.
Lane 4: Enriched cathepsin from mAb-1 product showing a band consistent with single-chain cathepsin D
(‘cat D’, at 48 kDa), and bands consistent with 2-chain cathepsin D (~14 and 34 kDa) by silver stain. A
strong signal for the single-chain cathepsin D (~48 kDa) was seen on the Western blot. Some residual
mAb-1 heavy and light chains were observed (~50 and 25 kDa).
Lane 5. Enriched cathepsin from clarified cell culture supernatant showing a strong band consistent with
single-chain cathepsin D (~48 kDa), and minor bands consistent with 2-chain cathepsin D (~14 and 34
kDa) by silver stain. A strong signal for the single-chain cathepsin D (~48 kDa) was seen on the Western
blot.
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