LC-MS Applications in Regulated Bioanalysis

Steve E. Unger

Worldwide Clinical Trials

Austin, Texas

1

Regulations

• Present requirements to perform bioanalysis for submission to regulatory agencies have evolved as the result of bioanalytical workshops held in 1990, 2000 and 2006

• Shah VP, et al. Pharmaceutical Research, 1992; 9: 588-592

• Shah VP, et al. Pharmaceutical Research, 2000; 17: 1551-1557

• Guidance for Industry: Bioanalytical Method Validation. Rockville, MD: US Department of Health and Human Services, FDA, CDER; 2001

• Viswanathan CT, et al. AAPS J. 2007; 9: E30-E42

• Bansal S. and Destefano A. AAPS J. 2007; 9: E109-E114

• International Conference on Harmonization. ICH Q2A: Text on Validation of Analytical Procedures. Federal Register. 1995; 60 FR 11260

• International Conference on Harmonization. ICH Q2B: Validation of Analytical Procedures: Methodology. Federal Register. 1997; 62 FR 27463

• Fast DM et al. AAPS J. 2009; 11: 238-241

Regulations (cont.)

• European Medicines Agency. Guideline on bioanalytical method validation. July 2011. CHMP/EWP/192217/2009

– Effective Feb 1, 2012

– EBF response: Van Amsterdam P et al. Bioanalysis 2013; 5: 645-59

• Canada will adopt EMA guideline

• FDA has issued a revised draft FDA guidance

– Draft Guidance for Industry: Bioanalytical Method Validation. September 2013 (Revision 1 is in 90-day review)

– Changes to standard curves (6 runs analyzed in duplicate)

• Brazil guidance incorporated testing for hemolyzed and lipemic plasma which EMA also adopted

Agência Nacional de Vigilância Sanitária (ANVISA). Guide for validation of analytical and bioanalytical methods. 2003

• Japan is working to update their guidance

• China plans a revision in 2015

Lipemia & Hemolysis

• Clinical chemistry analyzers routinely assess plasma or serum samples for lipemia and hemolysis

– Measure components directly and are sensitive to spectral interference

• LCMS assays extract and separate analytes from matrix, making them less prone to direct interference

– Focus on testing matrix effects for suppression from phospholipids or injected formulations (PEG)

– Inversion chromatograms or direct phospholipid monitoring in MD

– CV of the IS-normalized MF calculated from 6 lots of matrix ≤ 15%

• Good reason to always use a stable label IS

– Best is 13C, 15N but D may be acceptable

– Using stable label of parent to cover metabolite is bad practice

Stability

• Confusion from the following EMA statements:

– Ensure that every step taken during sample preparation and sample analysis do not affect the concentration of the analyte

– Stability should be ensured for every step in the analytical method

• Each step in a procedure is not individually tested for stability

– Would need to demonstrate by kinetic method

• Stability of stock and working solutions of the analyte and IS

– It is not needed to study the stability at each concentration level of working solutions and a bracketing approach can be used

• Stability of the analyte directly after blood sampling of subjects and further preparation before storage

– Often done as part of blood-plasma partitioning studies

– Blood stability by sampling and analyzing plasma to T0

– Spike in 37˚C blood, then room temperature or ice processing

– Decide whether stabilizer is needed for Vacutainer or in plasma tube

Comed stability

• Stability of the analytes in the matrix containing all the analytes

• Global CRO Council provided 103 nonproprietary and 60 proprietary compounds that show stability of a combination product can be accurately judged by individual assessments – Lowes, S et al. Bioanalysis 2011; 3: 1323–1332

• Passed ISR performed at considerable time intervals for a combination product serves the same purpose

• Inhibition of a common plasma enzyme would increase, not decrease, apparent stability in a mixture

• One instance would be an enzyme product on its substrate

• Generally done with combination assays for combo products

LCMS set-up

• Set of conditioning samples is injected using ULOQ – Prepared separately from run

– 10 injections limit the potential for drift of instrument response

– Equilibration of MS response and LC recovery

– If other procedures are required, they are defined in the method

• Carryover is assessed by injecting a control blank following injection of the last conditioning sample – ≤ 20.0% of the response of the lowest standard’s analyte peak

– ≤ 5.0% of the response of lowest standard’s internal standard peak

– Require changes to the system to eliminate or diminish the carryover prior to starting the run

– Also assessed within the run

System suitability

• Qualified and properly maintained instruments should be used for routine bioanalysis

• As part of qualifying instruments, system suitability ensures an instrument is operating properly at the time of analysis

• System is sufficiently sensitive, specific and reproducible for the current analytical run – Signal to noise ratio > 5:1

– No saturation (proportionality to ULOQ conditioning)

– Resolution of all critical pairs (Rs ≥ 1.5)

– Peak symmetry sufficient to allow proper integration and resolution

• System suitability tests are recommended to ensure success, but are not required, nor do they replace the usual run acceptance criteria

Data processing

• Software limitations to measuring both small & large peaks – Must deal with minor interferences and differences

• Have SOPs governing integrations and chromatogram review procedures

• Allow changes in parameters to optimize daily batches – Digital processing (smoothing/bunching, etc.) defined in validation

– Start with recommended parameters and document rational for change

– Settings must apply to the whole run (all sample types)

– Audit trail must record all decisions (report original and re-integrated results)

• No quantitative monitoring during run

• Avoid manual integrations – Questions on reintegration (used to drive results)?

– Inability to exactly reproduce manual integrations

• Require manager review of reintegration

• Avoid changes to integration when questioned – Selected for consistency across run

– Analytical is complete and properly QC checked prior to PK analysis

IS response plots

• Every laboratory should have IS response rules

– Each assay is different

– Having different criteria for each assay is too complex

• One standard defined by SOP

– Top boundary of 175% covers double spike of IS

– Lower boundary is often 50%

– Relative to overall mean IS response

– Judged prior to other rules (4/6-15/20) being applied

– May be different if using analog vs. stable label

• Differences across sample types indicate problems

Differential recovery or matrix effect on aging LTS problems

Inconsistent response across run

Global Bioanalytical Consortium Harmonization teams

A1 Scope and regulations

A2 Tiered approaches for method validation

A3 Method Transfer, partial/cross validations

A4 Reference standards and reagents

A5 Sample Management

A6 Stability

A7 Repeat analysis and ISR

A8 Documentation

A9 Analytical Instrument Qualification

A10 New Frontiers

A11 Biomarkers

L1 Large molecule specific run acceptance

L2 Large molecule specific assay operation

L3 Assay formats

L4 Reagents and their stability

L5 Automation practices in LM bioanalysis

L6 Immunogenicity

S1Small molecule specific run acceptance

S2 Small molecule specific assay operation

S3 Chromatographic run quality assessment

• Harmonization teams focusing on topics which apply to:

– Both chromatography based assays and LBA (all molecules)

– Chromatography based assays (small molecules)

– Ligand binding assays (large molecules)

FDA guidance on cross-validation

• Cross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies

• An example of cross-validation would be a situation where an original validated bioanalytical method serves as the reference and the revised bioanalytical method is the comparator

• The comparisons should be done both ways

• When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory to establish inter-laboratory reliability

• Cross-validation should also be considered when data generated using different analytical techniques (e.g., LC-MS-MS vs. ELISA) in different studies are included in a regulatory submission

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EMA guidance on cross-validation

• Where data are obtained from different methods within and across studies or when data are obtained within a study from different laboratories, applying the same method, comparison of those data is needed and a cross-validation of the applied analytical methods should be carried out

• Differences in sample preparation or the use of another analytical method may result in different outcomes between the study sites

• Cross-validation should be performed in advance of study samples being analyzed if possible

• For the cross-validation, the same set of QC samples or study samples should be analyzed by both analytical methods

• For QC samples, the obtained mean accuracy by the different methods should be within 15% and may be wider, if justified

• For study samples, the difference between the two values obtained should be within 20% of the mean for at least 67% of the repeats

• The outcome of the cross-validation is critical in determining whether the obtained data are reliable and whether they can be compared and used

How to perform a cross-validation or conformance test

• Must-do within a study using QC and incurred (study) samples

– Exchange and test blinded QC and incurred samples from both labs

– Use pooled samples to avoid replicate values of individual samples

– QC results within 15% of nominal

– ISR rules for incurred samples (2/3 < 20% difference)

• Helpful across studies when blending PK data from different assays/labs

– Statistical approach (ANOVA) assessing confidence intervals

– Best for comparing multiple assays and does not require a priori rules

– Requires a considerable amount of data and is time consuming

• Final phase of validation prior to starting sample analysis at a new lab

• Other approaches

– Comparison of means and %CV from replicate analysis

– Xu X et al. Bioanalysis 2013; 5: 83-90

– Plot assay results and look for unity slope, r2

– Bland J and Altman D. Int. J. Nursing Studies 2010; 47: 931–936

• A priori rules compare two assays with defined rules

• Establish a cross-validation or conformance test plan well in advance

16

Unexpected events

• A situation contrary to anticipated or expected results that could impact the quality or integrity of the data

• Routine in discovery but uncommon in regulated bioanalysis

– Method testing and validation improves quality prior to start of study

• Need to work through probable causes to assign a definitive cause

– Decide who has authority to run investigations and make decisions in SOP

• Can be noted anytime during the study

– Interference early in analysis

• Change method, revalidated and start over

– ISR failure late in sample analysis

• Execution error or fundamental problem with method?

– During data review

• Can be noted by anyone during the study

– Individual directing validation or analysis has bioanalytical responsibility

– Project Director communicates to Study Director or Study Monitor

Relevant guidance on unexpected events

• Guideline on the Evaluation of Control Samples in Nonclinical Safety Studies: Checking for Contamination with the Test Substance. 17 March 2005. CPMP/SWP/1094/04

• Guidance for Industry, Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production, October 2006

• Rocci M, et al. Confirmatory Reanalysis of Incurred Bioanalytical Samples. AAPS J. 2007; 9: E336-343

• Fast D, et al. Workshop Report and Follow-Up – AAPS Workshop on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples – Implications of Crystal City Recommendation. AAPS J. 2009; 11: 238-241

• James C, Hill H. Procedural Elements Involved in Maintaining Bioanalytical Data Integrity for Good Laboratory Practices Studies and Regulated Clinical Studies. AAPS J. 2007; 9: E123-127

• Reflection paper for laboratories that perform the analysis or evaluation of clinical trial samples. 28 February 2012. EMA/ISN/GCP/532137/2010

Assign risk to decide process

Level 1

Least likely to affect study data integrity

Usually the cause is immediately identifiable and no impact to study data is expected

No further investigation or corrective action is required

Level 2

Moderate potential to affect study data integrity

The Study Director and/or the Sponsor are informed in writing of the event

Level 3

Most likely to affect study data integrity

Sample analysis may be halted

A committee is convened to discuss all available information

Additional investigations and meetings may be required

Potential impact to study data requires further assessment

Study Director and Sponsor are notified as soon as details become available

Notification prior to completion of UEI to allow for Study Director and Sponsor input

Examples of high risk

• For bioequivalence studies, overall accuracy and precision of the mean at each run qualifying QC level exceeds 15% (excludes QC levels/preparations used to verify a dilution)

• Pattern of aberrant internal standard response between standards, QCs, and unknowns, including failure to meet internal IS rules

• Repeated chromatographic interference in any sample within a study • Failure of incurred sample reproducibility within a study • Anomalous results for unknowns that are not confirmed (dilution repeat,

PK requested, or client requested) • For samples collected in serial fashion, a result below the limit of

quantitation at a nominal time when a quantifiable result would be expected (e.g. a BLQ result between two quantifiable results)

• For samples collected in serial fashion, a quantifiable result at a nominal time when a non quantifiable result would be expected (e.g. a quantifiable result between two non quantifiable results)

• Any other phenomenon that may cause concern regarding the overall integrity of data

Probable causes → assignable cause

• Work through hypothesis to reduce possibilities to one definitive cause Failure of a chromatographic column to provide adequate separation Interference from metabolites or co-administered drugs Mistaken use of wrong calibration standard or IS solution Contaminated plasma, buffers, solvents, or glassware Evaporator or leaking 96-well sealing cap mats causing extract

contamination Aberrant QC as a result of sample switching that cannot be verified by

cross checking with vial identification in the sample tray Malfunctioning pipettes Sample inhomogeneity Improper sample preparation Improper batch size

• Lessons learned translate to corrective actions In-house UEI reports stored in Trackwise™

Justification for incurred sample re-analysis (ISR)

• There are a number of situations where the performance of standards and QCs may not adequately mimic that of study samples from dosed subjects

• Examples include:

– Metabolites converting to the parent species

– Protein binding differences in patient samples

– Differential recovery issues

– Sample inhomogeneity

– Mass spectrometric ionization matrix effects

• These factors can affect both the reproducibility and accuracy of the concentration determined in incurred samples

• While these effects are often characterized and minimized during method development, it is important to assure that they are under control when the method is applied to the analysis of incurred samples

• First reportable result was dogma of the day

– Allow analysts to test their work without need for PK repeats

What is different for incurred vs. QC samples?

• Metabolites

– Specificity established against known metabolites?

– Back-converting phase II conjugates (acyl glucuronides of parent)

– Interfering phase II conjugates (ether glucuronide with co-elution and in-source fragmentation)

– Isomeric/isobaric interference (common when assaying metabolites)

• Formulation

– Absorbed or IV formulations are abundant surfactants (e.g. PEG - ESI)

• Preparation

– Study sample has no spiking solution & are “pre-equilibrated”

• Population

– Recovery is poor in specific samples (condition; differential recovery)

– Unexpected interference due to co-medication

– Unusual abundance due to age, gender, disease state (renal/hepatic impaired)

• Stability

– Inter-conversion is concentration-dependent and QC concentration ratio does not properly test mass action (e.g. statins)

Selection of ISR sample

• ISR is conducted on individual samples

• Analyzed in singlet (n = 1) or how the sample was assayed originally

• Backup samples may be used for incurred sample reanalysis

• The incurred sample reanalysis will use the same dilution as the original assay if there is enough sample volume

• Pooled samples may be used for investigation purposes

• ISR can be performed with other study samples or as a separate analytical batch

• Samples and analyte of interest will be specified in the batch records for each run that includes ISR

Sampling & acceptance criteria

• Subjects must be selected randomly

• Representative of PK profile of individual analyte

• Minimum of 10% of total analyzed samples must be selected

• New FDA draft guidance is 7%

• Assign a minimum number for small studies (20 samples)

• For larger studies (> 1000 samples), 5% of total sample size

• ISR Samples must be analyzed within stability period

• For two-thirds of samples, the % difference between initial and ISR values must be: – ≤ 20% for small molecules

– ≤30% for large molecules)

Samples for ISR

• Samples with an original concentration greater than three times the lower limit of quantitation of the method (>3 x LLOQ)

• Samples within the previously established stability

• Samples with sufficient volume

• Applies for each of the analytes being evaluated in the sample and the number of study samples that qualify for ISR selection

• Samples selected will have an original reportable concentration

• For every subject/animal that has a sample selected for ISR, at least one sample will be selected at a timepoint close to the Tmax and one sample at a timepoint in the elimination phase

• If the Tmax and elimination phase are not obvious (e.g. steady state concentrations), representative samples may be selected

When to do it?

• GLP animal studies

• Only in samples from the first subchronic toxicity study for each species

• Only one time, per species, per assay method and per laboratory is necessary

• Clinical

• Recommended for each clinical study

• Decision not to perform ISR directed by the sponsor through the study protocol or written communication

Percent Difference (%Diff) = % 100

ions Concentrat Original and Assay ISR of Mean

ion Concentrat Original - ion Concentrat Reassay x

Failures

• Investigate and assess impact (analyst vs. method)

• Bias in runs compared (execution error) – Day-to-day bias in making standard curves

• Inhomogeneity of urine, tissue homogenates and blood – High renal clearance near solubility limit causes precipitation of

analyte onto urinary crystals

– Examine specimen (cloudy, turbid, not transparent) to indicate sampling errors

– Compare %CV of replicate injections of same extract and replicate analysis of same sample

– Aliquoting blood clotting on bench with high blood-plasma ratio

• Unstable, interconverting forms (acyl glucuronide)

• Stable interference (co-eluting ether glucuronide)

Impact assessment / CAPA

• GXP testing of (bio)analytical production runs

– Test run results for repeatability

– Stop production and investigate for cause

• Repeat analysis may implicate

– Results, runs, studies and/or assays

• Investigation for cause will determine direction

– Unique sample (fail sample result – not reportable)

– Improper execution of assay (fail run results)

– Continue use of assay in this or other studies

– Unique population (fail study results)

– Revise/re-validate a new assay (fail assay)

– If assay is implicated, can invalidate results from other runs or studies for which the assay was used

• Thoroughly test assay prior to use to avoid releasing an assay which is inadequate for study

– Ensure training and oversight of staff in execution of assay

Know your DMPK to protect your study

• Major CRO had performed two clopidogrel bioanalytical studies 8 to 10 years ago (pre-ISR)

• Satisfactory and reproducible results at US site

• Unsatisfactory and irreproducible results at Canadian site

• CRO stated that similar procedures were used at both sites

• Upon investigation, extraction procedures were different

• Trans-esterification of acyl glucuronide in methanol during extraction was cause of irreproducible results

• Parent methyl ester is prodrug with low plasma concentrations relative to acid and acyl glucuronide

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Why is metabolic specificity important?

• Program received from a client who had failures at another CRO

• Studies were conducted on a prodrug of phenazopyridine (3-phenyldiazenylpyridine-2,6-diamine; PAP) a urinary tract analgesic

• Six male humans each received a single oral administration of a dual labeled [14C]2-PAP prodrug in an AME study

• Total recovery was 89% (82 to 94%) with 73% excreted in urine

• PAP levels in plasma determined using an LC-MS/MS were higher than those determined from total radiochemical analysis (parent ~ 50% TRC)

• Repeat analysis for incurred sample reproducibility (ISR) failed

• Samples with longer times waiting for injection showed higher results

• Extract stability studies using QC’s showed no change in PAP levels

• Over 10-fold increase was seen when some study extracts were stressed

Proposed metabolite pathways of phenazopyridine dosed as a prodrug in humans

Why knowing metabolism can be critical

• Acidification of incurred sample extracts resulted in increased PAP levels

• A decrease of mono-hydroxylated PAP metabolite was observed with a subsequent increase in a peak that interfered with PAP

• Acid-catalyzed reduction of diazo bond yielded an isomeric interference with an improved MRM response

– Fragment monitored is cleavage of the N-N bond

– Swati P. S. et. al. Synthesis, Electrochemical and Antimicrobial Studies of 2-Phenylazo-1-naphthol-4-sulphonic acid. Int. J. ChemTech Res. 2011: 3; 1164-1171.

• A co-eluting, acid-catalyzed degradation product of a metabolite was identified by increasing the LCMS separation to 30 minutes

• A more selective and stable extraction reduced the amount of unstable mono-hydroxylated PAP metabolite resulting in reproducible results

• Concurrent AME study assisted troubleshooting bioanalytical procedure

Heme-mediated oxidation impacts stability in hemolyzed plasma

• Oxybutynin (OXY) is a muscarinic acetylcholine receptor antagonist dosed as its racemate

• Anti-muscarinic activity resides predominantly in the R-isomer

• Its primary indication is to relieve urinary and bladder difficulties, including frequent urination and incontinence

• Metabolized to N-desethyloxybutynin (DEO) by cytochrome P450

• Yaich M et. al. In-vitro cytochrome P450 dependent metabolism of oxybutynin to N-deethyloxybutynin in humans. Pharmacogenetics 1998: 8; 449-51

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LLOQ Chromatograms ChromTech Chiral-AGP, 5 µ , 100 x 4.0 mm

XIC of +MRM (4 pairs): 358.1/72.1 amu from Sample 6 (ATM104421006) of ATM1... Max. 2815.9 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0

500

1000

1500

2000

2500

2816

In

te

ns

it

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c

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3.13

1.62

1.04 1.240.88

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Printing Date: 04/13/2012

Workstation: AUSCPDD00058

Operator: Mike Brake

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XIC of +MRM (4 pairs): 330.1/96.1 amu from Sample 6 (ATM104421006) of ATM1... Max. 5521.3 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0

1000

2000

3000

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50005521

In

te

ns

it

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1.40

1.91

0.78

Printing Time: 10:42:46

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Workstation: AUSCPDD00058

Operator: Mike Brake

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XIC of +MRM (4 pairs): 369.1/72.1 amu from Sample 6 (ATM104421006) of ATM10... Max. 1.8e4 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

1.8e4

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1.55

2.94

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Operator: Mike Brake

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XIC of +MRM (4 pairs): 335.1/101.1 amu from Sample 6 (ATM104421006) of ATM1... Max. 3.0e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0.0

5000.0

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1.5e4

2.0e4

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Printing Time: 10:42:46

Printing Date: 04/13/2012

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Operator: Mike Brake

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R- and S-OXY

R- and S-DEO

R- and S-OXY D11-IS

R- and S-DEO D5-IS

35

0.03 to 1.20 ng/mL for R-OXY

0.06 to 2.40 ng/mL for S-OXY

0.10 to 4.00 ng/mL for R-DEO

0.05 to 2.00 ng/mL for S-DEO

What data can we report and why?

• Previously validated bioanalytical method for enantiomeric quantitation of R/S-OXY and R/S-DEO in human plasma was used

• A small (0.2%) number of hemolyzed samples were received

– 1% (~275 mg/dL) to 4% (~1100 mg/dL) blood content

– Determined by color (BD chart, HemoCue)

• QC samples prepared using as much as 5% hemolyzed plasma showed no negative bias for parent drug OXY but a negative bias for the DEO metabolites when measured against normal plasma standards

• Difference of -41% and -31% for R-DEO and S-DEO, respectively, was noted in hemolyzed plasma when stored at -20 ⁰C vs. -70 ⁰C

• No difference in recovery, matrix effect or in-process stability could be associated with hemolyzed plasma

36

Investigation and corrective action

• Potential causes included the release of intracellular RBC enzymes or heme-related material

• Oxidation by hemoglobin was tested by monitoring the expected N-oxide

• Large peak at 2.0 min was observed when hemolyzed plasma was slowly frozen at -20 ⁰C

• Ascorbic acid added to hemolyzed plasma before freezing stopped oxidation

– 0.1%, 0.5% and 1.0% ascorbic acid were tested

– 0.1% ascorbic acid added as an anti-oxidant is sufficient

• Specificity to oxidation (R-DEO > S-DEO and DEO > OXY) suggests hemogloblin rather than just heme is responsible

37

Challenges in metabolite quantitation

• Multiple metabolites

– Too similar (phase I) or dissimilar (phase II) • Differences in recovery, LC retention, polarity & ionization

– Isomers and isobars need lengthy separations

• Unstable metabolites

– Must stabilize sample to chemical and enzymatic degradation • Oxides, esters, amides, conjugates (sulfate, glucuronide, GSH)

– May need to measure precursor and product to infer reactive intermediate concentrations

• Range of exposures across species / doses

– Considerable re-analysis

– Samples assayed both with dilution for parent and without dilution for low level metabolites

• Stable isotope internal standards are rarely available

• Purity of metabolite reference compound (no COA)

• Failure rates increase and sensitivity decreases when larger numbers of metabolites are quantified

Assessing efficacy in clinical studies

• PK-PD – Metabolite is pharmacologically active

– Metabolite has significant plasma concentrations

– Major circulating active equivalents (AUCactivity)

– Combination validated assay (parent + active metabolites) for all clinical studies

• DDI – Metabolite represents a significant clearance of parent drug

– Drug interaction is likely Plasma concentration is comparable to P450 or transporter IC50

– Individual validated assay used only within DDI studies

– Exception is P450 clinical assay Cooperstown, Glaxo, Pittsburgh cocktails

Metabolites in Safety Testing (MIST)

• Differences in drug metabolism between animals used in non-clinical safety assessments compared to humans should be identified as early as possible

• To provide guidance for metabolite analysis, the MIST committee was formed • Published MIST position in Toxicol. Appl. Pharmacol. 2002;182: 188-196 • FDA response issued in Toxicol. Appl. Pharmacol. 2003; 190: 91-92 • CEDR issued a draft guidance in June 2005 • PhRMA response (August 2005) coordinated by Tom Baillie • PhRMA/FDA joint workshop in November 2005

– Six case histories from FDA and PhRMA to build consensus • Final FDA Guidance for Industry: Safety Testing of Drug Metabolites. February 2008

– 10% of parent human plasma exposure – Determined as AUC at steady-state (multiple dose)

• ICH Guidance M3(R2) Nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. June 2009 – 10% of total circulating equivalents

• FDA has accepted EMA guidance • FDA draft guidance requires fully validated assay for unique or disproportionate

metabolites (depending upon its activity)

Assessing potential for metabolism-mediated toxicity

• Metabolites may not be adequately assessed during standard non-clinical studies

– Occur only in humans (unique metabolite)

– Occur at much higher levels in humans than in the species used during standard non-clinical toxicology testing (disproportionate metabolite)

• Evaluated as early as possible during clinical development

• Animal and human plasma assays to determine exposures across species or genders to major metabolites whose safety margin < 1 in tox species

• No mention in MIST guidance on how to perform the bioanalysis

• Bioanalytical guidance from Crystal City III report (Section 6)

– Can be tiered (non-GLP) assays

– Metabolite screening studies to be performed in early drug development using bioanalytical methods with limited validation

– Validation criteria increasing as a product moves into clinical trials

– Scientifically-appropriate criteria, established a priori

Reference standards

• Synthesis of metabolite reference standards and associated stable label internal standards for regulated assays – Analytical characterization (mini-certificate of analysis) – IS needs only qualitative analysis, isotopic purity and stability – May be difficult to synthesize certain metabolites and biosynthesis does not

confirm structural assignment or yield large quantities – Expensive and costly

• Biosynthesis – Isolation of sufficiently pure material in microgram levels – In vitro (microsomes, microbial or expressed P450 systems) – In vivo (high dose rat BDC study, urine)

• Purity assessed using 1H or 19F NMR by reference to known parent – Mutlib A et al. Drug Metab Dispos. 2011; 39: 106-16

• Radiolabeled metabolites as calibrators of LCMS to estimate exposure – Zhang, D et al., Drug Metab. Lett. 2007; 1: 293-98

• Calibration of MS response – MS response calibrated from NMR or RAD – Used to estimate exposures in cold studies

Phase II conjugates

• Direct is preferred when: – One known metabolite is present (1-O-β acyl glucuronide)

– Concentrations are low

– One phase II conjugate has activity – Morphine 6-O-glucuronide vs. morphine 3-O-glucuronide

– Reference standard can be synthesized and purity assigned (COA)

– Enzymatic or chemical hydrolysis is either inconsistent/incomplete or degrades analyte product

• Indirect or total (conjugated) is preferred when: – Multiple conjugates are present

– Apomorphine sulfates and glucuronides

– Concentrations are abundant

– Conjugates are difficult to synthesis – Low yield from synthesis or bioreactors

– FDA requires total

Sourcing phase II metabolites

• If using an in vivo generator, purify urine or bile of associated endogenous compounds prior to spiking into plasma – SPE or LC fractionation

• High concentrations of endogenous sulfates or glucuronides can overwhelm the capacity of sulfatase or glucuronidase – Incomplete and inconsistent turnover of conjugates

• Unable to achieve consistent results when using human urine as source of apomorphine sulfates and glucuronides – J. Chromatogr. B. 1997; 702: 131–141

• Pooled human plasma resulted in a successful analysis – Excreta can concentrate systemic metabolites but will not represent

what is in plasma

Hydrolysis QC

• Hydrolysis QC samples added to normal QC samples • Establish their concentration during validation • Use Hydrolysis QC throughout sample analysis

– Source a sufficient quantity to support studies – Determine their stability – Anchor/qualify new batches as needed

• Added cost and time to perform analysis – Hydrolysis procedure added to the unconjugated (free) assay – Sample analysis is twice as long due

• Reasons to measure conjugates (UGT DDI, activity or toxicity) justify the time and effort – Humphreys, WG et al. Chem. Res. Toxicol. 2006; 19: 1564-69

Options to measure phase II conjugates

• Chemical hydrolysis with strong base / high temperature

• Enzymatic hydrolysis with β-glucuronidase or sulfatase

• Direct assay with reference standard – Best when measuring low levels (difference determinations

accentuate errors)

• Acid containing drugs – Prepare and characterize the 1-O-β-acyl glucuronide

– Generate isomers by increasing pH and temperature

– Characterize using LC/MS and/or LC/NMR

– All theoretical isomers should be observed in the chromatogram of the stress-degraded 1-O-β-acyl glucuronide

– Final separation need not resolve all isomers from each but must resolve the 1-O-β-acyl glucuronide analyte from all others

Muraglitazar

• PPAR (α, γ) in development at BMS until 2006

– Waites CR, et al. Toxicol. Sci. 2007; 100:248–58

• Rearrangement by intramolecular acyl migration yields the β-glucuronidase resistant 2-, 3- and 4-O positional isomers

– Transient ring opening with production of a reactive aldehyde group

– Ring closure leads to either the α- or β-configuration

• To define LC separation, prepare a mixture of all isomers by forcing rearrangement under stressed conditions

– Heat sample and continue to monitor rearrangement

• Ensure final separation maintains 1-O-β isomer from all others

– Phenomenex Luna C18, 3 mm x 150 mm, 3 µm column

• Ensure stability of 1-O-β isomer to rearrangement

– Not all isomers will have same MRM response

• Xue Y-J et al. Rapid Commun. Mass Spectrom. 2006; 20: 1776-86

Muriglitazar acyl glucuronide anomers and positional isomers generated by stress degradation (top) and IS (bottom)

Mesalamine (5-ASA)

• Mesalamine or 5-aminosalicylic acid (5-ASA) is an anti-inflammatory agent for the treatment of inflammatory bowel diseases

• Metabolized by acetylation to N-Ac-5-ASA • Amphoteric, high polarity and low MW complicate analysis • Most prior methods used pre-analytical derivatization • Anion mixed-mode polymeric SPE gave recovery > 87% • MF ranged from 0.95 to 1.05, including lipemic or hemolyzed lots • HILIC-separation (50 x 2.1mm, 5 µm) with Sciex API 5000

– 5-ASA-13C6 & NA-D3-5-ASA IS

• 4 to 2000 ng/mL for both (0.25 mL of human K2-EDTA plasma) • 5-ASA was unstable both in acetonitrile/water and hemolyzed plasma due

to oxidation to the quinoneimine • NA-5ASA was stable in both normal and hemolyzed plasma • The reducing reagent, Na2S3O5 was added to spiking solutions

• Ascorbic acid was added into plasma • Protected from light

LLOQ / 5-ASA, 5-ASA-13C6, NA-5-ASA, NA-D3-5-ASA

Improving sensitivity of bisphosphonates

• Three biphosphate drugs, residronate, alendroante and ibendroante

• Extraction using 96-well SAX with methylation in diazomethane ether

• HPLC using Ascentis Express HILIC (3 x 50 mm) & Sciex API 4000/5000

• By selecting the transitions of the quaternary amine product, assay sensitivity and selectivity were improved significantly

• Risedronate 0.05 - 50 ng/mL

• Alendonate 0.05 - 50 ng/mL

• Ibandronate 0.01 – 2.0 ng/mL

• Pentamethylated risedronate MRM extracted from plasma sample was more than 10-fold higher than its tetramethylated product

• Quaternary amine raises MRM sensitivity not only because it is a preformed ion but also yields a simpler fragmentation pattern

Vitamin K

• Natural biologically-active form of vitamin K1(phytonadione) is the trans (E) isomer

• The inactive cis (Z) isomer is found in synthetic vitamin K1, and may arise as a result of exposure to light

• A method for the determination of vitamin K1 isomers in human plasma samples was developed, validated and successfully used in clinical studies

• Assay range from a 0.5 mL human plasma sample was: – 0.500 to 150 ng/mL for E vitamin K1

– 0.300 to 90.0 ng/mL for Z vitamin K1

Vitamin K

• Specificity and selectivity tested in multiple lots – Each of six undepleted lots contained concentrations below LLOQ – Over-spike to provide stability QC samples

• LLE – Recoveries between 85-99% – Matrix between 0.87-1.01

• Sciex API 5000 – Positive APCI – m/z 451>187 – D7-stable label IS

• Gradient HPLC – Water/methanol/5 mM ammonium acetate – C30, 4.6 x 150 mm, 5 µm – Eluted between 9 to 10 min (Rs ~ 1.5) – 13 minutes injection cycle

Improving throughput in regulated bioanalysis While retaining quality

• Chromatography is our rate-limiting process in the LCMS lab

– MS systems achieve their job in milliseconds

– LC separations can take several minutes

– Differential ion mobility, MS3 or high resolution may help

• Global Bioanalytical Consortium has made recommendations for chromatographic figures of merit

– k’/k*, Rs, N and Symmetry Factor

• Require better separations for clinical studies

– Higher potential of interference in diverse populations

• UPLC

– 5 Waters Acquity and 10 Shimadzu Nexera with SIL-30 AS

– High resolution or high speed separation with improved mass flux sensitivity

• Multiplexed HPLC

– 5 Thermo LX2 with Aria V1.6.3

– Shimadzu SIL-20 AB and CTC DLW PAL

Biomarkers

• Validation of a biomarker method to support drug development did not fall within the scope of GLP, 2001 FDA Bioanalytical Method Validation guidance, 2011 EMA Guideline on Bioanalytical Method Validation nor Clinical and Laboratory Standards Institute (CLSI) guidelines

• Most laboratories followed their SOPs or method validation plans and the following position paper:

• Lee, J et al. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm. Res. 2006; 23: 312-328

• New FDA draft guidance includes guidance on endogenous compounds and biomarkers

– Standard and QC preparation for endogenous analytes

– Fit-for-purpose biomarkers

– Full validation of diagnostic kits

Standard and QC preparation

• Challenges in achieving specificity, selectivity and sensitivity, especially when endogenous levels are down-regulated

• Four approaches to the preparation of standards: – Authentic analyte in native matrix

Depleted population (M/F steroids; disease states)

– Authentic analyte in surrogate matrix

Buffer, bovine serum albumin, artificial CSF

– Surrogate analyte in native matrix

17β-estradiol biomarker for SERM in rat serum or brain

17β-E2-d5 used as calibrator; Ethinyl estradiol as internal standard

Petucci, CJ et al. J. Mass Spectrom. 2010: 45: 65–71

– Charcoal or chemical stripping, extraction and immunodepletion

• Stability indicating QC must be in native matrix – Anchor endogenous (QC-Low) and over-spike (QC-High)

Immunoassays

• Commercial assays are validated according to their intended use (fit-for-purpose) – Between laboratories comparisons serve to standardize results

• Manufacturer package inserts or published literature are taken into consideration when defining the degree of validation necessary to support its validation – Cross-reactivity (specificity) and quality of critical reagents is defined

by manufacturer

• Method validation plan is used to augment normal SOP

• Numerous kit assays exist for biomarkers of interest – Screen kits to determine their suitability for drug development

– Nowatzke, W et al. Bioanalysis 2010; 2: 237–247

– Provided kits are suitable, most cost effective means

LCMS analysis of peptide biomarkers

• Trend in biomarker quantitation is LC-MS bioanalysis of peptide or protein biomarkers

• Compared to ELISA, LC-MS assay development is relatively fast with no need to raise antibodies

• Characterization of the β-amyloid peptide (Aβ) population in biological matrices – Dillen, L et al. Bioanalysis 2011; 3: 45–55

– Ford, MJ et al. J. Neuroscience Methods 2008; 168: 465–474

• Immunoprecipitation with LC/MS

• Direct analysis suitable for peptides up to 10,000 Da – Insulin, C-peptide

• LC-MS assays can measure signature peptides as a surrogate for the proteins with similar sensitivity and specificity to immunoassays

Human CSF and plasma collection

• Continuous CSF collection at WCT San Antonio Clinic

– Study design often collects at predose, Day 1 and steady-state

• Six foot tubing serves as catheter for lumbar puncture collection

– Sterile Silicone or Tygon®

– Test for NSB losses prior to writing sample collection procedures

• Peristaltic pump draws CSF through tubing and into collection tube

– 2.5 mL purge at beginning of each collection to flush 1.8 mL of void volume

– CSF collected into Nalgene Cryoware 4 mL vials

– BioFrac Fraction Collector set to maintain CSF samples at < 4˚C

• Blood sample is collected into a K2EDTA Vacutainer on ice

– Plasma harvested in a refrigerated centrifuge

– Native plasma is used to determine stable analytes

– Plasma is treated with an acid preservative for unstable analytes

CNS drug development

• Strong interest in CNS drug development

• Mike Sullivan undertook the development and validation of several CSF and plasma assays to measure biogenic amines to FDA/EMA requirements

• Assist clients in development of PK-PD relationships – Penetration/target engagement by determination in CSF

– Validate animal pharmacology model in man

• Proper study design, sampling and analysis avoids errors – Supine vs. upright

– Effect of platelet binding

– CSF tubing and sample collection tube showed no loss with small molecule biogenic amines

Human plasma

Analyte LLOQ ULOQ Units Volume (mL)

5-HIAA 1 100 ng/mL 0.1 5-HT

(serotonin) 0.05 25 ng/mL 0.2

Dopac 0.4 40 ng/mL 0.1

HVA 4 400 ng/mL 0.1

Levadopa 0.2 10 ng/mL 0.2

Dopamine 0.1 5 ng/mL 0.2

NE 40 2000 pg/mL 0.1

DHPG 200 10,000 pg/mL 0.1

Five individual assays for NE/DHPG, DA/DOPA, DOPAC/HVA, Serotonin and 5-HIAA All use stable label internal standards and are validated to FDA/EMA requirements

Human CSF

Analyte LLOQ ULOQ units Volume (mL)

5-HIAA 5 500 ng/mL 0.1 5-HT

(serotonin) 0.05 25 ng/mL 0.1

Dopac 0.1 10 ng/mL 0.1

HVA 5 500 ng/mL 0.1

Levadopa 0.1 5 ng/mL 0.2

Dopamine 0.1 5 ng/mL 0.2

NE 40 2000 pg/mL 0.1

DHPG 200 10,000 pg/mL 0.1

Five individual assays for NE/DHPG, DA/DOPA, DOPAC/HVA, Serotonin and 5-HIAA All use stable label internal standards and are validated to FDA/EMA requirements

Serotonin (5-HT)

• 96-well SLE using EtOAc

• Often determined using LC-EC; useful for oxidation to scrub matrix

– Sodium hypochlorite (plasma)

– Hydrogen peroxide solution quenched with sodium bisulfite (CSF)

– Protect spiking solutions using ascorbic acid anti-oxidant

• Derivatization using acetic acid anhydride

• Enhance ionization and control fragmentation

• Improve the separation

• HPLC separation and positive electrospray MRM on Sciex API 4000 (plasma) or API 5000 (CSF)

5-HIAA

• Serotonin (5-hydroxytryptamine, 5-HT) is a low level neurotransmitter involved in many physiological functions

• A major inactive metabolite of serotonin, 5-HIAA, circulates in higher abundance, is excreted in urine, and is correlated with serotonin release

• Monitoring for 5-HIAA concentrations in bodily fluids can serve as a useful surrogate for direct analysis of serotonin

• Sodium hypochlorite is used to deplete endogenous 5-HIAA in matrix

• Acidification and EtOAc SLE is used for CSF, plasma and urine assays

• Derivatization using dansyl chloride at 60˚C is needed for CSF and plasma

• UPLC separation on Sciex API 4000 in positive electrospray MRM mode

5-HT 5-HIAA

DOPA & Dopamine in CSF

• Dopamine is a catecholamine neurotransmitter, responsible for conducting signals in the central nervous system as well as in the sympathetic nervous system

• L-DOPA is precursor to dopamine and has been used to treat Parkinson’s

• Catechols are sensitive to oxidation, requiring acid pretreatment

• Endogenous DOPA and dopamine required a synthetic solution of salts, urea and albumin as blank control matrix for CSF standards

• Lot screening provided sufficiently low levels in plasma standards

• Selective extraction on a 96-well Strata SCX with SPE derivatization using propionic anhydride gave required sensitivity

• UPLC separation on a Sciex API 5000 in positive MRM mode

DOPA Dopamine

Large molecule drugs and biopharmaceuticals

• Peptides, proteins and oligonucleotides have special requirements for their bioanalysis

– Potent, adsorptive and often unstable (disulfides, proteolysis)

– Sensitivity loss due to lack of distinguished fragmentation is common

• Protein therapeutics can have post-translational modifications

• Need to be selectively reduced in size to make them tractable to LC-MS

– Signature peptides (primary and secondary “epitopes”)

– Universal peptides (Fc) – discovery/preclinical assays

• Stabilization of peptides is often accomplished by using non-native amino acids or attaching fatty acids and polyethylene glycols to reduce renal clearance, proteolysis and immunogenicity

• Pegylated peptides represent particular challenges due to their heterogeneous nature and large molecular weight

– Direct detection is difficult due to broad isotope distribution

– Immunoprecipitation using anti-PEG antibodies with signature peptide

Unique features of measuring large molecules

• Special collection procedures (ice and non-absorptive tubes) and stabilizing agents to avoid proteolysis or oxidation of Cys thiols (DTT/IAA)

• When a stable label is unavailable, it may be possible to obtain a highly conserved peptide from a related species as a structural analog IS

• Losses due to nonspecific binding can be complicated when conformational changes result in greater adsorption

– Surfactants such as Tween-20 or Triton X-100 may be required

• Immunodepletion of abundant plasma proteins using protein columns, common to MS-based proteomics, can enrich samples but is expensive

• Use an antibody to immunocapture the desired peptide for target analysis

– Preferably the same antibody as was used in an immunoassay

– Allows specificity assessments in cross-validation

• Is it size, technique or heterogeneity of API that determines A/P rules?

Nowatzke, W. et al. Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs. Bioanalysis 2011; 3: 509-521

Peptides & proteins

• Due to their size, proteins are less amenable to direct quantification • Broad isotope distribution and presence of multiply charged species

distributes their ion abundance among several response peaks • Direct measurement of intact proteins may be made more specific using

high resolution instrumentation • Data processing algorithms can plot a summation of ions resulting from

individual isotopes and charged forms Individual ions need to be free of interference to maintain specificity

• Many proteins have abundant heterogeneity due to post-translational modifications (PTMs)

• LC-MS of enzyme digests have been used for quantification of proteins using a stable label or surrogate internal standard

• Signature peptide analysis is increasingly being used to measure proteins With immunocapture using a specific mAb, specificity and detection sensitivity

can rival ELISA (low-mid pg/mL) With less specific immunocapture (Anti-hu Fc), typical LLOQs are low ng/mL With traditional extraction, high ng/mL LLOQs are common

– Automation of processing is key to being cost-effective with ELISA

Hepcidin

• Amgen reported a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS

• 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism

• Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum

• 2.5 to 500 ng/mL

• Sciex AP 4000 using MRM of 4+ charge

• Oasis™ HLB SPE and UPLC reverse phase separation

• Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects

• Mean concentration was less than 10 ng/mL

• Mean serum concentrations in disease populations were:

– Sepsis = 252 ng/mL (n = 16, median 121 ng/mL)

– Chronic kidney = 99 ng/mL (n = 50, median 68 ng/mL)

Li, Hongyan et al. J. Pharmacol. Toxicol. Meth. 2009; 59:171-80

Endogenous and recombinant-methionyl human leptin

• Therapeutic recombinant-methionyl human leptin (MW 16155) has an identical 146-amino acid sequence with endogenous leptin (MW 16025)

• Involved in body weight regulation

• Immunoassays do not discriminate between r-metHu-leptin and endo-leptin

• Concentration measured in study subjects receiving r-metHu-Leptin can reach supra-physiological levels

• To determine which leptin species contributes to the elevated concentrations, an LCMS method of both leptin species was developed

• Endo-leptin and r-metHu-Leptin was enriched from plasma by immunocapture

– Anti-metreleptin antibody conjugated to agarose beads

• Onyx Monolithic C18, 3.0 X 100 mm LC coupled to a Sciex QTRAP 4000

• 15.6 to 1000 ng/mL

• Multiple charge state ions and specific MRMs were monitored to provide unambiguous differentiation between endo-leptin and r-metHu-Leptin

• Elevated leptin observed in subjects reflected accumulation of r-metHu-Leptin

Wang Y and Heilig JS. J Pharm Biomed Anal. 2012; 70: 440-6

Signature (surrogate) peptide from a PEGylated peptide

• MK-2662 is a glucagon-like peptide-1 (GLP-1) receptor agonist under development for the treatment of type-2 diabetes

• PEGylated oxyntomodulin (OXM) analogue contains a 38 amino acid peptide and a 40 kDa branched PEG covalently linked to the C-terminus (avg. MW 47,500)

• To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by 2D LC-MS/MS was developed

• Biotinylated anti-PEG antibody, bound to streptavidincoated magnetic beads, was used to capture MK-2662 and its 13C18, 15N2 stable label IS from human plasma

• After on-bead digestion with trypsin, injected on a 2D HPLC (Thermo BioBasic SCX 50 mm × 2.1 mm, 5 μm; Hypersil Gold PFP 50 mm × 2.1 mm, 5 μm)

• MS/MS detection of the surrogate N1-12-mer (HAibDGTFTSDYSK)

• m/z 672 → 223 from MH2+ on a Sciex API5000

• IAP assay was validated over 1-1000 nM

• Protein precipitation assay over 2-200 nM showed values obtained from IAP assay were 15-30% lower, supporting the more specific PEG recognition provided by IAP

Xu Y. et al. Anal. Chem. 2010; 82: 6877–6886

Identification and evaluation of an Fc universal surrogate peptide

• Fc universal surrogate peptide to for bioanalysis of diverse mAb and human Fc-fusion therapeutic proteins in preclinical animal studies – A universal method for immunoaffinity LC-MS/MS

– Potential for a dual peptide approach (universal & signature)

– Reliably produced with trypsin digestion and has a desirable amino acid composition

– Not found in the plasma proteins of animals used in preclinical studies

• Fc region sequences typically used in mAb and Fcfusion proteins based on IgG1 and IgG4 were obtained

– Trypsin digests analyzed for specificity

– Fifteen candidate peptides were identified

– Residues with Pro next to Lys/Arg and Cys removed

– Small (≤ 5 a.a.) and known Asp-glycosylation removed

Jemal M et al. Bioanalysis 2012; 4: 17-28

Finding the universal peptide

• Three tryptic peptides were selected from NCBI website: DTLMISR - found in monkeys and rabbits

GFYPSDIAVEWESNGQPENNYK

VVSVLTVLHQDWLNGK – selected universal peptide for IgG1 and IgG4

– Triply charged m/z 603.7 to doubly charged m/z 805.7

• Later found a similar peptide for IgG2 therapeutic proteins Highly conserved (L→V) VVSVLTVVHQDWLNGK

– Not found in plasma of commonly used preclinical species

– Triply charged m/z 599.0 to doubly charged m/z 798.4

• Adequate sensitivity to support animal studies – Blank monkey plasma spiked with 2 ug/mL IgG2 mAb was the most

abundant peak in the chromatogram

Ligand-binding MS and LBA to select Fc fusion proteins

• Most mAb are antagonists that block pathways contributing to disease

• Fusion proteins of agonist peptides attached to human Fc (“peptibodies”)

– Fuse agonistic peptides to carrier proteins or other polymers to extend the circulatory t1/2 of the peptides

– Bioengineered chimeric constructs that do not occur naturally

– Behave similarly to mAb with recycling through FcRn due to conjugation to human Fc

– Not expected that they will be afforded the same metabolic stability

• Fused peptide to non-glycosylated IgG1 Fc C-terminus via polyglycine (Gly5) linker

• Three peptibodies that target the thrombopoietin receptor Anti-human Fc

• Immunoaffinity capture followed by LCMS analysis

– Immunoaffinity pipet tips prepared by covalent attachment of anti-human Fc antibody to activated silica-based resin

– Interchain Fc hinge disulfides were reduced, then alkylated with iodoacetamide

– Affinity tips eluted with aq. ACN (3.3% FA) for QTRAP 4000 LC-MS analysis or with sinapinic acid in aq. ACN (0.8% TFA) for Autoflex II MALDI

Hall MP et al. AAPS J. 2010; 12: 576-85

Metabolite profiling for soft spots/determining assay specificity

• Five proteolytic points were identified for AMG531 in rats

• Two proteolytic products were identified for AMG195 (linear)

• AMG195 (loop) is the most stable construct in rats

• Bioanalysis included total radioactivity (125I-AMG531) and three immunoassays of varying specificity

– AMG531 bridging detected predominantly intact drug

– Fc-AMG531 detected intact drug and metabolites (similar to 125I-AMG531)

– Fc-AMG195 constructs

AMG531 in rat serum after IV dosing of AMG531

and 125I-AMG531 at 0.3 mg/kg LBA1 (open circles) LBA2 (filled circles)

Radioassay (filled triangles)

Antibody-drug conjugates (ADC)

• Toxins attached to antibody-drug conjugates (ADC) are tractable to LC-MS

• The antibody is immunocaptured and the toxin released by hydrolysis

• In the case of a peptide toxin, enzymatic hydrolysis is used and the peptide measured using LC-MS

• Pharmacokinetic profile of the toxin load vs. time is established

• Immunoassays of differing specificity can be used to assess antibody concentrations

• ADC development is now showing the long sought promise of antibodies for drug targeting

• LC-MS has a critical role in characterizing the “payload” of these drug delivery agents

Girish S et al. Cancer Chemother Pharmacol. 2012; 69: 1229–40 Xu K et al. Anal Biochem. 2011; 412: 56-66

Kaur S et al. Bioanalysis 2013; 5: 201-26 Gorovits B et al. Bioanalysis. 2013; 5: 997-1006

Selected LCMS peptide & protein references

• Electrospray ionization for mass spectrometry of large biomolecules

– Fenn JB et al. Science 1989; 246: 64–71

• Opioid peptides, hormone analogs, antimicrobial and anticancer peptides

– van den Broek, I. et al. J. Chromatogr. B. 2008; 872: 1–22

• Analysis of HIV fusion inhibitor Enfuvirtide

– Chang D, et al. J. Pharm. Biomed. Anal. 2005; 38: 487–96

• Analysis of protein drug Tenecteplase

– Buscher, B et al. J. Chromatogr. B 2007; 852: 631–34

• Immunoaffinity chromatography/protein digestion and LCMS

– Johannes S. Hoos et al. J. Chromatogr. B 2006; 830: 262–69

– Whiteaker JR et al. Mol. Cell. Proteomics 2010; 9: 184–196

• Endogenous neuropeptides

– Van Eckhaut A et al. Bioanalysis 2011; 3: 1271-85

• Plasma proteome (major plasma proteins) - SISCAPA

– Anderson, L and Hunter, C. Mol. Cell. Proteomics 2006; 5: 573-588