Determination of Pluronic F-68 in High Protein Matrices by HPLC-RAM-ELSD
Helen Chung, Melissa Khor, and Jinshu Qiu*
Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320

Column TypeHi SEP

Cadenza
HS-C18AV-2Runtime

30 min30 min35 minFlow Rate

0.2 mL/min

1.0 mL/min

0.4 mL/min

ObservationNo
separation

Poor
reproducibility

Goodseparation

Selection

NoNo

Yes

Mobile Phase Selection
For Mobile Phase (MP) A, 100 mM ammonium
acetate and 0.1% HAc were examined. A blank peakinterfering with the PF-68 peak was seen withammonium acetate, but not with 0.1% HAc (Fig. 1).
Fig. 1. Selection of MP A

52000

PF-68 is a low-foaming, non-ionic surfactant that is widelyused in mammalian cell culture processes. Because PF68 is not a generally recognized as safe reagent, its
clearance during the purification processes should bedemonstrated. PF-68 is often determined by acolorimetric method. This type of method requires tedious
sample preparation procedures to isolate the PF-68 fromthe proteins present in the samples. However, theCadenza HS-C18 column has the capability to exclude
proteins while separating small molecules. The column isa hybrid octadecylsilyl stationary phase that has
hydrophilic groups on the outer surface and hydrophobicgroups within the pores. A sample with a high protein
concentration can be injected directly on the column
without a clean-up procedure. The objective of this study
was to develop an HPLC method with this column for
determination of PF-68 in samples with high protein
concentrations.

Materials and Methods
HPLC System:
Detection:
Column:Agilent 1100 pump and autosampler
ELSD
Cadenza HS-C18 (150 x 3.0mm, 3µparticle size)
Mobile Phase: A: 0.1% HAc
B: 0.1% HAc in acetonitrile
MP Gradient:0 – 5.0 min, 0%B; 5.1 – 10.0 min,
10%B;10.1-15.0 min 45%B; 15.1-25 min
100%B; 25.1-35 min 0%B
Col. Temp.:
Ambient
Injection Vol.:
15 µL
Flow Rate:
0.4 mL/min
Runtime:35 minSampler Temp.: 4ºC
Dilution Soln.: 0.1% HAc

50000

Limit of Quantitation (LOQ). The LOQ value wasdetermined to be around 10 mg/L using a signal tonoise ratio (10:1) approach (Fig. 6). For verification,
the PF-68 standard was spiked into a sample to afinal concentration of 10 mg/L and the spiked
sample was measured. The precision and accuracy
at the LOQ level are shown in the insert of Fig. 6.
Good accuracy and precision results confirmed thatthe LOQ was 10 mg/L.
Fig. 6. The 10 mg/L PF-68 signal and its precision
and accuracy in the sample matrix

mAU

PF-68
PF-68

Sample with PF-68

8000

Sample buffer

6000

ADC1 A, ADC1 CHANNEL A (2009\HC090..._PLURONIC\HC090217RO_HS-C18_PLURONIC 2009-02-18 15-13-06\HC000019.D)
mAU

4000

119000

PF-68

48000

2000

11800046000

102030
ADC1 A, ADC1 CHANNEL A (2008\HC081..._PLURONIC\HC081015RO_HS-C18_PLURONIC
2008-10-15 12-46-27\HC000014.D)
ADC1 A, ADC1 CHANNEL A (2008\HC081..._PLURONIC\HC081015RO_HS-C18_PLURONIC 2008-10-15 12-46-27\HC000011.D)

400minmAU

00.1% Acetic Acid

1600005101520*ADC1 A, ADC1 CHANNEL A (R:\CHEM32\..._PLURONIC\HC090327RO_HS-C18_PLURONIC 2009-03-27 10-48-04\HC000019.D)*ADC1 A, ADC1 CHANNEL A (R:\CHEM32\..._PLURONIC\HC090327RO_HS-C18_PLURONIC 2009-03-27 10-48-04\HC000020.D)

2530min117000mAU
10000

150000Sample without PF-68
PF-68

8000

PF-68

PF-68

140000

RepPF-68 Recovery in
Sample Matrix

I78%

II

89%

116000

6000

Blank

Sample with LOQ Spike

4000

115000III

1300002000

96%Ave.

88%

RSD

10.4%

1200001140000510152025min

0Two HAc levels (0.1% and 0.5%) with ACN in MP B
were also evaluated for PF-68 separation. A minor
peak interfering with the PF-68 peak was found with
0.5% HAc, but not with 0.1% HAc (data not shown).
Therefore, 0.1% HAc and ACN in 0.1% HAc wereused for MP A and MP B, respectively.
MP Gradient Selection
Several gradients were used to separate PF-68. It
was found that the gradients had a profound effect on
the PF-68 signal. Chromatograms of four gradientsare shown in Figure 2. Gradient 3 was selected (seethe gradient in Materials and Methods Section).
.
Fig. 2. Effect of MP gradients on PF-68 signal

51015202530min

5Response Curve, Range and Linearity. The
responses of the standards from 10 to 100 mg/L
were fitted to a quadratic equation. All the r2 valuesof the tested standard curves were > 0.995. Atypical standard curve is shown in Fig. 4. Linearity
was evaluated by determining the correlation
between the recovered and expected concentration
for the PF-68 standards spiked into the tested
samples (5 levels with 3 replicates each). The r2 for
Samples A and B was > 0.998 (Fig. 5).
Fig. 4. A quadratic standard curve
Pluronic,
Area = 166.067637*Amt^2 +8478.993*Amt -63469.533
Area*ADC1 A, ADC1 CHANNEL A (2008\HC081..._PLURONIC\HC081015RO_HS-C18_PLURONIC 2008-10-15 12-46-27\HC000012.D)*ADC1 A, ADC1 CHANNEL A (2008\HC081..._PLURONIC\HC081016RO_HS-C18_PLURONIC 2008-10-16 08-26-54\HC000011.D)*ADC1 A, ADC1 CHANNEL A (2008\HC081..._PLURONIC\HC081022RO_HS-C18_PLURONIC 2008-10-22 09-33-10\HC000014.D)*ADC1 A, ADC1 CHANNEL A (2009\HC090..._PLURONIC\HC090122RO_HS-C18_PLURONIC 2009-01-22 09-05-28\HC000023.D)

2000000

mAU

1500000

Gradient 3202530min

Robustness. DOE with two factors was used toevaluate the method robustness (Table 3). The
statistical analysis showed that small variations inthe compositions of the MP and column temperature
did not affect the analytical results (P>0.05),
indicating the robustness of the method.
Table 3. The method robustness evaluation
Composition

Factor-1

0PF-68, mg/L
01

0.09% 0.10% 0.11%

33.5

33.3

33.5

0.28

Col. Temp

20C

33.5

33.3

33.5

0.88

25C

1P-value-1

MP A-HAc

30C

3500000

Table 4. PF-68 Conc. at different column pools

21

Gradient 2

60000041000000

Gradient 4800000

15PF-68 clearance in purification processes
The results show that PF-68 is cleared to below theLOQ by column A (Table 4).

25000001200000

1000000

10Rel. Res%(1): 10.022

5Correlation: 0.99996

00400000

50Amount[mg/L]

Gradient 1
200000In-process

Col. A LoadCol. ACol. B

UFDF

PF-68, mg/L

690

< LOQ

< LOQ

< LOQ

Fig. 5. Method linearity and accuracy

00

5

1015

20

25

30

35

40

PF-68, mg/L
Sample A, 20x Dil. Sample B, 5x Dil.

Results and Discussion

100minTable 2. Effect of the HAc level in the dilution solution
HAc LevelColumn Selection
Three types of protein exclusion columns were evaluated.
The Cadenza HS-C18 column was found to be suitablefor separation of PF-68 (Table 1).

Method Characterization
Specificity. The method specificity was examined bycomparing chromatograms of the sample and its
buffer without PF-68 for the PF-68 containing
sample, or chromatograms of the sample with andwithout PF-68 spike for the sample without PF-68(Fig. 3). No significant interference from the two
sample matrices was found.

*ADC1 A, ADC1 CHANNEL A (R:\CHEM32\..._PLURONIC\HC090325RO_HS-C18_PLURONIC 2009-03-25 13-16-47\HC000022.D)*ADC1 A, ADC1 CHANNEL A (R:\CHEM32\..._PLURONIC\HC090217RO_HS-C18_PLURONIC 2009-02-18 15-13-06\HC000026.D)

100mM
ammoniumacetateBlank peak

PF-68

54000

Repeatability and Intermediate Precision (IP). The
RSD of the spike recoveries (n=15) calculated from
Fig. 5 was used for the repeatability determination.
The precision was 7.7% for Sample A and 4.5% for
Sample B. The IP was evaluated by 2 analysts, 2instruments, 2 columns over 5 days. The RSD(n=15) was 5.9%. The results indicated acceptable
precision.

Fig. 3. Specificity of the method

ADC1 A, ADC1 CHANNEL A (2008\HC080115_PLURONIC_HS_C18\HC080115 2008-01-15 12-43-38\HC000008.D)
ADC1 A, ADC1 CHANNEL A (2008\HC080115_PLURONIC_HS_C18\HC080115 2008-01-15 12-43-38\HC000009.D)
mAU

56000

Introduction

Effect of HAc Level in Dilution Solution
HAc was used in the sample dilution solution todissociate PF-68 from proteins. Three acid levels(0.1, 1 and 2%) were examined. Samples werediluted ≥ 5x. Different acid levels did not affect thedetermination of PF-68 (Table 2). Since both of themobile phases contained 0.1% HAc, this concentration was selected for a better comparability.

14.446 - Pluronic

An HPLC method was developed to determine Pluronic
F-68 (PF-68) in in-process samples from monoclonal
antibody (MAb) preparations. The method uses aCadenza HS-C18 column, a restricted access media(RAM) stationary phase, that permits the direct injectionof proteinaceous samples after a single sample dilution.
The HPLC system consisted of a pump, an autosampler,a column switching valve, and an evaporative lightscattering detector (ELSD). PF-68 was separated withtwo mobile phases, 0.1% acetic acid (HAc) and 0.1%
HAc in acetonitrile, with step gradient elution. Proteins
eluted in the void volume and were diverted into a wasteline, while PF-68 eluted at 14.5 min. A quadratic equation
was used to fit the standard curve. The present method
offers the advantages of simple sample preparation (asingle dilution) and sensitivity (10 mg/L quantitation limit).

Table 1. Columns for PF-68 separation

Recovered PF-68 Conc., mg/L

Abstract

Conclusion

Sam ple Ay = 1.0598x - 1.426
R2 = 0.9989

908070A simple and sensitive method for determination ofPF-68 was developed using the HS-C18 column andELSD detection.. The results of specificity, linearity,precision (repeatability and IP), accuracy, LOQ, androbustness showed that the method was suitablefor testing PF-68 clearance in MAb preparations.

605040Sam ple B
y = 0.9725x + 0.9303
R2 = 0.9991

3020
1000.1%33< LOQ1%33< LOQ2%32< LOQ

0204060
Expecte d PF-68 Conc., m g/L

80100Accuracy. Using the data shown in Fig. 5, themethod accuracy was calculated from the slope ofthe regression * 100%. The accuracy was 106% forSample A and 97% for Sample B.