PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com

Introduction 1

5

Six-plex MS/MS method to measure I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 enzyme activities in DBS

Joe Trometer, Anna Potier, Jason Cournoyer, and Mack Schermer; PerkinElmer, Waltham, MA 02451 Alyssa Vranish and Jim DiPerna; PerkinElmer, Bridgeville, PA 15017 Yang Liu, Fan Yi, Naveen Kumar Chennamaneni, Zdenek Spacil, Arun Kumar, Joyce Liao, Michael H. Gelb, C. Ronald Scott and Frantisek Turecek; University of Washington, Seattle, WA 98195

The mucopolysaccharisodes (MPS) family of lysosomal storage disorders (LSDs) is caused by defects in the metabolic breakdown of glycosaminoglycans (GAGs). Our previous work demonstrated the ability to distinguish samples with low enzyme activities for I2S (MPS II), NAGLU (MPS IIIB), GALNS (MPS IVA) and ARSB (MPS VI) using a single 3.2 mm dried blood spot (DBS) punch and one incubation cocktail. Adding to this MPS family, the current work will focus on also measuring the enzyme activity of GUSB (MPS VII) and TPP1 (CLN 2). This new six-plex still requires only a single DBS that is incubated overnight at 37 °C followed by a post-incubation workup that is less than 30 minutes per plate. Sample-to-sample time using MS/MS analysis can be as low as 2 minutes, which allows the possibility to obtain more than 4320 results per day if desired. Method performance studies show good linearity for each enzyme in their respective activity range. Furthermore, a study consisting of several hundred presumed healthy neonates, confirmed low I2S/NAGLU/GALNS/ARSB/GUSB/TPP1 activity and CDC control DBS showed excellent resolution and clear distinctions between the different enzyme activity levels.

Summary

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Materials and Assay Procedure 2

Formulation of 96 sample S+IS vial (3.3 mL) assay cocktail

Substrates MW µM mg/vial Int. Standards MW µM µg/vial

I2S-S 767.2 500 1.19 I2S-IS 648.3 5 11

NAGLU-S 622.8 500 1.03 NAGLU-IS 422.6 5 7

GALNS-S 781.9 1000 2.52 GALNS-IS 689.9 5 11

ARSB-S 753.9 1000 2.42 ARSB-IS 661.8 5 11

GUSB-S 609.7 500 1.01 GUSB-IS 440.6 10 15

TPP1-S 638.4 200 0.42 TPP1-IS 358.3 15 18

Buffer Composition

Reagents MW mM

Ammonium Acetate (pH 5.0) 77.1 60

Cerium Acetate 317.3 7

NAG-thiazoline 219.3 0.1

MSMS

3.2 mm DBS punch

to 96-well plate

Quench & Mix

37°C, 400 rpm

18 ± 2 hours

Remove seal, add 100

µL

50:50 MeOH:EtOAc

Mix with pipette

Transfer Liquid & Extract

Transfer Top

Layer

Transfer 200 µL top layer

to sampling plate

Room temp (25°C) at

400 rpm 10

min

Shake &

Incubate

Transfer to 96 deep well plate

(DWP)

Add 400 µL EtOAc then 200 µL 0.5

M NaCl Solution Mix with pipette

Separate Layers

Centrifuge DWP 5 min,

700 x g

Dry

Evaporate at

40°C, 10-15 min Reconstitute with 100

µL 60:40 H2O:ACN with

0.1% formic acid

Add Flow Solvent

Add 30 µL

cocktail Seal plate

Punch

Shake

Add Cocktail

1 2 3 4

5 6 7 8 9 10uL injection

using X-Select

column

The assay procedure is detailed in the workflow diagram in Figure 1. The formulation of the incubation cocktail is in Table 1 and the structures of the substrates and IS are in Figure 2.

Table 1. 6-plex incubation cocktail components

Figure 1. 6-plex Assay Protocol

Substrates Internal Standards

Samples were analyzed using a Waters® Xevo® TQD with an X-select CSH C18 column and a VanGuardTM CSH precolumn. A flow solvent consisting of 60:40 H2O:Acetonitrile with 0.1% formic acid at 0.5 mL/min showed good separation between the internal standard and residual substrate peaks for each analyte.

To assess assay performance, a study consisting of DBS from de-identified presumed healthy subjects (N=616) collected in August 2016 and DBS with confirmed low (N=29) activity for I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 were run over 3 days (~3 plates per day). Each plate had 2 blanks (punched blank filter paper) and two replicates of each control level. Assay precision was estimated using the 18 control replicates each of low/mid/high over the 3 days.

Methods 3

Dot plots of the results for the six enzymes are shown in Figure 3. Multiple replicates (N=18) of the low, mid and high control DBS showed good precision in the assay for all six enzymes. Table 2 shows that all controls had %CV ≤ 11% except the low control for NAGLU and TPP1.

The data shows clear distinction between the presumptive normal and the confirmed low activity DBS suggesting that a cut off can easily be made to routinely differentiate low activity from normal neonatal DBS. Furthermore, all confirmed low activity samples were less than 10% of the average presumptive neonatal population and also less than the lowest presumptive normal activity for each enzyme, as shown in Table 2.

Results 4

A new tandem mass spectrometric multiplex assay for measuring I2S, NAGLU, GALNS, ARSB, GUSB and TPP1 enzyme activities in DBS was demonstrated. This method was able to clearly differentiate between the activities in samples collected from presumed healthy subjects and in DBS samples from individuals confirmed to have low enzyme activity. Good precision and linearity of each enzyme was demonstrated using control DBS. • We thank the Mayo Clinic, Greenwood Genetics and Serv.

Genet. Med. HCPA for providing confirmed low activity DBS samples.

All DBS samples were collected following IRB approved protocols.

Type Enzyme Activity (mM/hr)

I2S NAGLU GALNS ARSB GUSB TPP1

Low Control 0.78 0.21 0.46 1.68 8.51 5.33

%CV 7% 24% 9% 10% 11% 22%

Mid Control 6.89 2.68 3.04 11.6 25.7 19.9

%CV 5% 10% 6% 8% 7% 10%

High Control 13.0 5.85 6.14 24.3 45.1 24.9

%CV 5% 10% 6% 11% 7% 6%

Ave. Normal Neonatal 13.8 2.56 2.52 11.0 27.7 20.4

Lowest Normal Neonatal 6.74 1.17 0.72 3.33 14.8 11.6

Highest Confirmed Low 1.22 0 0.24 1.13 0.11 0.64

Table 2. Average activities for control DBS and DBS from presumptive normal neonatal DBS for the enzymes I2S, NAGLU, GALNS, ARSB, GUSB and TPP1. The lowest normal neonatal is the lowest activity measured of the 616 DBS analyzed and the highest confirmed low is the highest measured activity in the confirmed low-activity cohort.

Figure 3. Dot plots for the enzymes measured in DBS from the presumptive normal neonatal, DBS from individuals with confirmed low enzyme activity and low, mid and high controls.

R² = 0.995

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Observed Enzyme Activity (mM/hr)

I2S

R² = 0.9967

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Observed Enzyme Activity (mM/hr)

NAGLU

R² = 0.9955

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Observed Enzyme Activity (mM/hr)

GALNS

R² = 0.9911

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ARSB

R² = 0.9961

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Observed Enzyme Activity (mM/hr)

GUSB

R² = 0.9804

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Observed Enzyme Activity (mM/hr)

TPP1

Figure 4. Linearity series for the enzymes measured in DBS.

I2S

NAGLU

GALNS

ARSB

GUSB

TPP1