1. Alz. Assoc. CSF Reference Method Sub-group
Notes of 21nov2011
Alz. Assoc. CSF Reference Method Sub-group

2. Goal
Quantify concentration of analyte as accurately as possible in the reference material to then be used to compare other methods and platforms available…

3. Methods to evaluate (for pros/cons)
MRM published by Waters et al… (need cite)
Absorbance based methods
PPD (Randy Jenkins)- Assay from ICAD poster
Proteome Sciences - “validated” ab42 assay
C2N – quant ab42 and “total ab” assay
Immunoassay based approaches
MSD, Myriad RBM, INNX/Fujirebio
Manu Vandijeck had a new methodogy from Ab42 std working group…
Note: need request validation reports from each.

4. Reference Methods Issues (2)
Issue: are we considering what is available today or what may need to get developed
Consideration of pre-analytic methods and their impact on the analytical methods
Oligomer vs monomer of Ab42
What is the minimum validation required? What is the quality standard?
What is overall target goal? FDA clearance or approval, operate in research space only?
Resources to achieve the goals?
Suggest pull EMA opinions based on BMS submission regarding CSF biomarkers
MSD-RUO initially, require more than MSD alone if tied to drug or indication, working on how take to next level, what are clinical study design and samples for a PMA . . .
Invite DX companies: OCD, Roche to the “table”, CRO have done “validated” Pfizer/ICON, BARC, and Covance
Issue: is method/assay validated and available? Where should it be done?

5. TC Notes 17jan2012 3 methods for 3 analytes: ab42, tau, pTau
Possible methods: MS, immunoassay methods, HPLC, spectrophotemetry basis
Method have or method to develop? May differ depending on the analyte.
If method reference is available, who should conduct the method? Commercial company or international organization? (NIST, etc)
Case Example of Testosterone/Hormone: done by special laboratory in academia (Holland) as a reference method for the world … (??)
MS is one of the candidate methods (e.g. PPD or others), needs to be data driven decisions to assess the possible reference methods. Need to push to develop data itself.
Q: Which technique are reference methods? 3 options … (KB)
Suggestion: to gather the available data on each possible reference method on an analyte by analyte basis in priority order (AS)
Suggestion (HZ): could collaborate on Kaj’s reference method paper with deadline of April to review candidate reference methods
Create 3 by 4 table of analyte by method (MS, immunoassay methods, HPLC, spectrophotemetry basis… (MC)
Note CAMD meeting with FDA to discuss suitability of CSF markers (MRI and CSF), Feb. 28/29th, , AA would like to push to get some progress before (MC).
Document the progress or state of methodology would be worthwhile… (LS) longer term is reference materials and reference methods
Rec’d to focus on Ab42 and tau, then circle back later to pTau which appears more difficult with multiple isoforms (HV, Charlotte ?), pTau offers clinical differential value
Suggest: stepwise order, Ab42, then Tau, then pTau in this order of focus (?)
For CAMD FDA mtg, would like to show FDA there are approaches to standardization (this groups work and plan), we are moving forward (MC)
Longer term goal is harmonization, but this is longer term and uncertain as to when it will occur (LS), should be transparent about this gap of harmonization
Look in other fields, go for serum proteins or hormones as examples, assays in field for long time and still not harmonized across assays… (KB)
Next steps: table with 3 analytes, 3 or 4 methods
Suggest start with Ab42 (KB)
MS, immuno assay, HPLC, possible for spectro … should look at data directly (RB)
Promising paper to include Lei Wang (Biomarker … Theresa will forward for inclusion).

6. TC Notes 17jan2012 (2)
Agree focus on Ab42 3 methods for 3 analytes: ab42, tau, pTau
MS papers on:
Waters published in Analytical Biochem (Erin ? At Waters , presented at ASM last week and published)
PPD poster at ICAD, based on Waters method with important modifications
ASM meeting abstracts
PPD works in GLP environment, subjected to some validation, should include them in the process (OL), deeply involved in method for over 2 yrs now
Penn has spoken extensively with Rand Jenkins at PPD (LS) and now has the PPD methods installed in the Penn lab…
Action items: collect papers, validation reports, etc on Ab42 methods available . . .
Next call on Jan 31. Must be able to get materials to Maria in advance of next call
Ab42 MS : ? Kaj and Henrik collate works, Les to join
Ab42 Immunoassay candidate methods: ? Amsterdam to take lead (RB and Charlotte, Hugo?)
Ab42 HPLC: ? Method for preparation of sample, not detection … total vs denatured Ab42?
Ab42 SDS/Page and Western blot : ? Piotr etal working on this …
Ab42 Spectrophotometery : ?
Possible need for two independent (but linked) steps in the reference method:
Sample prep / handling / extraction – e.g. HPLC
Quantitation / detection method – MS, immunoassays, put spectrophotometry on hold
Or do we just focus on a single method ? For review of what exists for Ab42, this is only what is available, be pragmatic on what exists without need for further development (OL)
Clearly need Analytical performance characterized and ironed out before any clinical performance evaluation or assessment is considered (0L)
Step 1 of AB42: focus on MS assays as they exist today?
Suggest review published criteria for a reference method: AACC, WHO etal; look at criteria/characteristics (come to agreement) and then evaluate putative reference methods against them (RB)

7. TC Notes 31jan2012 (1)
Review of past actions since last meeting-data / materials shared
Abstract from PPD method, Waters paper out from Pfizer group (suggest present to the group), PPD additional information – Bill comfortable with distribution
What the plan is for analytical validation, # analytes, grouping, etc… ?
U Penn able to replicate PPD work flow nicely. Focus on Ab42 important, at sacrifice of other analyte performance and 2-42 are also at reasonable concentrations.
U Penn focusing on AB1-38, AB1-40, AB1-42 via MS in an adapted form of the PPD based work flow.
INNX assays: yes, they are specific to the n-term aa1, rec’d AB1-42.
Q re K Blennow data on Abx-40/Abx-42 as similar to 1-40, 1-42? A: maybe due to CSF samples, could need to compare on the same technology platform.

8. TC Notes 31jan2012 (2) PPD Poster discussion follows:
Lot 1,2,3 are individual human lots of CSF
Authentic CSF materials with spiked material and look at recovery to determine Bias
Q: what was spiked, in what liquid were the spiked analyte prepared in?
DMSO stock solution, then dilute into aCSF solution for nominal solution, to then spike this nominal solution into aCSF and the method of standard additions in hu CSF.
Note: purchase peptides AnaSpec but 1-40, 1-42 from R-peptide, non amyloid from Phoenix pharma.
Extraction recovery is from previous expts is from 70-80%, calculated into via non-radio internal standard adjusts for differential extraction recovery.
Stable isotope labeled internal standards to adjust for extraction differences: 5 standards are IS labeled, repeater pipettes within 3%.
Omar clarifies, use of internal standards via repeaters are well established methods
Challenge in LC and MS to get broad coverage, about to break in class 14/15/16/17 for one method, another method 34/37/38/40/42/43/39 would be another MS class … assay direction
PPD doing 34-43, then back for 14-17, perhaps consider zero in on ab1-42 only
Rat serum for non-specific blocker, now prefer BSA, can BSA be in tube ahead of time
Pre-analytical variables should be considered
Les U Penn to try the SUH AA QC samples program , PPD as well..
Possible Next steps:
Organize what we know amongst the 3 methods, Les/Omar to generate slide(s)
Draft validation plan to review at next meeting (Holly/Adam)
Need specify: the peptide vendors, extractions, spikes, solvents, how many analytes to be measured in the MS reference method
then go thru an extensive validation exercise
then talk about next steps to get IFCC to recognize a reference method; are we measuring the same thing, correlations between MS and immunoassay results

9. TC Notes 8feb2012 Validation Plan from Holly/Omar:
Les / Omar to report on 3 method comparison – next meeting
Rand on additives: Some use Tween to reduce binding to tubes, while others use low binding tubes without additives
Holly: CSF ab42 MS well developed, we need consortium analytical validation, calibration or sample analysis
Level of ab42 MS analytical performance:
Robustness of variability
6-7 runs of variability
LLOQ/ULOQ
Specificity : focus on 1-42, do have interference with other peptides
What are ideal upper/lower limits for the assay… 50 pg/ml LLOQ; ULOQ?
Les: pg/ml is a good dynamic range
Establish concentrations for reference material going forward
Chromatography piece, insure not carryover,
Understand matrix effects, to the extent they do or don’t, spike recovery, dilution linearity, parallelism
Derivatize reagents – not in this method
Solid phase extraction, little columns in 96 well array
Long term stability, -80 for sure, -20 if some site will only have this
Stock equivalency checks – robustness of reagents
Key topics: Precision, stability , matrix issues, robustness of reagents
Rand Q: are we going to delve into pre-analytical issues? (suggest leave to the side)
Compare MS to immuno-assays? correlations? How operationalize?
Maria: how operationalize? Who draft plan, who agree to the plan, who run them, who fund them
PPD, Covance, BARC

10. TC Notes 8feb2012 (2) Tau based MS reference method?
Q: Do we want an antibody to concentrate all 6 isoforms?
Q: C13-leu ab42? Do we want to compare to the Wash U approach?
BMS possibly to come forward with where MS tau methodology (Tentative)
Bob Martin: MS paper to see different isoforms of tau in CSF (will try to get paper circulated)
CSF tau? It is fragmented and all isoforms are represented…
Ab42 Next Steps:
Rand to share ab42 single analyte validation plan from PPD
Team to nail down the actual MS methodology – PPD, Waters, U Penn agree on method
Team to develop and agree to single analyte validation plan
Then move forward with PPD method validation at
Rand and PPD team
Les S and U Penn team
Henrik to work with Les and Rand for SUH involvement (have 1-42 assay running presently, have not done prelim clinical performance assessment)
Covance (Bob Martin) express interest to participate
Caprion ? Will be open if they have interest as well
Les provides caution: Need realistic expectation and timelines for this work
Action item 1: Les/Henrik/Rand/Omar put method slide compare methods – mar 15.
Action item 2: Rand/Les/Henrik/Erin Chambers (Waters) – come up with aligned method – mar 15.

11. TC Notes 8feb2012 (3)
Action item 3: Validation plan from Holly and Omar to be brought back to team for next call
Henrik update on reference methods in biomarkers in medicine paper
Henrik to invite co-authors, others to send Henrik an if have interest…
They (SUH) will draft, others to give input and ultimately publish
CAMD meeting with FDA end of February to discuss CSF biomarker use in drug development
Holly and Les to participate
Qualify ab42, t-tau, p-tau for use to enrich clinical trials in predementia context of use
Hope to rally around prognostic use as stepping stone to companion Dx approach
Approvable assay needs an independent method to measure calibrators, standardize components within the field, come up with certified ref material as “standard of truth”
Do we have tests ready today? The field has RUO tests today with good clinical performance, but gaps exist, but they are precision measures.

12. TC Notes 16mar2012 (1)
Les Shaw (Penn) presented: on MS method comparison excel table done collaboratively with Omar Laterza (Merck)
Goal: develop Ab42 standard ref material for CSF … then for tau
Gothenburg working with IFCC etc
Not only have the material but also the ref method to assign values in 2-3 pools of CSF covering a range of ab1-42 concentrations.
Considerations for reference methodology MRM HPLC tandem MS, development spear headed by Erin (at Waters, on call), high conc Quan HCl treatment to release forms of abeta, sample clean up with mini cartridge mix bed extraction system, samples inject in MS/MS for MRM to quantify ab1-42 in csf, having released from all sources of aggregation
Peptides: range from 1 to quite a few, most common are 38, 40, 42
Internal stds: all non-radio isotopic labelled peptides
Conc int stds: 1-2 ng/ml
CSF volume: start from 50 ul upto 250 ul
Calibrator diluent : for immunoassay is critical re matrix effects, also true for MS, so calibrator diluent is very important… Erin started with rat plasma… Rand at ICAD described use of 4 mg/ml bovine serum albumin, play with add back some rat plasma
Les finding improved precision with rat plasma added in..
All use mixed bed micro columns and high conc guanidimium treatment
Liquid C systems do differ based on configuration
Dilution and injection: dilution is important, then inject uL of sample
Solvents; fairly comparable

13. TC Notes 16mar2012 (2) Flow rate eluting column Run time : 8-10 min
LLOQ: from 25 to 100 pg/ml, Penn now at 50 pg/ml
Ab42 accuracy of calibrators:
Spike in pure ab42 std peptide into huCSF: recovery % with bias less than 10%
Precision of QC: not as good as like, but getting better as learn to tweak and optimize
Calibrator precision: getting better with rat serum added to 4% BSA
June Q: is there loss thru micro extraction column? A: Erin: typically see recovery about 85-90% thru the solid phase extract cartridges… for calibrators in absence of diluents, goes down to 50%, but with BSA and rat plasma gets better… adding the right percentage is important to get best recovery… more going on than we are fully aware of.. For aCSF to fully mimic huCSF, nec to add-in other components… other interactions beyond proteins, in case of plasma; calibrators had to sit on bench for 30min to equilibrate to mimic huCSF.
Just add carrier protein to reduce non-specific absorption..t hen found plasma added benefits than simply protein alone, improved accuracy and precision.
PPD work (Bill): HSA and other fractions (final 400 mg/L), hu IgG (50 mg/L final) was nearly double, IgG added is doubled as well for internal std work at PPD.
Plasma : danger is sources could be variable, differing backgrounds, would want manufactured equivalent to plasma… overall cry for aCSF …

14. TC Notes 16mar2012 (3)
aCSF : ok in many instances, but Ab42 has protein binding and protein aggregation needs something else
Erin criteria: Need meet +/- 15% for all pts but lowest which is 20%, she likes 7-8% overall better goal; Les likes 5-7% overall, PPD (Bill) feels may be tough: he likes more typical acceptance criteria of 20 and 25% ... For the candidate ref method to assign values as accurate as possible to a small set of 2 – 3 pools of huCSF
Erin at Waters: we see average accuracy numbers are in single digits …
Penn: need clean system after month or two.. When cleaning is done, can’t run an assay at that point.. Run repeatedly CSF samples as if we released hot spots and need fill them in…
PPD: ion optics do get contaminated more easily, typically Q-jet needs to be cleaned …
Omar raise Q: how to assess amongst these MS methods to pick a reference method ?
Omar further refine Q: is challenge to unify all methods to perform in a similar fashion or take each independently, then select the ref method, or unification?
Les: combination of the two approaches…
Holly’s view: not see a lot of shades of difference, working group getting towards agreement, hope to get to an interlab study… instruments are different.
Platform?: IRMM (Ingrid Zeegass?) stated (to Henrik Z) it does not have to be identical platform, could be run on different MS instruments, she said “Yes” possible…
Henrik Z: Working group at IFCC, no news yet when formed, but we can interact with them when form, hope to send overview paper to folks…

15. TC Notes 16mar2012 (4)
Next steps? Les, get further discussion on diluents constituents, ultimate maybe human protein based, HSA, IgG, other constituents, concentrations of glucose, etc …
Bill (PPD) efforts for aCSF: increased HSA and IgG, each lot of rat plasma is different… EDTA for anti-coag for rat plasma… Heparin tried?
Next steps ?: Les, continue discussion off line, key items to settle on … work off-line…
Not flexible: Sample pre Tx, extraction, nature of the calibrators, assay accuracy
April 6 to-do:
les update on diluents
Henrik on working group for IRMM, update on publication
Omar, Adam, Randy, have discussion on assay specification -- ID critical parameters
Omar interested to get validation reports from the labs if at all available.

16. TC Notes 16mar2012 (1)
Action item 3: Validation plan from Holly and Omar to be brought back to team for next call