[ CARE AND USE MANUAL ]
Biopharmaceutical Training Kit
CONTENTS
I. INTRODUCTION
II. STANDARD PREPARATION
III. ADDITIONAL RESOURCES
IV. ORDERING INFORMATION
The Biopharmaceutical Training Kit comes in two separate boxes. The Biopharmaceutical Training Standard Kit (see Table 1) contains
the standards. This kit is shipped at ambient temperatures, however upon receipt of this kit, please store at -20 °C to maximize long
term stability. The Biopharmaceutical Training Prep Kit (see Table 2) contains the materials needed for solubilization and standard prep.
This kit is shipped at ambient temperatures and can be stored at room temperature. Once any component is opened, use right away for
optimum results.
I. INTRODUCTION
The Biopharmaceutical Training Kit is designed to provide trainers
and customers with high quality standards that can be utilized as a
starting point for running biopharmaceutical applications on their
Waters UPLC inlet with Tof or Q-Tof mass spectrometry detection
systems. The training kit pulls together many of the individual
standard samples that are often used in demos and training classes,
including reference compounds for calibration and lockmass. T his
kit provides a foundation for biopharmaceutical LC-MS analysis and
can be used for optimization of methods prior to running valuable
samples to ensure system performance and confidence in results.
Table 1. Kit Components for Biopharmaceutical Training Standards (P/N 186006921)
Part # Contents Description of Components Acid
186006921-1 1 x 15 mL Horse Heart Myoglobin 2.5 mg 15 mL Clear Nalgene Bottle
1860 0 6921-2 1 vial Intact mAb Mass Check Standard 1 mg
1860 0 6921-3 1 vial Glu-fibrinopeptide B 0.5 mg 4 mL Amber Screw Top Vial
1860 0 6921-4 1 vial MassPREP Enolase Digest 1 nmol
1860 0 6921-5 1 vial Reverse Peptide Ser-Asp-Gly-Arg-Gly 1.0 mg of each 4 mL glass amber bottle with screw top
1860 0 6921-6 1 vial Reverse Peptide Gly-Arg-Gly-Asp-Ser 1.0 mg of each 4 mL glass amber bottle with screw top
Amount
per Vial
Container
TruView™ Max Recovery with non-slit PTFE/
silicone Septa (p/n 186005668CV)
TruView Max Recovery with non-slit PTFE/
silicone Septa (p/n 186005668CV)
Table 2. Kit Components for Biopharmaceutical Prep Kit (P/N 186007517)
Part # Contents Description of Components Acid
186007517-1 1 x 2 mL Formic Acid 2 mL 2 mL Ampoule
186007517-2 1 x 30 mL Sodium Cesium Iodide 2 μg/μL 30 mL Nalgene
290002010 3 30 mL Nalgene Fludics Bottle (with cap) Empty 30 mL Nalgene Fludics Bottle (with cap)
Amount
per Vial
Container
[ CARE AND USE MANUAL ]
Table 2. Additional Components Required (not included in kit)
Solvents Containers Pipettes (and tips)
5 x 1.5 mL Waters disposable
LCMS-grade acetonitrile
®
LCMS-grade or Milli-Q
3% hydrogen peroxide, approximately
2 mL (quality that is available and can
be purchased at local drug or grocery
store will suffice)
Caution: Use solvents of the appropriate
grade. Waters recommends Fisher Scientific
LC/MS grade high purity solvents.
water
glass vials (with caps)
Recommended Part # 186000327c
2 x 20 mL scintillation vials with
teflon-lined screw caps or 50 mL
polypropylene falcon tubes for
making up and storing diluents
2 x 100 mL graduated
measuring cylinders
Caution: All glassware must be clean,
rinsed with LCMS-grade methanol and
dry prior to use. Do not use any detergents.
20 µL pipette
200 µL pipette
1,000 µL pipette and 5,000 µL pipette
Caution: All pipettes used to make up diluents
or samples must be calibrated and within
specification. Use appropriate pipettes and
tips during the sample dilution steps.
II. STANDARD PREPARATION
Section 1. Diluent Preparation
a. 90:10 water:acetonitrile + 0.1% formic acid
1. Using a 5,000 µL pipette, add 8.990 mL of LC/MS grade
or Milli-Q water to a clean vessel such as a 20 mL glass
scintillation vial or a 50 mL polypropylene Falcon tube.
2. Using a 1,000 µL pipette, add 1.000 mL of LC/MS grade
acetonitrile. Vortex.
3. Using a 20 µL pipette, add 10.00 µL of formic acid.
4. Vortex.
5. Label as “90:10 H2O:ACN + 0.1% FA”.
b. 75:25 water:acetonitrile + 0.1% formic acid
1. To a clean, dry 100 mL graduated cylinder, fill to the
25.00 mL mark with LC/MS grade acetonitrile.
2. Add LC/MS grade or Milli-Q water to the 100.00 mark.
3. Using a 200 µL pipette, add 100 µL of formic acid.
c. 0.2% formic acid
1. Using a 5,000 µL pipette, add 9.980 mL LC/MS or Milli-Q
water to a clean vessel such as a 20 mL glass scintillation vial
or a 50 mL polypropylene Falcon tube.
2. Using a 20 µL pipette, add 20.00 µL of formic acid.
3. Vortex.
4. Label as “0.2% FA”.
d. 50:50 water:acetonitrile + 0.1% formic acid
1. To a 100 mL graduated cylinder, add acetonitrile to the
50.00 mL mark.
2. Add LC/MS or Milli-Q grade water to fill to the 100.00 mark.
3. Using a 200 µL pipette add 100 µL formic acid into the
graduated cylinder.
4. Sonicate for 5 minutes.
4. Vortex and sonicate for 10 minutes.
5. Label as “75:25 H2O:ACN + 0.1% FA”.
5. Label as “50:50 H
O:ACN + 0.1% FA”.
2
Biopharmaceutical Training Kit
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[ CARE AND USE MANUAL ]
Section 2. Sample Preparation
a. Sodium Cesium Iodide:
1860 07517- 2 1 x 30 mL
Sodium
Cesium Iodide
2 μg/μL
1. Use without dilution for on-board sample fluidics for
calibration and LockMass reference solutions for analyzing
intact proteins for SYNAPT® G2, G2-S and Xevo® G2, G2-S Tof,
and Q-Tof instruments.
b. Horse Heart Myoglobin solution (10 pmol/µl):
186 006921-1 1 x 15 mL
Horse Heart
Myoglobin
2.5 mg
1. Using a 5,000 µL pipette, add 15.000 mL of 90:10
water:acetonitrile + 0.1% formic acid to the bottle containing
2.5 mg horse heart myoglobin (186006921-3).
2. Vortex, and sonicate for 10 minutes. Solution concentration is
167 ng/µL (10 pmol/µL).
3. Using a 1,000 µL pipette, add 500 µL of the stock solution into
a 1.5 mL Waters vial.
30 mL
Nalgene
15 mL Clear
Nalgene
Bottle
d. Glu-fibrinopeptide B solution (200 fmol/µL):
186 006921- 3 1 vial Glu-fibrinopeptide B 0.5 mg
290002010 1
30 mL Nalgene
fludics bottle
(with cap)
Empty
4 mL Amber
Screw Top Vial
30 mL Nalgene
Fludics Bottle
(with cap)
1. Using a 5,000 µL pipette, add 2,000 µL of LC/MS grade or
Milli-Q water to vial containing 0.5 mg of glufibrinopeptide B.
2. Vortex and sonicate for 5 minutes. Stock is 160 pmol/µL.
3. Using a 200 µL pipette, add 25.0 µL of stock to one of the
empty 30 mL Nalgene bottles provided in the kit.
4. Using a 5,000 µL pipette, add 19.975 mL of 75:25
water:acetonitrile + 0.1% formic acid to the bottle.
5. Vortex and sonicate for 5 minutes. Solution is 200 fmol/µL.
6. Label as “200 fmol/µL GFP in 25% ACN + 0.1% FA”.
e. MassPREP Enolase Digestion (2.5 pmol/µL):
TruView Max Recovery
with non-slit PTFE/
silicone Septa
(p/n 186005668CV)
186 006921- 4 1 vial
MassPREP
Enolase
digest
1 nmol
4. Label the vial “Myoglobin 10 pmol/µL”.
5. Inject 1-10 µL for LC/MS analysis of the intact protein,
depending on the sensitivity of the instrument.
c. Intact mAb Mass Check Standard solution (1 µg/µL):
TruView Max Recovery
1 mg
(p/n 186005668CV)
with non-slit PTFE/
silicone Septa
186 006921-2 1 vial
Intact mAb
Mass C heck
Standard
1. Using a 200 µL pipette, add 100 µL LC/MS grade or Milli-Q
water directly to the vial.
2. Vortex and sonicate for 5 minutes. Stock is 10 µg/µL.
3. Using a 20 µL pipette, add 20 µL of stock into a Waters
1.5 mL vial.
4. Using a 200 µL pipette, add 180 µL of 90:10
water:acetonitrile + 0.1% formic acid to the vial, vortex.
Concentration is 1 µg/µL.
5. Label the vial “mAb 1 µg/µL”.
1. Take vial of MassPREP™ Enolase Digest and using a 200 µL
pipette, add 200 µL of LC/MS grade water to the vial.
2. Vortex and sonicate for 5 minutes. Stock concentration is
5 pmol/µL.
3. Using a 200 µL pipette, take 100 µL out of the stock solution
and pipette into a Waters Max Recovery Vial (p/n 186000327c)
or equivalent.
4. Using a 200 µL pipette, add 90 µL of 0.2% formic acid to the vial.
5. Using a 20 µL pipette, add 10 µL of LC/MS grade acetonitrile
to the vial.
6. Vortex. Final concentration is 2.5 pmol/µL.
7. Label as “Control Enolase 2.5 pmol/µL”.
8. Inject 5-10 µL for UPLC®/MSe peptide map analysis, depending
on instrument sensitivity.
6. Inject 5-10 µL for LC/MS analysis of the intact protein,
depending on the sensitivity of the instrument.
Biopharmaceutical Training Kit
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[ CARE AND USE MANUAL ]
2.5 pmol/µL Optional Analyte Enolase digest (Comparative)
sample preparation:
1. Using a 200 µL pipette, transfer remaining 100 µL
of Enolase digest to a Waters Max Recovery vial.
2. Using a 20 µL pipette, add 10 µL of 3% hydrogen
peroxide solution.
3. Vortex.
4. Incubate at 40 °C for 30 minutes.
5. Quench after 30 minutes by using a 200 µL pipette
to add 80 µL of 0.2% formic acid to the vial.
6. Using a 20 µL pipette, add 10 µL of LC/MS grade
acetonitrile to the vial.
7. Vortex. Final concentration is 2.5 pmol/µL.
8. Label as “Analyte Enolase 2.5 pmol/µL.
e
9. Inject 5-10 µL for UPLC/MS
peptide map analysis,
depending on instrument sensitivity.
f. Reverse Peptide Mix (100 pg/µL):
186 006921- 5 1 vial
186 006921- 6 1 vial
290002010 2
Reverse Peptide
Ser-Asp-Gly-Arg-Gly
Reverse Peptide
Gly-Arg-Gly-Asp-Ser
30 mL Nalgene
fludics bottle
(with cap)
1.0 mg
of each
1.0 mg
of each
Empty
4 mL Glass
Amb er Bottle
with Screw Top
4 mL Glass
Amb er Bottle
with Screw Top
30 mL Nalgene
Fludics Bottle
(with cap)
5. Using a 200 µL pipette , transfer 200 µL of Ser-Asp-Gly-Arg-
Gly 1 mg/mL solution into one of the empty, clean 30 mL
Nalgene bottles provided in the kit.
6. Using a 200 µL pippette, transfer 200 µL of Gly-Arg-Gly-Asp-
Ser 1 mg/mL solution directly into the same 30 mL Nalgene bottle.
7. Using a 5,000 µL pipette, add 19.600 mL of 50:50
water:acetonitrile + 0.1% formic acid to the bottle.
Note: Add both peptide solution aliquots directly to the 30 mL
bottle, then add 19.600 mL 50:50 water:acetonitrile + 0.1% formic
acid to the 30 mL bottle, using a 5,000 µL pipette (eg. Add 3 x
5,000 µL aliquots, followed by a single 4,600 µL aliquot of diluent).
8. Vortex and sonicate for 5 minutes. Label this solution as
“10 ng/µL reverse peptide” standard.
9. Using a 200 µL pipette, transfer 200 µL of the 10 ng/µL
reverse peptide solution into one of the empty clean 30 mL
Nalgene bottles provided in the kit.
10. Pipette 19.800 mL of 50:50 water:acetonitrile + 0.1% formic
acid to the bottle.
11. Vortex and sonicate for 5 minutes.
12. Label this solution as “100 pg/µL reverse peptide” standard.
III. ADDITIONAL RESOURCES
Additional literature for some of the products found in the kit can
be located on the waters website at www.waters.com.
1. Using a 1,000 µL pipette, add 1,000 µL of 50:50
water:acetonitrile + 0.1% formic acid to contents of 1 mg
Ser-Asp-Gly-Arg-Gly (186006921-7) in a 4 mL amber bottle.
2. Vortex and sonicate vial for 5 minutes.
3. Using a 1,000 µL pippette, add 1,000 µL of 50:50 water:
acetonitrile + 0.1% formic acid to contents of 1 mg Gly-
Arg-Gly-Asp-Ser (186006921-8) in a 4 mL amber bottle.
4. Vortex and sonicate vial for 5 minues.
Table 1. Kit Components
Description Part Number
MassPREP Enolase Digestion Standard
Care and Use Manual
Intact mAb Mass Check Standard Care
and Use Manual
Reverse Peptide Kit Care and Use Manual 720004421en
Biopharmaceutical Training Kit
715001598
720004420EN
4
[ CARE AND USE MANUAL ]
V. ORDERING INFORMATION
Many of the individual components in this kit can be ordered separately.
Product Description Part Number
Biopharmaceutical Training Kit 176003354
Biopharmaceutical Training Standards 1860 0 6921
Biopharmaceutical Training Prep Kit 186007517
Sodium Cesium Iodide 700002646-8
Horse Heart Myoglobin 700004749
Intact mAb Mass Check Standard 186006555
Glu-fibrinopeptide B 700004729
MassPREP Enolase digest 1860 02325
Reverse Peptide Kit 700005089
Waters, The Science of W hat’s Possible, SYNAPT, Xevo, and
UPLC are registered trademarks of Waters Corporation. TruView
and MassPREP are trademarks of Waters Corporation. All other
trademarks are property of their respective owners.
©2014 Waters Corporation. Produced in the U.S.A.
January 2014 720004859EN K P-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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