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Validation of LC-MS/MS Methods for the Determination of Large Molecules: Promises and Challenges
Mark Rose, Linh Tran, Amir Sharifi, and Hongyan Li Amgen Inc., Thousand Oaks, CA
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1. Why do we need validated LC-MS/MS methods for LM? 2. Key components of a robust validation
a. Extraction b. LC-MS/MS
3. Validated methods in the literature 4. Amgen case study 5. Other considerations
a. Structure/Modality b. Project stage
6. Bioanalytical strategy - decision tree 7. Regulatory concerns
Presentation Outline
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Why do we need LC-MS/MS methods for large molecules?
1. When reagents are unavailable
2. When reagents are non-specific
3. Cases where complimentary specificity is required.
4. Non-traditional molecules a. Antibody drug conjugates
b. Bi-specific molecules
c. Endogenous biomarkers
5. When ADA's interfere with the LBA
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Biotherapeutics are becoming more complicated.
• Multiple constructs with multiple sites for catabolism or degradation
• All exhibit different in vivo PK properties
Bioactive Peptide 20kDa PEG
Peptide
Fc-bivalent peptide
Fc-monovalent peptide Fc-bivalent
peptide/ Anti KLH Ab
Fc Loop-peptide
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1
10
100
1000
10000
100000
0 48 96 144 192 240 288 336 384 432 480 528
Seru
m c
onc
(ng/
mL)
Time (hr)
Total antibody concentration
Intact conjugate concentration
Construct Development Requires a Comprehensive Understanding of Catabolism
Measuring just the antibody with an LBA would give a different conclusion of stability.
LC-MS/MS LBA
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1
10
100
1000
10000
100000
0 48 96 144 192 240 288 336 384 432 480 528
Seru
m c
onc
(ng/
mL)
Time (hr)
Total antibody concentration
Intact conjugate concentration
A Stable Peptide Warhead Increases Circulation Half-life of the Intact Molecule
Selective assays are important for optimizing complex conjugates
LC-MS/MS LBA
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Clean-up Strategies and Achieved LC-MS/MS Sensitivity – 46 Publications
7
Bioanalytical LC–MS/MS of protein-based biopharmaceuticals Irene van den Broeka, et.al., J. Chrom. B, (2013) 929 ,161– 179
IC: Immunocapture, PP: Protein Precipitation, SPE: Solid Phase Extraction, LC: Liquid Chrom., SEC: Size Exclusion Chrom.
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Detecting Hepcidin (DTHFP ICIFC CGCCH RSKCG MCCKT) Using LC-MS/MS (m/e 698.1 →354.1)
8 8
Quad 1 Quad 2 Quad 3
HPLC
Electrospray
Channel Electron Multiplier
N2-CAD
500 600 700 800 900 1000 1100 1200
698.1[4+]
930.8[3+]
D T H F P I C I F C C G C C H R S K C G M C C K T
y19
b3
y21y22
b4
y23
b24b6 b23b5
y20
D T H F P I C I F C C G C C H R S K C G M C C K T
y19
b3
y21y22
b4
y23
b24b6 b23b5
y20
300 400 500 600 700 800 900 1000 1100 1200
b3354.1
b4501.2
Hepcidn[4+] 698.1
b5598.2
y23[4+]644.3 y24[4+]
669.2
y21[3+]763.2
y22[3+]812.6
y23[3+]858.5
y19[2+]1039.5
y21[2+]1144.8
Hepcidin DTHFP ICIFC CGCCH RSKCG MCCKT
Highly Specific Detection – So what’s the problem?
m/e m/e
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Farrah et. al., A High-Confidence Human Plasma Proteome Reference Set with Estimated Concentrations in PeptideAtlas,, Mol. Cell. Prot. (2011) 10 (9) 1-14.
20,433 distinct peptides from1929 identified protein
sequences
Endogenous Proteins Identified in Plasma
Conclusion: Having an extraction method specific to your analyte is important.
!The same twenty amino acids rearranged > 20,000 ways!
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Examples of Qualified or Validated Methods Published Since 2012 Year Author Analyte Internal
Standard LLOQ Matrix Extraction
Technique 2012 Hongyan Li
(Amgen) mAb • SIL Peptide
• SIL Flanking Peptide
• SIL mAb
100 ng/mL Rat and Monkey Serum
Magnetic Beads
2012 Mireia Fernandez Ocana (Pfizer)
Free and bound mAb
SIL Flanking Peptide
7.03 ng/mL Human Serum Magnetic Beads
2013 Hao Jiang (BMS)
Coadministered mAbs
SIL Flanking Peptide
• 5 ug/mL • 25 ug/mL
Monkey Serum Pellet Digestion (no extraction)
2013 Daniel Wilffert (U. of Groningen)
rhTRAIL SIL rhTRAIL 20 ng/mL Human and Mouse Serum
IMAC Chromatography
2013 Guowen Liu (BMS)
mAbX SIL Peptide 1 ug/mL Mouse Serum Pellet Digestion (no extraction)
2014 Ichio Onami (Chugai Pharm Company)
RANKL Lysozyme 3.13 ng/mL Mouse Serum Magnetic Beads
2014 Hao Jiang (BMS)
• ADA • mAb • hu IgG
SIL Flanking Peptide
• 1 ug/mL • 1 ug/mL • 1 ug/mL
Human Serum BEAD Extraction and Acid Dissociation
Not many validated LM LC-MS/MS methods published during the last two years.
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General Observations for Recently Validated Methods
§ Precision and accuracy are dependent on choice of internal standard and extraction/enrichment method. – Non-specific extraction method = high LLOQ – Very specific extraction method = low LLOQ – Appropriate internal standard = good precision and accuracy
§ Method formats affect LLOQ – Pellet digestion methods can be validated when a high LLOQ is
sufficient. – Affinity extraction is required for methods with LLOQ < 0.5 ug/mL
11
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Limited Sample Preparation (Pellet Digestion) with LC-MS/MS Analysis
Biological sample
Denature, Cys reduction
Tryptic digestion
UPLC-MS/MS
Protein Precipitation
Add SIL-Peptide internal standard
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Validated Methods Using Pellet Digestion and LC-MS/MS
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Fully Validated LC-MS/MS Assay for the Simultaneous Quantitation of Coadministered Therapeutic Antibodies in Cynomolgus Monkey Serum
mAb-A LLOQ : 5 µg/mL mAb-B LLOQ: 25 µg/mL Intra-assay Precision: <10% Accuracy: < 5.4%
Pellet digestion: a simple and efficient sample preparation technique for LC–MS/MS quantification of large therapeutic proteins in plasma
Protein 1 (150 kDa) LLOQ : 2.6 µg/mL Intra-assay Precision: <16.2% Accuracy: < 10%
Acceptable precision and accuracy possible without immunoaffinity reagents - High LLOQ
Zheng Ouyang, et.al., Bioanalysis (2012) 4(1), 17–28
Hao Jiang, et.al., Anal. Chem. (2013) 85, 9859−9867.
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More Sensitive LC-MS/MS Assays for mAbs Incorporate Immunoaffinity Extraction
Biological sample
Denature, Cys reduction
Tryptic digestion
UPLC-MS/MS
Immunoaffinity protein capture
Add SIL-Peptide internal standard
Streptavidin Tips
Streptavidin Magnetic Beads
Format Chosen at Amgen for Validation
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LC-MS/MS Method Validation for the Determination of Hu-IgG2 mAb in Preclinical Serum
• Column: Phenomenex Aeris Peptide XB C18 , 3.6 mm, 2.1 x 100 mm
• Mobile phase: • A: 5% ACN/water/0.1% formic acid • B: 95% ACN/water/0.1% formic acid
• Gradient elution: 5 min • Injection volume: 10 uL
Standard calibration curve range: 50 – 10,000 ng/mL
Enzyme digestion LC-MS/MS Immunoaffinity Extraction 60 min 45 min + 10 min 5 min/injection
DP CE EP CXP(volts) (volts) (volts) (volts)
VVSVLTVVHQDWLNGK 599 798.8 100 65 21 10 12VVSVL*TVVHQDWL*NGK 603.7 805.8 100 65 21 10 12
Compound Q1 Q3 Dwell time (ms)
XIC of +MRM (2 pairs): 599.000/798.800 Da ID: Hu-G2-Fc-VVSV from Sample 55 (50) of 02Aug2013AMG589_cyno_manprep_pre-qual_w-ba... Max. 690.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0
100
200
300
400
500
600
690
In
te
ns
it
y,
c
ps
2.60
2.88
2.78
XIC of +MRM (2 pairs): 603.670/805.800 Da ID: Hu-SILAC-Hu-G2-VVSV from Sample 55 (50) of 02Aug2013AMG589_cyno_manprep_pre-qu... Max. 3.1e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
In
te
ns
it
y,
c
ps
2.60
XIC of +MRM (2 pairs): 599.000/798.800 Da ID: Hu-G2-Fc-VVSV from Sample 55 (50) of 02Aug2013AMG589_cyno_manprep_pre-qual_w-ba... Max. 690.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0
100
200
300
400
500
600
690
In
te
ns
it
y,
c
ps
2.60
2.88
2.78
XIC of +MRM (2 pairs): 603.670/805.800 Da ID: Hu-SILAC-Hu-G2-VVSV from Sample 55 (50) of 02Aug2013AMG589_cyno_manprep_pre-qu... Max. 3.1e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
In
te
ns
it
y,
c
ps
2.60
LLOQ: 50 ng/mL
SIL-VVSV IS
Slide: Hongyan Li
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LC-MS/MS Method Validation for the Determination of Hu-IgG2 mAb in Preclinical Serum
Hu-IgG2 Conc. (ng/mL)
Calibration Standards
QC Samples QC Sample Description
50 X X LLOQ QC (validation only)
100 X 150 X Low QC 250 X 500 X 750 X Mid QC
1000 X 2500 X 5000 X 7500 X High QC
10,000 X X ULOQ QC 100,000 X Dilution QC
Validation Format Fundamentally Similar to LC-MS/MS and LBA Assays
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LC-MS/MS Method Validation for the Determination of Hu-IgG2 mAb in Preclinical Serum
Hu-IgG2 Method validation parameters using LC-MS/MS assay with immunoaffinity extraction
• Accuracy • Precision • Sensitivity • Stability • Reproducibility
• Calibration curve and regression • Dilution linearity and hook effect • Selectivity • Matrix effect
Parameter Cyno Rat Mouse Validated
Range 50-10,000 ng/mL 50-10,000 ng/mL
50-10,000 ng/mL
Precision % CV and % Bias (intra-assay)
<9.8% and -6.4 to 9.3%
<7.6% and -5.0 to 4.6%
<11.3 and -12.6 to 6.0%
Precision % CV and % Bias (inter-assay)
<7.0% and -1.1 to 8.5%
<6.5% and -2.0 to 0.0%
<10.5% and -5.9 to 0.2%
Validation results were similar to standard small molecule LC-MS/MS assays
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Considerations in Validating a Large Molecule LC-MS/MS Assay
1. Large and small molecule assay components require a hybridization of regulatory guidances. a. Large molecule, e.g., affinity extraction and large molecule
standardization b. Small Molecule, e.g., UPLC, MS/MS, solid phase extraction
2. Little regulatory interactions reported thus far concerning validated large molecule LC-MS/MS – data gathering required.
3. Caution recommended due to the multistep complicated nature of the assays
4. Some validation experiments seem scientifically unwarranted, but will still be performed until a knowledge base is obtained.
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Hybrid Parameters for Validating a mAbs Method
Validation Parameter Conditions Specific Details n Acceptance Criteria
Accuracy & Precision
(A&P) Calibration Standards (CS)/ QC Preparation
CSs and QCs are prepared separately. Freshly prepared CSs used for each analytical batch. Single use QCs prepared, and stored at -60° to -80°C.
- 3 A&P batches from a minimum of 2 separate days. - 8 freshly prepared non-zero CSs in duplicate (n=2) in each analytical batch. - n=6 at each QC level (in each analytical batch)
Minimum 75% of CS ≤ ± 20.0%* of target conc. (*≤ ± 25.0% at LLOQ). At least one calibrant at the LLOQ and ULOQ must meet the acceptance criteria. Mean intra- and inter-assay bias ≤± 20.0%*, CV ≤ 20.0%*. (*25.0% at LLOQ).
Selectivity Analyze with IS (blank) and without IS (double blank). 6 different sources/subjects/animals. - n=1 for each lot (B)
- n=1 for each lot (DB).
For 5 of 6 sources, no response >20.0% of mean response of LLOQ. For IS, no response >5.00% of mean IS response.
Matrix Effect Spike 6 different sources of biomatrix at the LOQ QC conc.
Evaluate LOQ in 6 different sources/subjects/animals.
analyze in triplicate (n=3) for each source
For 5 of 6 sources, the measured conc. for 2 of 3 (>66%) samples for each source ≤ ±20.0% of target spiked conc..
Dilution Accur.
Dilution accuracy for each dilution factor used will be assessed using QC samples. Analyze 6 replicates for the dilution scheme.
Dilution factor of 10, 100 and 1000 for dilution QC at100,000 ng/mL. n=6 for each dilution factor Mean intra-assay bias ≤ ±20.0%,
CV≤ 20.0%.
Benchtop Stability
(BTS)
2 conc. (low and high QC), n≥6 for each conc. Store at least 4 hours at RT. A minimum of 5 of 6 results (>80%) must be available at each level.
Low and high QC samples are thawed at room temperature and kept at this temperature for at least 4 hours. The stability samples are processed and quantitated using freshly prepared calibration samples.
n=6 for each level (HQC and LQC) and for each condition
The mean measured conc. at each level ≤ ±20.0% of target (nominal conc.). CV ≤ 20.0%.
LBA Based Common to Both LC-MS/MS Based
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Validation Parameter Conditions Specific Details n Acceptance Criteria
Long Term Stability
(LTS)
- 2 conc. (low and high QC). Analyze n≥6 for each conc. at each stability time-point. - Samples must be analyzed using freshly prepared QSs; batches contain QCs to accept/reject. - A min of 5 of 6 results (>80%) available for evaluation at each level
- Bulk HQC and LQC levels will be prepared, aliquoted and stored at -60° to - 80°C. - Each condition is subjected to only 1 freeze/thaw cycle. - Storage 3 weeks, 1 month, 3 months, and 6 months (or as needed to support study samples).
n=6 for each level (HQC and LQC) and for each condition
The mean measured conc. at each level ≤±20.0% of target (nominal conc.). CV ≤20.0%.
Storage at -15° to -25°C targeted this must meet acceptance criteria. for approximately 4 weeks
- HQC and LQC from bulk preparation will be used for this experiment. - HQC and LQC aliquots will be transferred to -15° to -25°C for approximately 4 week storage stability.
Freeze/Thaw Stability
(FTS)
- 2 conc. (LQC and HQC), 3 F/T cycles (min 12 hours frozen for each cycle). - Analyze n≥6 for each conc. Samples must be analyzed using freshly prepared CSs. A min of 5 of 6 (>80%) must be available for at each level.
- Bulk HQCs and LQCs will be stored at -60° to -80°C. - All F/T conditions will be assayed on the same plate.
n=6 for each level (HQC and LQC) and for each condition
The mean measured conc. at each level ≤±20.0% of target (nominal conc.). CV ≤20.0%.
Carryover Two control blanks, each injected after a ULOQ CS in one batch.
If this spec. cannot be met, alternative spec to control carryover may be employed.
No response >20.0% of the mean response of LLOQ CS.
Autosampler Reinjection
Reproducibility
Store a batch consisting of a minimum of CSs and a total of 18 QCs (n=6, low, mid, high conc.). Inject after preparation, storage at the autosampler temperature for at least 24 hours and re-inject.
Allows for reinjection of samples when the original batch fails for instrumental or reasons unrelated to sample preparation.
1 A&P batch
Both original and re-injected batches meet routine acceptance criteria
LBA Based Common to Both LC-MS/MS Based
Hybrid Parameters for Validating a mAbs Method
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Hybrid Parameters for Validating a mAbs Method Validation Parameter Conditions Specific Details n Acceptance Criteria
Interference Anti Hu-IgG2 PAb
- Approximate Molar Ratios: 0.01:1, 0.05:1, 0.1:1, 1:1, 1:5, 10:1, 100:1.
- QC level of Hu-IgG2 will be used. - Prepare all conditions in 100% cynomolgus
monkey serum
n= 1 per conc., including blanks (positive and negative controls).
%Bias will be calculated from nominal spiked Hu-IgG2 (MQC level).
Parallelism If ADA is observed, incurred samples with ADA will be tested.
- Perform at least 4 dilutions, and each dilution is prepared in 100% cynomolgus monkey serum. - Conc. after dilutions must cover span of standard range. - Used pooled incurred samples
n=1 for each dilution
- %Diff at each dilution will be calculated from the undiluted pooled incurred samples. - For the sample to show parallelism, the measured conc. for 2 of 3 (>66%) dilution factors is tested. ≤±20.0% of target conc.
Hook Effect 3 conc.
- A sample will be prepared at 2 mg/mL. - The sample will be serially diluted to conc. of 100,000, and 10,000 ng/mL in 100% cynomolgus monkey serum. - All diluted and the neat samples will be assayed.
n=1 for each dilution
- %Bias for sample at 10,000 ng/mL must meet acceptance criteria from nominal. - All other samples must be AQL for no Hook effect.
Robustness
Use of multiple instruments Two different LC-MS/MS systems. 1 A&P batch
n=6 of each level
- Minimum 75% of CS ≤ ± 20.0%* of target conc. (*≤ ± 25.0% at LLOQ). - At least one calibrant at the LLOQ and ULOQ must meet the acceptance criteria.
Different affinity capture incubation times
- 3 incubation times: Long (75 min), middle (60 min), and short (45 min). - Run with Dilution Linearity run.
n=6 for each level (HQC and LQC) and for each condition.
The mean measured conc. at each level ≤±20.0% of target (nominal conc.). CV ≤20.0%.
LBA Based Common to Both LC-MS/MS Based
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Choosing Between Ligand Binding Assays and LM LC-MS/MS in an Overall BA Strategy
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Discovery and Pre-GLP 1. Fast moving 2. Multiple compounds /false
starts 3. Inability to generate highly
specific reagents 4. No need for parallel
processing
Early Regulated Studies 1. Limited studies on a
single molecule 2. Some tolerance for
resource spend 3. Parallel processing
desired
Late Stage Regulated Studies 1. Large studies on a single
molecule 2. Less risk involved for investing in
specific and rugged methods 3. Many samples requiring a high
throughput method. 4. Parallel processing and analysis
preferred. 5. Outsource-able methods
Time-bound Factors 1. Program stage 2. Reagent availability 3. Sample numbers 4. Outsourcing strategy
Time-independent Factors 1. Type of matrix 2. Clearance mechanism 3. Catabolic sites/degradation 4. Molecule complexity (ADA, conjugates) 5. ADA interference 6. Pegylation 7. Required sensitivity
LBA or LM LC-MS/MS
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Bioanalytical Platforms Evolve with Project Stage
Launch Filing Phase 3 Phase 2 Phase 1 Preclinical Lead opt Hit-to- Lead Screen Discovery
Antibody Drug Conjugates:
Large Molecule LC-MS/MS
Monoclonal Antibodies:
Fusion Proteins:
Ligand Binding Assays Some confirmatory use of LC-MS/MS
Time dependent transition from 100% LC-MS/MS to 100% LBA
Key Factors to be Considered: 1. Attributes of the modality 2. Cost 3. Speed 4. Risk tolerance 5. Outsourcing strategy
No transition due to complexity of the molecules
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The Argument Against Preclinical Use of LC-MS/MS for mAbs
Generic Human IgG PK Assays Used for Rat Serum
Beth Leary, et.al., J. Immunol. Meth. (2013) 4(1), 28–36
Is there a justification to use LM LC-MS/MS for mAbs development? Perhaps not a compelling one.
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LBA vs. LM LC-MS/MS – Decision Tree
25
Conventional LC-MS/MS 1. Highly specific 2. Well established 3. Sensitivity may be limiting
(LLOQ > 10 ng/mL), so Inappropriate for potent compounds, low expressing biomarkers
MW> 10,000 Da LM BA
Required
MW< 10,000 Da
LBA Reagents Available
No
LBA 1. ELISA and GYROS 2. Sensitive 3. Lack of specificity may be limiting 4. High throughput 5. Cannot do multiplexing 6. Better for free/bound measurements.
Affinity LM LC-MS/MS 1. Some form of affinity extraction
required (mAb, IMAC, Prot A,G). 2. Expensive instrumentation 3. Specific 4. Multiplexing possible 5. Resolution may be limiting 6. Limited validation experience
Direct LM LC-MS/MS 1. Simple procedure 2. Sensitivity may be
limiting 3. Expensive
instrumentation 4. Limited validation
experience
Reagent Pairs
Yes
No
Yes
Other Affinity Capture
No
Yes
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LM LC-MS/MS Features that Would Cause a Regulatory Concern
There does not seem to be anything special about LM LC-MS/MS that would cause a regulatory concern.
Feature Risk Precedent
Standardization Low/Medium Ligand Binding Assays
Multistep Method Medium SM Derivatization Methods
Stability Low Ligand Binding Assays
Specificity Medium Ligand Binding Assays
Paradigm Change for Assay Format Medium Ligand Binding
Assays
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Acknowledgements
Tim Heath Peng Luan
All work was funded by Amgen Inc.
