a unique lc-ms assay for host cell proteins(hcps) in...
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©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