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PURPOSE

To develop and validate a routine UPLC based

method for the separation of C-terminal lysine

truncation variants in a chimeric monoclonal

antibody based on IEX gradients generated by

automatic mobile-phase blending

Robert Birdsall*, Thomas E. Wheat, Julann Miller, and Weibin Chen Waters Corporation, 34 Maple Street, Milford, MA 01757

RESULTS

CONCLUSION

Automated method development using

Auto Blend Plus Technology shows highly

reproducible performance.

The integrated platform is suited for robust

and routine method development and can

greatly improve the separation results and

enhance the throughput of the analysis.

W3051 AUTOMATIC METHOD DEVELOPMENT FOR ROUTINE CHARGE VARIANT ANALYSIS OF BIOTHERAPEUTICS BY ION-EXCHANGE CHROMATOGRAPHY

Mobile phase reservoirs:

column

Salt Water

Base Acid

Figure 1. Reservoir Setup. Illustration of a typical Auto Blend

Plus™ Technology reservoir setup.

Day Peak 1 Area

Peak 2 Area

Peak 3 Area

Peak 4 Area

Peak 5 Area

Total

1 252260.0 788195.0 504001.0 296130.0 1052614.0 2893200.0

2 203498.0 660040.0 427519.0 237894.0 940898.0 2469849.0

3 214836.0 686459.0 437254.0 255744.0 974813.0 2569106.0

mean 223531.3 711564.7 456258.0 263256.0 989441.7 2644051.7

SD 25517.4 67665.7 41632.2 29835.9 57276.6 221402.7

% RSD 11.42 9.51 9.12 11.33 5.79 8.37

Test

pH

Buffer

mix 1

Buffer

mix 2

Buffer

mix 3 Mean

Std.

Dev.

%

RSD

5.13 5.10 5.02 5.10 5.07 0.05 0.91

6.12 6.19 6.05 6.19 6.14 0.08 1.32

7.10 7.23 7.06 7.23 7.17 0.10 1.37 Figure 3. Robust Separations with Salt Gradients. The Auto

Blend Plus™ Technology delivers robust separations of C-terminal lysine truncation variants in a chimeric mAb using a

25 minute 25 mM to 65 mM NaCl gradient.

The effects of protein mass load was evaluated

for robustness. The reproducible retention

times and linear response in the Area vs. Mass

Load are indicators of a robust method.

METHODS

An optimized ion exchange method for the

characterization of C-terminal lysine truncation

variants of a chimeric monoclonal antibody was

developed that incorporates a solvent

management system using pure solvents and

concentrated stocks (Figure 1) which will

herein be referred to as Auto Blend Plus™.

Conditions:

LC System: ACQUITY UPLC H-Class System with Auto Blend Plus Technology

Detector: ACQUITY UPLC TUV (Flow cell 5 mm) =280 nm

Column: Protein-Pak Hi Res SP, 4.6x100 mm, 7µm

Temperature: 25 °C Sample Temperature: 4 °C

Injection Volume: 3 µL Flow Rate: 0.500 mL/min

Mobile Phase A: 100 mM MES monohydrate Mobile Phase B: 100 mM MES sodium salt

Mobile Phase C: 1000 mM sodium chloride Mobile Phase D: 18 MΩ H2O

Buffer Conditions: 20 mM MES buffer

Automated algorithms for solvent delivery

Figure 6. Intra-system pH Precision. Separation of C-terminal

lysine truncation variants with three different preparations of MES buffer using Auto Blend Plus™ Technology. Covariance

ranged between 0.91 % – 1.37 % R.S.D.

The optimized method was tested for

robustness using pH, precision, and mass load

as test parameters.

Figure 2. Empirical Table. An example of the empirical

table feature for the Auto Blend Plus™ Technology. Nine

standards were used to calibrate the Auto Blend Plus™

Technology system over a pH range of 5.13 7.09.

The AutoBlend Plus™ Technology delivers

robust separations of C-terminal lysine trunca-

tion variants in a chimeric mAb using salt

gradients (Figure 3) or pH gradients (Figure 4).

The separation method were developed by

systematically varying the salt or pH gradient

generated by The AutoBlend Plus algorithm

(Figure 5).

Figure 4. Auto Blend Plus™ Technology Flexibility. The Auto

Blend Plus™ Technology delivers comparable separations of

C-terminal lysine truncation variants in the same chimeric

mAb using a 15 minute pH gradient from pH 6.50 to pH 7.20 in a 40 mM MES buffer.

Figure 5. The Auto Blend Plus™ Interface. An example of

the Auto Blend Plus™ Technologies intuitive interface for

programming pH or salt gradients. The software algorithm

automatically calculates the percentage of acid and base required for the specified pH or ionic strength.

Intra-system pH precision was tested across

three different MES buffer systems and

determined to be within acceptable range.

The Auto Blend Plus Technology was evaluat-

ed over 40 injections to test reproducibility.

Covariance of individual and total area were

determined to be within acceptable range.

Buffer

mix 1

Buffer

mix 2

Buffer

mix 3

Retention Time [min]

Table 1: Intra-system pH results

+1 Lys

+2 Lys

Retention Time [min]

Chimeric

monoclonal

antibody

+0 Lys

+1 Lys

Retention Time [min]

+2 Lys

Chimeric

monoclonal

antibody

Salt

gradient

+0 Lys

pH

gradient

Day 1

(injection 1)

Day 2

(injection 20)

Day 3

(injection 40)

Table 2: Method precision results

Figure 7. Method Reproducibility. Separation of charge vari-

ants of a chimeric monoclonal antibody at three different time intervals over three days. Integration intervals represent the

five peak areas calculated in Table 2. Individual peak area co-variance was determined to be 11.42 %, 9.51 %, 9.12 %,

11.33 %, and 5.79 %. Total peak area covariance was deter-mined to be 8.37%.

Precision pH Mass load

20 µg

60 µg

100 µg

140 µg

180 µg

Protein Mass Load (µg)

( x1

06

)

To

tal

In

teg

rate

d A

rea R2 = 0.9999

Retention Time [min]

A)

B)

Figure 8. Mass Load Effects. A) Reproducible retention times

were observed over a 9-fold increase in mass load ranging from 20-180 µg of protein. B) The total peak area was

integrated as a measure of precision as shown in the plot of integrated area vs. protein mass load.

100 mM

NaH2PO4

100 mM

Na2HPO4

1000 mM

NaCl H2O

measure pH populate table at

initial setup

flow

low medium

high

measure pH

Protocol: 1) Prepare stock solutions

2) Cross calibrate AutoBlend Plus™

collect

3) Prepare and collect test solutions from instrument

4) Compare

Prepare solutions

The AutoBlend Plus algorithm automatically

calculates the percentage of acid and base

required for the specified pH from the known

pKa of the buffer system or by using an

empirical calibration table (Figure 2).

table populated once at

initial set-up