©2009 Waters Corporation

A Unique LCA Unique LC--MS Assay for Host Cell MS Assay for Host Cell Proteins(HCPsProteins(HCPs) in Biologics) in Biologics

Catalin Catalin DoneanuDoneanu, Ph.D., Ph.D.Biopharmaceutical Sciences, WatersBiopharmaceutical Sciences, Waters

September 16, 2009September 16, 2009

Mass Spec 2009Mass Spec 2009

Host Cell Proteins (Host Cell Proteins (HCPsHCPs))

Recombinant Proteins produced in host cells

Proteins from cells can co-purify with therapeutic protein of interest

—e.g. Chinese Hamster Ovary (CHO) cell proteins in recombinant monoclonal antibody therapeutics

Purification steps should remove contaminants. Low levels can remain because of:

—Poor process control

—Process changes: can affect HCP

pattern and abundances

Biopharm International 2008, 13, Number 6, 38-45

Guidelines Governing Guidelines Governing HCPsHCPs

Safety drives the need for removal/minimization

—Link between HCPs and immunogenicity

European regulations in effect since 2007

—‘6.2 Validation of the purification procedure - …. The ability of the purification process to remove other specific contaminants such as host-cell proteins …should also be demonstrated’

—ICH Guidelines: 2009 review in progress (http://www.emea.europa.eu/pdfs/human/bwp/BWPworkprogramme.pdf)

Importance of Importance of HCPsHCPs: : Drug Approval or FailureDrug Approval or Failure

2008: a human growth hormone was approved by FDA, after initial denial

— “The cause of immunogenicity was linked to excess host cell protein contamination, which was resolved by the

manufacturer with additional purification steps”. (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2638545)

2006: an interferon biosimilar was rejected by EMEA— “The reasons for the rejection by the EMEA included quality and

clinical differences between [the biosimilar product] and the reference product, … inadequate validation of the process for the finished process and insufficient validation of immunogenicity testing.”

Challenges of HCP AnalysisChallenges of HCP Analysis

Thousands of possible protein contaminants

HCPs can be present at extremely low levels— Typically ppt to ppm (relative to biotherapeutic)

— Guidelines suggest monitoring to ppm (1-100ppm)

Developing methods is expensive and time consuming

Biopharm International 2008, 13, Number 6, 38-45

Narrow dynamic range (<100)

Comparison of Current HCP MethodsComparison of Current HCP Methods

Requirements for HCP Identification and Requirements for HCP Identification and Quantification AssayQuantification Assay

Ability to detect protein impurities down to 0.001%

(5 orders of magnitude)

A non-targeted, unbiased approach for HCP identification and monitoring

Fast, high-throughput measurements

HCP Analysis WorkflowHCP Analysis Workflow

Workflow Overview:

—Enzymatic digestion of sample into peptides

—2D-LC/MSE with IDENTITYE to DISCOVER contaminant proteins

—2D-LC allows more sample loading

—Develop specific host cell protein databases

—(Top3 peptides for absolute quantitation, label-free *)

—Data mined for MRMs using VERIFYE

—Transfer to Tandem Quad for targeted quantitation (e.g. using isotopically labeled peptides)

* Silva et al. Moll Cell Proteomics, 2006, 5, 144-156.

MSPrecursor

MSE

Fragments

Retention Time

MSMSE E Acquisition : Alternating Low/High Acquisition : Alternating Low/High Energy ScansEnergy Scans

Comprehensive Peptide Ion Comprehensive Peptide Ion Accounting: IDENTITYAccounting: IDENTITYEE

Geromanos et al. Proteomics 2009, 9, 1683 – 1719.

0-56% B in 70 minutes 20 mM NH4OH pH 10

1

100

%

Bovine_Hemoglobin_Digest_Stored_091803_1 1: Scan ES+ TIC

4.51e928.55

18.75

17.36

16.3010.99

8.91

4.704.29

6.29

11.4013.24

11.9314.09

23.86

22.79

22.39

19.6119.93

27.0026.68

26.51

26.06

35.0530.68

31.41

34.27 36.19

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00Time1

100

%

TIC4.37e8

18.95

15.79

8.53

6.775.705.214.10

14.0310.38

9.64

13.2111.73

16.41

18.58

18.21

25.68

22.39

21.0419.89 24.20

29.41

29.0025.92

41.26

35.85

35.48

37.65

39.5841.92

pH 10

pH 2.6

neutral acidic

basicacidic

basic

pH 10.020 mM ammonium formate0-42% acetonitrile in 15 min

pH 2.60.2% Formic acid

0-42% acetonitrile in 90 min

Gilar M. et. al, J. Sep. Sci. 2005, 28, 1694-1703

2D2D--LC Separation using High/Low LC Separation using High/Low pH Reversed PhasespH Reversed Phases

Fluidic Configuration for 2D-Chromatography and Online Dilution: Sample Loading

WASTE

TRAP

BSM1

0.3 x 150 mm

ASM

BSM2

TEE

1.0 x 50 mm

5 µm XBridge

1.7 µm BEH

MS

A: 0.1% FA, pH = 2.4

A: 20 mM Amm Formate, pH = 10.0

0.1% TFA, pH = 2.1

HTM Valve

Injection Valve

0.5 x 20 mm

100 µL/min

10 µL/min

4 µL/minB: ACN

B: 0.1% FA in ACN

Fluidic Configuration for 2D-chromatography and Online Dilution: Peptide Elution

WASTE

TRAP

BSM1

0.3 x 150 mm

ASM

BSM2

TEE1.0 x 50 mm

5 µm XBridge

1.7 µm BEH

MS

A: 0.1% FA, pH = 2.4

A: 20 mM Amm Formate, pH = 10.0

0.1% TFA, pH = 2.1

HTM Valve

Injection Valve

0.5 x 20 mm

100 µL/min

10 µL/min

4 µL/minB: ACN

B: 0.1% FA in ACN

A

B

C

A – direct injection of 1 picomole of ENL digest on a 300 µm x 150 mm BEH column

B – “simulated 1D” run using a single elution step (50% Eluent B) – 1 picomole ENL digest

C – Fraction 3 of the 2D-LC run – 60 fmoles ENL digest on column

All separations were performed using a 30 min gradient (3-40% ACN, 0.1% FA)

Same

Chromatographic

Performance

Chromatographic PerformanceChromatographic Performance

Mass chromatograms of T43 peptide from ENL digest;

60 fmoles of digest were loaded in each 2 D-LC experiment.

This peptide (VNQIGTLSESIK) eluted only in Fraction 3.

24 h later

48 h later

Reproducibility of 2D ChromatographyReproducibility of 2D Chromatography

1D Chromatography1D ChromatographyNo Fractionation,60 No Fractionation,60 μμg Sample Loadedg Sample Loaded

2D Chromatograms2D Chromatograms5 Fractions, 60 5 Fractions, 60 μμg Sample Loadedg Sample Loaded

10.8 % ACN

50 % ACN

14.0 % ACN

16.7 % ACN

20.4 % ACN

Protein Name

Species Accession No.

MW (kDa)

Protein Conc. in Sample

(fmol/ul) (ppm) (Ratio) [log(DR)]

mAb Humanized n/a 145.2 32,000 n/a

LA Bovine P00711 16.3 1,000 3,500 285 2.5

ADH Yeast P00330 36.8 200 1,600 625 2.8

PHO Rabbit P00489 97.3 80 1,675 600 2.8

BSA Bovine P02769 69.3 20 300 3,300 3.5

ENL Yeast P00924 46.8 4 40 25,000 4.4

Dynamic Range (DR)

MIXMIX--5 proteins spiked in a 5 proteins spiked in a mAbmAb

Controlling False Positive IdentificationsControlling False Positive Identifications

Random peptide sequences added as Decoy strategy to ensure that identified peptides are real

— 13,600 entries from Swissprot (mouse and hamster proteins)

— 6 protein sequences from LA, ADH, PHO, BSA, ENL, porcine trypsin,

— 2 sequences from heavy and light chain sequences of MAB

— Equal number of random sequences (13,608)

— Total number of protein sequences: 27,216

False Positive Rate of Protein Return: 5% (user adjustable)

Concentration range 10 to 100 ppm

Lower confidence hits (nearing random) not reported

ENL was detected in all three 2D-LC/MSE replicates when present at a concentration of 40 ppm.

HCPHCP’’ss can be identified over 5 orders can be identified over 5 orders of magnitude in concentrationof magnitude in concentration

MS Spectrum of T43 ENL peptide in MS Spectrum of T43 ENL peptide in the the mABmAB digestdigest

T43

(A) direct injection of 1 pmole of ENL digest on a 300 µm x 150 mm BEH column

Simultaneous protein identification and quantitation

HCP’s Identified in the mAbBiosimilar

RSD = 8 %

Reproducible Chromatography

LC Conditions:

— Column: 2.1 x 150 mm BEH130 C18, 1.7 µm particles

— Flow rate: 300 µL/min

— Gradient: 3% to 40% ACN in 10 min

— Sample: 200 fmoles ENL digest on column

TGNPTVEVELTTEK

Reproducibility of the MRM assayReproducibility of the MRM assay

Two MRM transitions from the same peptide

Example of MRM InterferenceExample of MRM Interference

Summary on the Advantages of the Summary on the Advantages of the HCP AssayHCP Assay

— Confident Identification of individual HCPs

— Quantitation of each identified HCP

o Label-free in the discovery stage

o Using isotopically labeled peptides (Tandem Quadrupole)

— Much faster development time than immunoassays

— Provide a multi-purpose platform for many other tasks

— Sensitivity comparable to ELISA assays

— Applicable to subunit (recombinant) vaccines

The 2D-LC/MSE setup is able to identify low abundance protein contaminants present in biopharmaceuticals over 5 orders of magnitude

A high-throughput MRM assay on a tandem quadrupole can quantify these protein impurities (absolute quantification can be done using isotopically labeled peptides)

The combination of 2D-LC/MSE and tandem quadrupole MS provides a total system solution for HCP analyses

Conclusions and Future Directions

AcknowledgmentsAcknowledgments

Keith Fadgen

Martha Stapels

Weibin Chen

St John Skilton

Jim Kehoe

Scott Berger

Jeff Mazzeo