Care and use manual – Waters UPLC Intact Mass Analysis Application Kit User Manual

Page 8

MS Conditions
The MS parameters listed below can vary depending upon the
physical characteristics of the analyzed proteins, flow rate, and
MS probe position.

Note: Desolvation temp, source temp, and desolvation gas values used in the
above table are based on 0.5 ml/min flow rate.
* Waters SQD parameters can be optimized using IntelliSmart™ software.

Tips for Success
Results obtained with the UPLC Intact Mass Analysis Application
Kit can vary depending upon the physical characteristics of the
analyzed proteins as well as the performance characteristics of
the LC-MS system, injector, and injection wash protocol used.
Additional sawtooth gradient cycles can be applied following
the analysis gradient to regenerate the column back to pre-
injection conditions. Suggested needle wash solvents and column
temperature for antibody LC-MS analysis are listed below:

■

Needle Weak Wash Solution Composition: 0.1% formic acid
in water

■

Needle Strong Wash Solution Composition:
65% acetonitrile/5% IPA/30% water in 0.1% formic acid

■

Column temperature: 80 °C

Separation of light and heavy chains of an antibody can be
obtained by optimizing gradient slope (gradient slope is measured
as percent of organic such as acetonitrile per mobile phase
volume passed through the column), flow rate, initial gradient
strength and column temperature. Set the column temperature to
80 °C. Use the gradient method in Table 5 as a starting point for
optimizing the separation of light and heavy chains of an antibody.
Follow the steps below to further optimize the separation of light
and heavy chains.

1. Find a gradient slope that provides an acceptable resolution

of your reduced antibody.

2. Use the result from the initial separation to design a gradient

(focus gradient slope) that improves the resolution between light
and heavy chains by focusing the gradient (narrow gradient).

3. Then adjust the gradient initial strength (while keeping the

gradient slope constant) to reduce the analysis time without
sacrificing the resolution.

Conditions

LCT Premier

SYNAPT

HDMS

SQD*

Ionization

Mode

ESI Positive

Capillary

Voltage

3–3.2 kV

2–3 kV

4.2–4.5 kV

Cone

Voltage

40–50 V

35–37 V

39–45 V

Desolvation

Temp

350–450 °C

Source

Temp

120–150 ° C

120–150 °C

Desolvation

Gas

800 L/Hr

600–800 L/Hr

Acquisition
range (m/z)

600–5000

(Intact)/

600–3000

(Reduced)

600–5000

(Intact)/

600–3500

(Reduced)

600–2000

(Reduced)

Ion Guide I

80–100 V

(Intact)/5 V

(Reduced)

Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com

[ CARE AND USE MANUAL ]

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