Tools to Detect sub-1ppm Host Cell Proteins in Biological Products at Every Development Stage Milla Neffling, Ph.D. SCIEX, Warrington, UK
Jan 23, 2018
Tools to Detect sub-1ppm Host Cell Proteins in Biological Products at Every Development StageMilla Neffling, Ph.D.
SCIEX, Warrington, UK
2 © 2015 AB Sciex
SCIEX has a Strong Track Record with HCP Detection
In a Single-One Hour Run, SCIEX can:
PROFILE the HCP complement up to 1000s of proteins to sub-ppm level
IDENTIFY HCPs without bias [without inclusion/ exclusion lists]
Provide a CATALOG of HCPs for a process
Provide precursor and fragment information to allow easy MONITORING
Easy transfer to (QQQ or QTRAP®) ABSOLUTE QUANTITATION of HCPs
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HCP
SVA
Modified peptide
Nothing Hides
from SWATH™
Host Cell Protein Studies Using Unbiased Methodology
One data set
can be used for
multiple tasks
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Time ROI for Single-Digit PPM Detection
Post Purification
Fraction 01 – 2Hrs
Fraction 02– 2Hrs
Fraction 03– 2Hrs
Fraction 04– 2Hrs
Fraction 05– 2Hrs
Fraction 06– 2Hrs
Fraction 07– 2Hrs
Fraction 08– 2Hrs
Fraction 09– 2Hrs
Fraction 10– 2Hrs
Post Purification One Run – 60 Minutes
Total Time: ~ 1 Hour Total Time: ~ 20 Hours
2D Method
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Return on Investment Based on Representative Example
System Cost of MS per
Day(Instrument,
service, operator,
overhead)
# of cell
lines (Assume
~1000
Proteins)
# of Runs Time
Taken
Analysis cost
2D
Method
1000 USD 10 10 Cell lines
X 5 Samples
X 10 fractions
X 3 replicates
= 1500 Runs
1500 Runs
X 2Hr Ea
=3000 Hr
=125 Days
1000
USD/Day
X125 Days
= $125,000
SCIEX
1D
Method
1000 USD 10 10 Cell lines
X 5 Samples
X 3 replicates
= 150 Runs
150 Runs
X 1 Hr Ea
=150 Hr
=6.25
Days.
1000
USD/Day
X6.25 Days
=$6,250
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HCP analysis via SWATH™ Acquisition- Study Design
• 48 model HCPs digested and analyzed by IDA MS and MS/MS to
generate a peptide library via ProteinPilot™ software searches at upper
concentration.
• SWATH® acquisition used for all subsequent concentration levels for
quantitative analysis
• A range of concentrations of model proteins were spiked into 10 ug of
IgG1 product digest.
DigestLCMSMS Run (60’)
Generate Library
Analyze Dilution Series
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UniProt Protein Name [Synonym] MW (Da)PPM At Lowest
Dilution UniProt Protein Name [Synonym] MW (Da)
PPM At Lowest Dilution
Gelsolin 82,954 7.56 Ubiquitin-conjugating enzyme E2 C [UbcH10] 20,473 1.87Lactotransferrin 78,289 7.14 Peptidyl-prolyl cis-trans isomerase A [Cyclophilin A] 17,947 1.64Serotransferrin [Apotransferrin] 75,143 6.85 Tumor necrosis factor [TNF-alpha] 17,350 1.58Serum Albumin 66,393 6.05 Myoglobin C 17,051 1.55Catalase 59,583 5.43 Interferon gamma (IFN-gamma) 16,879 1.54Histidyl-tRNA synthetase [Jo-1] 58,223 5.31 Leptin 16,024 1.46Antithrombin-III 49,033 4.47 Cytochrome b5 16,021 1.46Microtubule-associated protein tau [Tau protein] 46,810 4.27 Hemoglobin beta chain 15,867 1.45Creatine kinase M-type [CK-MM] 43,070 3.93 Superoxide dismutase [Cu-Zn] 15,800 1.44Small ubiquitin-related modifier 1 [SUMO-1] 37,420 3.41 Gamma-Synuclein 15,363 1.40Annexin A 5 35,782 3.26 Hemoglobin alpha chain 15,127 1.38NAD(P)H dehydrogenase [quinone] 1 [DT Diaphorase] C 30,984 2.82 Fatty acid-binding protein 14,716 1.34Carbonic anhydrase 2 29,095 2.65 Lysozyme C 14,692 1.34Carbonic anhydrase 1 28,738 2.62 Alpha-lactalbumin 14,070 1.28Ribosyldihydronicotinamide dehydrogenase [quinone] [Quinone oxidoreductase 2] [NQO2] 25,817 2.35 Thioredoxin 12,424 1.13Glutathione S-transferase A1 [GST A1-1] 25,482 2.32 Platelet-derived growth factor B chain 12,286 1.12Glutathione S-transferase P [GST] 23,220 2.12 Beta-2-microglobulin 11,729 1.07C-reactive protein 23,030 2.10 Cytochrome c[Apocytochrome c] 11,608 1.06Ubiquitin-conjugating enzyme E2 I [UbcH9] 22,907 2.09 Ubiquitin 9,387 0.86Ubiquitin-conjugating enzyme E2 E1 [UbcH6] 22,222 2.03 Neddylin [Nedd8] 9,071 0.83Peroxiredoxin 1 22,106 2.01 Interleukin-8 8,381 0.76BH3 Interacting domain death agonist [BID] 21,978 2.00 Complement C5 [Complement C5a] 8,266 0.75GTPase HRas [Ras protein] 21,292 1.94 Insulin-like growth factor IA 7,643 0.70Retinol-binding protein 21,065 1.92 Insulin-like growth factor II 7,464 0.68Ubiquitin-conjugating enzyme E2 C [UbcH10] 20,473 1.87 Epidermal Growth Factor 6,211 0.57
48 Proteins Ranging From
7.56 ppm Down to 0.57
PPM at the Lowest
Dilution Level
The Number of Proteins Analyzed is Not Limited
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SWATH™ HCP Analysis - Optimized LC Method
LCMSMS Run (60’)
Divert
Valve
50 Micron Peeksil
25 Micron Peeksil
To Waste
Pump
Column Oven@ 55℃
Autosampler
Time %B
0 5
4 5
49 35
50 90
55 90
56 5
60 5
Flow Rate 7 ul/min Easily Transferable Method!
0.3x 150mm ChromXP™ C18 Column
10 ug Product on Column
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SWATH Acquisition: 45 Minute Run @ 7 uL/min on 0.3mm x 15cm column
visualized in PeakView® software
HCP Analysis via SWATH™ - Visualization Informatics
Ion Library
Listing - Proteins
Replicates of
Chromatographic
Runs
Overlaid Fragment
Ion Display Linked
to Peptide Display
Spectrum Display
Linked to Peptide
Display
Ion Library Listing
- Peptides
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Statistical Analysis for Tracking and Trending
MarkerView™ software: trending HCPs across different runs
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8.55 ppm4.28 ppm2.14 ppm1.07 ppm
Beta-2 Microglobulin
PEPTIDE: VNHVTLSQPK
137 ppm68.4 ppm34.2 ppm17.1 ppm
Example Data: 1 ppm Detection
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Complement C5 [Complement C5a]
90 ppm45 ppm22.5 ppm11.25 ppm
5.63 ppm2.81 ppm1.40 ppm0.70 ppm
PEPTIDE: AFTEC[MSH]C[MSH]VVASQLR
Example Data: sub-1 ppm Detection
Methods Developed for all Departments
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HCP Workflows to Support Different Groups
Sample-Limited
Environments
Routine
High-Throughput
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MicroFlow HCP Analysis: CASSS Mass Spec Conference
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HCP Workflows to Support Different Groups: High Flow
• Typically load 30-40 ug product per run
• 2mm x 250mm C18 Column or UPLC
column
• Gradients between 60 and 90 min long
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High Flow HCP Analysis: WCBP Conference
Poster presented at WCBP 2015, Washington DC
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HCP Workflows to Support Different Groups: Ultra low Flow
• Early development sample-
constrained (~1 ug)
• Very low flow rate = Lower ion
suppression.
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Analyzed using CESI-MS on a TripleTOF®5600 System
Transitions 536.3 (MS) and 814.5 (MS/MS) m/z
Concentration (ppm)
316 ppb
Linear scale
Myoglobin Peptide VEADAGHGQEVLIR (+3)
CESI-MS works on
all TripleTOF®
Systems
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MSMS Quantitation of All Product Peptides and Modifications
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MSMS Quantitation of All Product Peptides and Modifications
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Conclusions
Unbiased and Comprehensive Analysis
Sub-Single Digit PPM HCP Detection
Simple, 1D Generic Methodology
Substantial Return on Investment