Beyond a C of A, correlating results from HRMS CRC with assay performance
02May2017 Thomas D. Oglesby
Critical Reagent Management
2
© 2015 All Rights Reserved | CONFIDENTIAL
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Tracking of reagent suitability information is challenging – relies upon information provided by supplier combined with internally generated documents
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Can all critical Reagents managed in a LIMS system?
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COAs, Datasheets, Product inserts are uploaded to the system
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Expiration/re-tests date are managed electronically with an email notification system but still requires retesting
Reverse phase microflow: HRMS iKey BEH C4 300Å; 150 uM x 50 mm Flow 4 uL/min; H2O/ACN/0.1% FA gradient Waters G2-XS Time of Flight mass spectrometer in sensitivity mode High Sensitivity – information rich spectra Less than optimal LC peak shapes and separations from impurities such as PEG and BSA Potential MS artifacts?
Slide 4 www.aaps.org/nationalbiotech
Native Bevacizumab (top) Biotinylinated (bottom) on Waters iKey UPLC/G2-XS QToF (MS spectra)
Analytical SEC/HRMS BEH 200Å diol UPLC; 4.6 x 150 mm Flow 300 uL/min; 100 mM Ammonium Formate 200 ul/min split to Waters PDA detector: 100 ul/min to MS Post column addition to MS flow: 100 ul/min ACN with 0.8% FA Waters G2-XS Time of Flight mass spectrometer in sensitivity mode Increase confidence in identification of aggregates Confirms any potential MS artifacts
Slide 7 www.aaps.org/nationalbiotech
Analytical SEC: parallel UV and HRMS Thyroglobulin, IgG, BSA, Myoglobin, Uracil 150K 670K
66.4K
16.9K 112
Typical SEC/HRMS result for a Sulpho tagged mAb UV 280 nm
Labeled mAb
Aggregates
HRMS
Characterization of Reagents – MSD Sulfo-Tag (Ruthenium) labelling of Avastin (Challenge Ratio 5:1)
=1027
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Average Molar incorporation of Ru SulfotagDemonstration of calculation of DAR for ADC’s and Glycoforms
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Beyond the CofA
Slide 12 www.aaps.org/nationalbiotech
S/N (PCH/NC)
Performance of Various Biotin Labeled Lots of GLP-1 analog in ECL ADA assay
MS Comparison of Biotinylation Lots – Lot 3 has large abundance of N-terminal labeling CAR 1
Lot 1 CAR 0
Biotin = 2
Unlabeled
Lot 2
Lot 3
14
Biotin = 3
CAR 2
CAR = 3 CAR 3
The PK assay that should work NHS Ruthenium Tag
Infliximab
Human anti-Infliximab (Monovalent Fab)
TNF-α (Target)
Dissociated Fab Fragment Ruthenylation a Fab fragment is the result of the digestion of an antibody, it contains a small part of the heavy chain and a light chain (between 20 and 30kDa each). In this example the fragment has dissociated (loss of disulfide bonds) giving the two constituent parts i.e. 22kDa and 26kDa. The ruthenylated spectra is on top where we can see the addition of one and two rutheniums. In the 47kDa range we can see where the 22kDa piece has associated with another 22kDa piece to form a dimer. This is confirmed from the addition of two rutheniums (2054Da) for each peak.
+1 Ruthenium
Ruthenylated
22,560 Native 26,400
22,560 Da Dimer
22,560 Da Dimer + 2 Rutheniums
22,560 Da Dimer + 4 Rutheniums
Analytical SEC/HRMS of Fab 66K
17K
Preparations partially reduced
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Not as Stable as you thought? Freshly Thawed mAB
Thawed and Refrigerated mAB
Observed loss of ~24kDa
Too much BS, Ey?
BSA
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What the …. Raw Spectra: Deglycosylated (Top) versus Unaltered (Bottom) Native mAB
Did you buy the mAb? 143,575 da
144,520 da
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Primary Analytical Standard Purity?
Critical reagent management Be Proactive, not Reactive!
O’Hara et al., AAPS Journal, Vol. 14, No. 2, June 2012
Determination of Optimal ADA Reagents 4
Ruthenium CR=2:1 Ruthenium CR=4:1
3.5
Signal to Noise (PCH/NC)
Ruthenium CR=8:1 Ruthenium CR=12:1
3
Ruthenium CR=20:1 2.5
2
1.5
1
0
2
4
6
8
10
12
Biotin Challenge Ratio (CR)
14
16
18
20
Scan of CR2 and CR20 Mass Trace 380.17 3.3 e5
2.4 e6 Way too much free Biotin still present in final reagent prep
Element C
H
Cl
Nom. Mass 12
1
35
Isotope
Nom. Mass
13C
13
14C
14
D, or 2H
2
T, or 3H
3
37Cl
37
Serves both internal and external requests
Lessons Learned By Comparing Conjugate Ratios (CAR) obtained by LC/MS to assay performance we can set parameters for an acceptance criteria for a critical Reagent Future lots of a Reagent can be tested to conform to the acceptance CAR criteria or current lots may be recertified by LC/MS comparison Understanding the rates and extents of conjugation by LC/MS can enlighten method development and provide chemical/physical data to enhance troubleshooting initiatives. Do you know what you have in the vial? Crucial to start with CRC when the reagents come in the door! Cradle to grave means just that… why not start at method development?
Acknowledgements • Anthony Mancino • Matthew Sciscione • Lee Andrews
Thomas D. Oglesby Beyond a C of A, correlating results from HRMS CRC with assay performance Critical Reagent Characterization, Tuesday May 2
• Cradle to Grave… characterize and correlate the biophysical properties of a reagent to its performance in the assay. • Hi Res MS provides detailed information that can be tracked and replicated.