tools to detect sub-1ppm host cell proteins in biological products at every development stage

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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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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Conclusions

Unbiased and Comprehensive Analysis

Sub-Single Digit PPM HCP Detection

Simple, 1D Generic Methodology

Substantial Return on Investment

tools to detect sub-1ppm host cell proteins in biological products at every development stage

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