LakePharma offers a full line of protein analytical services. Latest advances in liquid chromatography and mass spectrometry techniques enable LakePharma to perform high resolution characterization of protein samples at a reasonable cost for all researchers.
LakePharma's protein analytical services can identify a protein by molecular weight, sequence confirm the primary structure of a protein and detect post translational modifications, characterize a protein by aggregation, thermostability, size, charge, and pI, measure a protein's purity/impurity, determine the amino acid sequences of a purified protein sample or discover the amino acid sequence of an antibody, compare one protein or antibody sample to a second sample or biosimilar molecule, and more.
3. 3What does PAS do?
PAS offers analytical characterizations, enabling informed decision
making in advancing drug candidates to development.
Discovery
Engineering
Development
4. 4How does PAS facilitate drug development programs?
• Antibody de novo sequencing
• Affinity screening (off rate) by Octet
• Epitope binning
• Thermostability screening
• Poly-specificity screening
• Intact mass analysis
• Sequence liability analysis
• Affinity characterization & FcgR
and FcRn binding profile
• Epitope binning
• Potency assay
• Peptide mapping and by Mass Spec
• PTM and glycan profile by Mass Spec
• Custom quantification for SCC
• Formulation study
Discovery DevelopmentEngineering
Developability Assessment Stability Study Formulation Study
5. 5Developability Assessment
In silico DNA Sequence check and Protein Sequence Hot Spot
Analysis, followed by Mass Spec verification
✓ DNA codon preference
✓ Protein sequence hot spot analysis
Productivity Readiness Check
✓ Transient production in HEK293 or CHO system
Integrity and Stability Check (to demonstrate whether the antibody
is stable biochemically)
✓ Intact Mass/Peptide Mapping/ PTM by Mass Spec
✓ DSF/DLS – Thermostability assessment
✓ Aggregation/Fragmentation/PTM and Glycan profiling over 2
weeks incubation under stressed conditions
✓ SE-HPLC/LabChip CE-SDS
PK Readiness Check
✓ Poly-specificity ELISA, Surface hydrophobicity assay –
Specificity requirement
P o ly -S p e c ific ity E L IS A
OD450
B
s
A
b
R
e
f
1
R
e
f
2
R
e
f
3
R
e
f
4
P
C
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
1 5 0 u g /m L
5 0 u g /m L
1 7 u g /m L
2 n d A b o n ly
HPLC
DSF/SLS
DLS
CE-SDS
mass
23140 23160 23180 23200 23220 23240 23260 23280 23300 23320 23340 23360
%
0
100
PP9650_DG_R_20170426_SEC 399 (3.577) M1 [Ev-623677,It19] (Gs,0.800,1034:3697,0.20,L33,R33); Sb (15,2.00 ); Cm (395:399) 1: TOF MS ES+
9.13e623206.4
23185.0
23264.6
23228.2 23245.8
mass
48400 48450 48500 48550 48600 48650 48700 48750 48800 48850 48900 48950 49000 49050 49100 49150
%
0
100
PP9650_DG_R_20170426_SEC 345 (3.114) M1 [Ev-383616,It27] (Gs,0.800,1132:2662,0.50,L33,R33); Sb (15,2.00 ); Cm (342:347) 1: TOF MS ES+
6.15e648600.0
48570.5
48783.5
48658.5
48630.0
48722.0
48691.0 48754.5
48843.0
48814.5
Heavy ChainLight ChainIntact Mass
6. 6Stability Study
▪ Conditions
– Non-stressed
– Thermal stress
– Oxidative stress
– pH stress
▪ Assays
– PTM by mass spec
– DSF/DLS
– LabChip CE-SDS (R, NR)
– SE-HPLC
– Binding assays
Non-stressed Thermal stress Oxidative stress
Loading Sample ID Sample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2
PPxxxx Non-stress Ag xx 9.2E-10 3.5E+05 3.2E-04 0.0311 0.9981
PPxxxx Heat Ag xx 1.1E-09 3.7E+05 4.1E-04 0.0307 0.9980
PPxxxx Oxidation Ag xx 9.9E-10 3.6E+05 3.5E-04 0.0256 0.9982
PPxxxx kinetics test by Octet with thermal and oxidative treatments
PTM (Glycosylation) Analysis
Peptide b/y Found Modifiers RT (Min) Intensity (Counts) Assigned Intensity (%) Mass Error (ppm)
NST 0 Glycosylation Man5 N(1) 3.3 17716 38.9 1.1
NST 0 Glycosylation G1F N(1) 3.5 107417 42.3 2.4
NST 0 Glycosylation G0 N(1) 3.6 1081 0 1
NST 1 Glycosylation G0F N(1) 3.6 174615 21.7 2.2
NST 1 Deamidation N(1) 23.5 4008 0.1 7.1
7. 7Formulation Study
▪ Formulation Buffers
– 3 recommend antibody formulations
– 6 recommended protein formulations
– Custom formulations
▪ Assays
– PTM by mass spec
– DSF/DLS by UNcle
– CE-SDS (R, NR) by LabChip
– SE-HPLC
– Binding assays
DSF/DLS: PPxxxx in formulation 6
PPxxxx formulation thermostability test by DSF/DLS
Formulations
Tm1
(°C)
Tagg 266
(°C)
Pk 1 Mode Dia
(nm)
PDI
1 20 mM L-Histidine buffer pH6, NaCl 150mM, TWEEN20 0.05% 52.8 47.4 323.6 1.553
2 20 mM L-Histidine buffer pH6, NaCl 150mM, TWEEN20 0.05%, sucrose 5% 53.8 44.4 323.6 1.966
3 20 mM L-Histidine buffer pH6, NaCl 150mM, TWEEN20 0.05%, Arginine 0.2M 51.0 45.3 6.7 0.299
4 20 mM HEPES buffer pH7, NaCl 150mM, TWEEN20 0.05% 54.5 49.2 6.7 0.356
5 20 mM HEPES buffer pH7, NaCl 150mM, TWEEN20 0.05%, sucrose 5% 55.3 50.8 174.0 3.389
6 20 mM HEPES buffer pH7, NaCl 150mM, TWEEN20 0.05%, Arginine 0.2M 52.9 48.7 7.2 0.176
7 20 mM Tris buffer pH8, NaCl 150mM, TWEEN20 0.05% 53.1 49.1 6.2 2.286
8 20 mM Tris buffer pH8, NaCl 150mM, TWEEN20 0.05%, sucrose 5% 54.3 50.0 7.2 1.479
9 20 mM Tris buffer pH8, NaCl 150mM, TWEEN20 0.05%, Arginine 0.2M 52.7 47.6 6.7 0.125
9. 9Characterization from Sequence to Structure
Primary Sequence Level
✓ Intact mass, sequence confirmation and PTM analysis by Mass Spec
✓ Antibody de novo sequencing by Mass Spec
✓ DNA codon check (optimization and cryptic sites) and signal peptide optimization
✓ Protein sequence liability analysis
✓ N-terminal sequencing
✓ Fragmentation by LabChip CE-SDS
✓ Amino acid analysis
Secondary Structure Level
✓ Circular Dichroism (CD)
✓ Fourier transform infrared spectroscopy (FTIR)
Tertiary Structure Level
✓ Aggregation propensity by SE-HPLC and DLS
✓ Thermostability by DSF and DSC
✓ Charge variant profiling by iCE3 and Glycan profiling by Mass Spec
✓ Structure-Function studies including affinity characterization by BLI and SPR, and Fc gamma receptor panel and
FcRn binding profile
✓ Epitope binning