host cell protein analysis of biotherapeutics using … cell protein analysis of biotherapeutics...

Host Cell Protein Analysis of Biotherapeutics Using LC/MS

Alex Zhu, Ph.D.

Applications Scientist

Agilent Technologies Inc.,

June, 2016

6/21/2016

For Research Use Only. Not For Use in Diagnostic Procedures.

1

Host Cell Proteins (HCPs) in Protein Therapeutics

6/21/2016

For Research Use Only. Not For Use in Diagnostic

Procedures.

2

Host cells

Therapeutic

antibodies

HCPs

Harvest

supernatant

Downstream

processing (e.g.

Affinity purification)

Residual HCPs could:

• Reduce drug efficacy

• Induce adverse patient

immunoreactions

Wash +

Elution

Traditional HCP Analysis Workflows

Golden Standard in Industry: anti-HCP enzyme-linked

immunosorbent assay (ELISA)

• Strengths:

- Very sensitive (ppb detection limits)

- High level of reproducibility

- High-throughput (plate format, automation)

• Challenges:

- Lack of specificity, no identification of individual HCPs

- Lack of coverage for non-immunoreactive HCPs

- Quantitation is based on a cohort of HCPs

6/21/2016


Procedures.

3

LC/MS as a Solution for Host Cell Protein Analysis

6/21/2016


Procedures.

4

Advantages:

• Can identify individual HCPs including non-

immunoreative proteins

• Doesn’t require protein specific antibodies

• High sensitivity (low ppm)

• Provide both qualitative and quantitative

information

HCPs can be analyzed with 1D or 2D LC/MS

Increasing demand on high flow instead of low flow

workflow

General HCP Identification Workflow

6/21/2016


Procedures.

5

Sample Preparation

• Sample Extraction

• Peptide digestion

• Peptide fractionation

LC/MS Analysis

• 1D or 2D LC separation

• MS analysis (Iterative directed MSMS, 6550)

Data Processing

• Database search and identification (Spectrum Mill)

LC/MS HCP Analysis

• 1D-LC/MS

• Offline 2D-LC/MS

- Fraction Collector (High pH or

SCX)

- Automatic peptide

fractionation using AssayMAP

(High pH or SCX)

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Procedures.

6

1D LC/MS

High pH RPFraction 0







Challenge for LC/MS analysis of HCPs

• Low abundance of HCP peptides co-elute with very intense “product” mAb

peptides

• Need broad dynamic range and better separation such as 2D-LC

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Procedures.

7

Broad Dynamic Range (>4 Orders)For Co-Eluting Peptides

June 21, 2016


Procedures.

8

TIC

Peptide Precursor Intensity

TPVSEKVTK 494.7869 5.33^5

LTPSPR 335.6981 2.49^8

WYPGSVKR 517.7762 1.39^6

KTAEWLTK 528.7610 9.46^3

WYQQKPGK 345.5188 7.36^6

Different Modes of MS-based Approaches

• Shotgun (Data-dependent or discovery)- Selection of peptide ions detected in the sample and their fragmentation based on signal

intensity.

• Targeted- Detection of a set of predetermined fragment ions from precursor ions that are anticipated,

but not necessarily detected, in a survey scan.

• SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra)

- The first quadrupole sequentially steps in precursor windows across the mass range ofinterest and passing the ions into the collision cell recursively during the LC separation.

• MSE

- DIA approach that acquires MS1 and MS2 mass spectra at the same time.

• Directed- The hallmark of directed MS is the selection and fragmentation of a predetermined set of

peptide ions detected in a survey scan.

• Iterative Directed - Recursive directed MS analysis.

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Procedures.

9

Iterative Directed MS Analysis of HCPs

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Procedures.

10

x10 7

0

0.5

1

1.5

2

2.5

Counts vs. Acquisition Time (min)5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

MassHunter Qualitative Analysis

-“Molecular Feature Extraction”MassHunter Acquisition

- “Preferred List”

Iterative Directed MS Analysis of HCPs

6/21/2016


Procedures.

11

For Research Use Only. Not For Use in Diagnostic Procedures.

x107

0

0.5

1

1.5

2

2.5

0

0.5

1

1.5

2

2.5

Counts vs. Acquisition Time (min)5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

Counts vs. Mass-to-Charge (m/z)1065 1070 1075 1080 1085 1090 1095 1100 1105 1110 1115 1120 1125 1130 1135

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

Counts vs. Mass-to-Charge (m/z)1065 1070 1075 1080 1085 1090 1095 1100 1105 1110 1115 1120 1125 1130 1135

Spectrum Mill

“AMRT Export”

Iterative Directed MS is Superior to Data-dependent Acquisition Method, 1D-LC/MS Workflow

6/21/2016


Procedures.

12

Total HCPs Identified

(≥ 2 peptides): 10

73Data-dependent

Directed MS

A B

Heavy chain

Light chain

90.4%

92.2%

98%

97.2%

Data-

dependent

Directed

MS

mAb Sequence Coverage:

Offline 2D-LC/MS Workflow for Identifying HCPs in Purified Antibodies

6/21/2016


Procedures.

13

Trypsin-digested

mAb samples

High pH-

RPLC

gradient

SCX

gradient

Collect

fractions

RP-LC/MS/MS

Analysis on

6550 Q-TOFor

Fraction collector or AssayMAP

Data-dependent vs. Directed MS, 2D-LC/MS

6/21/2016


Procedures.

14

Total HCP peptides identified

(Protein IDs ≥ 2 peptides): 54

1715 22SCX

High pH

B

Total HCP peptides identified

(Protein IDs ≥ 2 peptides): 242

8567 90SCXHigh pH

D

Total HCPs identified


1421 32SCX

High pH

Directed MS method

C

Data-dependent method

Total HCPs identified


66 7SCX

High pH

A

Peptide Intensity Reproducibility

6/21/2016


Procedures.

15

0

20

40

60

80

100

120

IGYPAPNFK

GISSV

PTK

LVQAFQ

FTDK

LQDMSIR

SIETDK

LTPEEIER

DTIVPSR

LASA

YGATQ

LRLG

SLVK

DDVALK

LGPLVEQ

GRQR

GLFIIDDK

ADEG

ISFR

TIAQDYG

VLK

MGAPEA

GMAEYLFDK

YFLH

QSH

EER

SSQVPPMLQ

DTD

KTK

LMDDLD

RLD

GSTnFTR

DLTLCAHK

ILDANVVCR

GDHVTN

LRAEA

ESmYQ

IkVIIH

PDYR

SELAVSSELDLLK

ELTIGSK

LVnGASA

SEGR

NTV

VPTK

STItLD

WK

VLSEERDQLLSR

NAPEYDGGK

ATA

VMPDGQFR

LALD

IEIATYR

LHIGNYnGTA

GDALR

LYTSSTWSTLVTD

RnITAIVK

MVNDAEK

VYEGER

PLTK

VEIFYR

TIVPSR

QNYH

QDSEAAINR

ITITNDQNR

ISSV

PTK

TTTA

LTTA

SPSQ

VR

SQVPPMLQ

DTD

KTK

LANGYKFLSPGR

LPVADR

ELGDHVTN

LRSCYFDRDDVALK

LGADmED

LRNR

SVDEILR

HTLGHSES

ITAIVK

SSSLPK

VDVDCMPVVR

LQDAEIAR

Peptide Intensity Reproducibility (3 Replicate Runs)

(High pH RP Fractionation + RP LCMS)

54 unique HCP peptides identified

33 peptides (61.1%) < 20% RSD

51 peptides (94.4%) < 50% RSD

RS

D%

From Identification to Quantitation

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Procedures.

16

Method Validation/QuantitationDiscovery

DMRM Chromatograms for Selected HCP Peptides

6/21/2016


Procedures.

17

5x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

Counts vs. Acquisition Time (min)4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

TIC

Overlaid MRMs

ALPAPIEK

NQVSLTCLVK

DSTYSLSSTLTLSK

SSSLPKFLEQQNQVLQTKVTDFGPK

AQELDPAGHK ETLSESVK SVNQSLLELHK

GAPGGSLR

2x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

8.5

Counts vs. Acquisition Time (min)3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2

2x10

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

Counts vs. Acquisition Time (min)38.2 38.4 38.6 38.8 39 39.2

Calibration Curves

6/21/2016


Procedures.

18

Concentration (fmol/µl)

KRT1

FLEQQNQVLQTK

R2 = 0.999

7.4 ppm

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 100 200 300 400 500 600

x106

Zoom

0

0.5

1.0

1.5

2.0

2.5

3.0

0 10 20 30 40 50 60

x105

Resp

onse

0

2

4

6

8

10

12

0 100 200 300 400 500 600

Intermediate filament containing protein

TNAENEFVTIK

R2 = 0.999

x105

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 10 20 30 40 50 60

x105

Zoom

37.33 ppm


Resp

onse

SPTAN1

SLSAQEEK

R2 = 0.9936

1690 ppm

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 100 200 300 400 500 600

x103

0

0.05

0.10

0.15

0.20

0.25

0.30

0 10 20 30 40 50 60

x103

Zoom


Resp

onse

KRT18

LAADDFR

R2 = 0.9991

2.8 ppm

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 100 200 300 400 500 600

Zoom

0

1.0

2.0

3.0

4.0

5.0

6.0

0 10 20 30 40 50 60

x105


Resp

onse

x106

Summary

• A combination of directed MS method with offline 2D fractionation yielded the

highest number of HCP IDs.

• Multiple first dimension fractionation strategies may better facilitate

comprehensive identification of HCPs.

• Highly sensitive quantification was achieved at low ppm levels of HCPs on the

QQQ with high flow 1D-LC/MS

6/21/2016


Procedures.

19

Validation/QuantitationDiscovery

Acknowledgement

Jordy Hsiao

Jing Chen

Zach Van Den Heuvel

Steve Murphy

Te-Wei Chu

Greg Staples

6/21/2016


Procedures.

20

host cell protein analysis of biotherapeutics using … cell protein analysis of biotherapeutics...

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