Polysorbate 80 Profiling by HPLC with Mass and Charged Aerosol Detection

APPLICATION NOTE 73979

Polysorbate 80 profiling by HPLC with mass and charged aerosol detection

Authors: Mauro De Pra, Denis A. Ispan, Katherine Lovejoy, Sylvia Grosse, Stephan Meding, Frank Steiner Thermo Fisher Scientific, Germering, Germany Keywords: Vanquish Flex, Vanquish Duo, ISQ EM, CAD, inverse gradient, Tween, polysorbate, surfactants, formulations, charged aerosol detection, single quadrupole, mass detection, ultra high performance liquid chromatography, excipient

Application benefits • HPLC-CAD method can detect batch-to-batch • Mass detection with ISQ EM enables identity assignment discrepancies of the main species in polysorbate 80

• Polysorbate 80 quality issues in raw material are detected • The CAD and ISQ EM mass detector are easy to operate, before the surfactant is used in the drug formulation even by operators without previous experience with nebulizer or MS based detectors • Optimized reversed-phase gradient enables fingerprinting of polysorbate 80 Goal • Charged aerosol detector (CAD) provides high-sensitivity Provide an HPLC method suitable for fingerprinting of detection polysorbate 80 samples and detect variability between different suppliers, grades, and production batches. • Inverse gradient enables uniform response under gradient conditions and eliminates the need of individual standards Introduction shed some light on this topic. For instance, a variation Polysorbate (PS) is a non-ionic surfactant widely used in the ester population may affect the critical micelle in pharmaceutical, biopharmaceutical, cosmetic, and concentration, which in turn can affect the solubility of free beverage formulations. Several types of polysorbate fatty acids2 present in the PS as degradation impurities. are available, with PS 20, PS 60, and PS 80 being More often, the different behaviors resulting from lot-to-lot most frequently used in pharmaceutical products. All variability of PS cannot be easily explained. Nonetheless, commercially available PS are complex mixtures of several understanding the quality of PS 80 by analyzing the raw hundred molecules, including low-level components. material is a potential time- and cost-saving approach, This complexity is a consequence of the inherently as it would decrease the need of costly root-cause heterogeneous raw materials used for the synthesis and analyses when formulations obtained with a particular the synthetic pathway that leads to the final product. batch of PS do not meet the required standards. A full Considering PS 80, the product is obtained by esterification quantitative characterization of a PS sample is, with the of oleic acid with sorbitan polyoxyehthylene (POE) current standard approach, an extremely complex task (Figure 1). The oleic acid originates from natural sources that consumes considerable analytical resources. While and contains other fatty acid impurities including, but quantification of every component of a PS is not required, not limited to, palmitic, linoleic, and stearic acids. These a simple analytical technique capable of profiling the impurities will participate in the esterification reactions, main features of the PS sample and enabling lot-to-lot thereby increasing complexity. The additional presence of comparison is highly desirable. the precursor and side product of sorbitan, respectively sorbitol and isosorbide, along with different degrees of In this work, we propose an HPLC-based approach ethoxylation of main and by-products, results in a mixture to monitor the characteristics of PS 80 samples that of hundreds of components. provides operational simplicity while generating a high degree of information. The reversed-phase method is highly optimized to ensure sufficient separation of different HO(CH CH O) (OCH2CH2)zOH 2 2 w compound classes within a reasonable run time. For detection, the Thermo Scientific™ Vanquish™ Charged (OCH CH ) OH 2 2 y Aerosol Detector, which is the instrument of choice for O detection of PS and other surfactants,3,4,5,6 is used.

(OCH2CH2)xOH The Thermo Scientific™ Vanquish™ Duo UHPLC System for Inverse Gradient seamlessly incorporates two independent HO(CH2CH2O)w O flow-delivery systems that simultaneously deliver the analytical and compensation gradients. Setting up the compensation flow is facilitated by a user-friendly wizard O (OCH CH ) OH 2 2 x in Thermo Scientific™ Chromeleon™ Chromatography Data Figure 1. Structure of POE sorbitan (top) and POE isosorbide (bottom). These species are found in polysorbates along with their fatty System (CDS). The inverse gradient option allows acid esters. The nature of the fatty acid, the degree of esterification, and CAD-based quantitation without standards even under the number of oxyethylene units contribute to the overall complexity of gradient conditions.7 polysorbates.

The Thermo Scientific™ ISQ™ EM single quadrupole The control of PS as chemical raw material for mass detector features intuitive setting of the detection (bio)pharmaceutical formulations is difficult because of parameters through the AutoSpray algorithm. It provides the complexity described above. Nevertheless, such a mass range up to m/z 2000 allowing for the mass control is needed since variations from lot to lot are detection of high-molecular weight PS species such as expected to occur. Variation of the relative population singly-charged di-ester and doubly charged tri- and tetra- of esters, and polyoxyethylated polyols, can affect the ester ions. Obtained masses of the variety of PS species behavior of PS 80 as an excipient in biotherapeutic allows the deduction of the identity of main components, formulations. The link between the composition of PS complementing the quantitative, mass selective CAD and its properties is still not fully understood,1 although analysis with qualitative information. there have been hypotheses put forth that attempted to

2 Experimental Instrumentation Chemicals • Thermo Scientific™ Vanquish™ Flex UHPLC system and • Deionized water, 18.2 MΩ·cm resistivity or higher Thermo Scientific Vanquish Flex Duo UHPLC system consisting of: • Fisher Scientific™ Acetonitrile, Optima™ LC/MS grade (P/N A955-212) – Vanquish Dual Pump F (used in CAD experiments) (P/N VF-P32-A) • Fisher Scientific™ Formic acid, Optima™ LC/MS grade (P/N A117-50) – Vanquish Quaternary Pump F (used in ISQ EM experiments) (P/N VF-P20-A) • Fisher Scientific™ Isopropanol, Optima™ LC/MS grade (P/N A461-212) – Vanquish System Base (P/N VF-S01-A-02) • Fisher Scientific™ Ammonium formate, Optima™ – Vanquish Sampler FT (P/N VF-A10-A) LC/MS grade (P/N A115-50) – Vanquish Column Compartment H (P/N VH-C10-A-02) – Thermo Scientific™ Vanquish™ Charged Aerosol Three PS 80 samples were purchased from Croda Inc. and Detector H (P/N VH-D20-A) one sample from Avantor (Table 1). • Thermo Scientific™ ISQ™ EM single quadrupole mass

Table 1. PS 80 samples spectrometer (P/N ISQEM-ESI)

Identification • Vanquish Duo for Inverse Gradient Kit (P/N 6036.2010) Product name used in Vendor Product name code the document Sample and solvents preparation Super Refined™ Polysorbate SR48833 SA1 PS 80 samples were prepared in 25 mL volumetric flasks 80-LQ-(MH) diluted with deionized water at a final concentration of Croda Super Refined™ Polysorbate SR40925 SA2 Inc. 80 POA-(LQ)-(MH) 2.5 mg/mL for sample SA1, which was used for the Tween™ 80 HP-LQ-(MH) SD43361 SA3 dilutions, and at 1 mg/mL for all the other samples. Polysorbate 80, N. F. Multi- Avantor Compendial J.T. Baker 4117-02 SB4 Eluent A (Table 2) was prepared by dissolving ammonium TWEEN 80 HP-LQ-(MH) formate in deionized water at the concentration of 5 mM. Afterwards, the pH of the eluent was adjusted to pH 4.8 with formic acid. Sample handling • Fisher Scientific™ Fisherbrand™ Mini Vortex Mixer Method parameters

(P/N 14-955-152) Table 2. LC method • Glass Vials (amber, 2 mL), Fisher Scientific™ Parameter Value (P/N 03-391-6) Thermo Scientific™ Accucore™ C18 Column • Vial Caps with Septum (Silicone/PTFE), Fisher Scientific™ 150 × 2.1 mm; 2.6 µm, P/N 17126-152130 A – 5 mM ammonium formate, pH 4.8 (P/N 13-622-292) Mobile phase B – 50/50 isopropanol/acetonitrile (v/v) • Thermo Scientific™ Orion™ 3-Star Benchtop pH Meter Time (min) %B Time (min) %B (P/N 13-644-928) 0 9 26 85 3 9 35 100 • Thermo Scientific™ Finnpipette™ F1 Variable Volume Gradient 10 22 45 100 10 57 46 9 Pipettes (P/N 14-386-309N) 21 69 56 9 21 84 The Fisher Scientific product codes can be unique to different countries; Flow rate 0.4 mL/min the codes given above should be compatible across the EU and USA. Autosampler temp. 6 °C 50 °C forced air mode, fan speed 5 Column temp. 50 °C active pre-heater Injection volume 10 µL Injection wash solvent 10/90 water/isopropanol (v/v)

3 Table 3. CAD detector settings Results and discussion The HPLC method was developed and optimized using Parameter Value CAD to maximize the number of observed peaks while Evaporator temperature 50 °C maintaining a reasonable run time below one hour. The Data collection rate 20 Hz profiles obtained by CAD for PS 80 from different vendors Filter 3.6 s and batches are shown in Figure 2 and Figure 3, where Power function 1.5 both standard and inverse gradient methods were applied. The chromatograms clearly showed four different groups Table 4. ISQ EM mass detector settings of peak clusters. This grouping will be used for further Parameter Value discussion and comparison of elution profiles. Even Ionization mode HESI though the profile of the different PS 80 samples is similar, Easy mode. Setting for sensitivity was 1; differences in the relative intensity are present. For both Source setting setting for mobile phase volatility was 3; inverse gradient and standard gradient, the main peaks in setting for thermally labile sample was 1 Groups 2 and 3 differ in height depending on the sample: Method type Scan mode, profile the peak heights for SB4 and SA3 are the lowest, and Polarity Positive Mass range full scan m/z 350–2000 quite comparable to each other; the most intense peaks Source CID voltage 0 V in Group 2 and Group 3 are detected for SA2. More Dwell time 0.5 s differences between samples are present. For instance, one peak in Group 3 and two peaks in Group 4 are Software detected only for samples SA2 and SA1 (Figure 2). Chromeleon 7.3 CDS was used for data acquisition and analysis. Thermo Scientific™ FreeStyle™ 1.6 application was used to calculate theoretical values of monoisotopic and most abundant isotope masses.

10.5 Group 1 Group 2 Group 3 Group 4 10.0

9.0

8.0

7.0

6.0 pA 5.0

4.0

3.0

2.0

1.0

0.0

0 5 10 15 20 25 30 35 40 Minutes Figure 2. Overlaid chromatogram of the PS 80 samples described in Table 1. CAD detection with standard gradient. Sample concentration 1 mg/mL. Other conditions are described in Table 2 and Table 3. Signals are corrected by matrix injection (matrix: 10 µL water). Orange trace: sample SA1; blue trace: sample SA2; pink trace: sample SA3; black trace: sample SB4.

4 (A)

2.80 Group 1 Group 2 Group 3 Group 4 (A) 2.50 2.80 Group 1 Group 2 Group 3 Group 4

2.25 2.50 2.00 2.25 1.75 2.00 pA 1.50 1.75 1.25 pA 1.50 1.00 1.25 0.75 1.00 0.50 0.75 0.25 0.50 0 0.25

0 5 10 15 20 25 30 35 40 0 Minutes Figure 3A. Overlaid chromatogram of the PS 80 samples described in Table 1. CAD detection with inversed gradient. Sample concentration 1 mg/mL.(B) 0 Other conditions5 are described in10 Table 2 and Table15 3. Signals are corrected20 by matrix injection25 (matrix: 1030 µL water). Orange35 trace: sample SA1;40 Minutes blue0.70 trace: sample SA2; pink trace: sample SA3; black trace: sample SB4. Group 1

(B) Group 1 0.600.70

0.500.60

0.400.50

pA 0.40 0.30

pA 0.200.30

0.100.20

0.10 0

0 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.25 12.50 13.81 0 Minutes

0 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.25 12.50 13.81 Minutes Figure 3B. Detailed view of Figure 3A. Zoom in Group 1. Orange trace: sample SA1; blue trace: sample SA2; pink trace: sample SA3; black trace: sample SB4.

5 The ISQ EM single quadrupole mass detector was used to POE isosorbide oleic acid esters. The components were elucidate the nature of the species eluting in the different detected as singly, doubly, or triply ammoniated adducts, groups. Figure 4A represents the total ion chromatogram depending on the species. The expected mass differences (TIC) obtained in positive ionization mode. The peak of one ethylene oxide unit for the singly, doubly, and triply annotation is based on the analysis of the averaged spectra charged ions are m/z 44.0, 22.0, and 14.7, which fit to the across selected time windows (Figure 4B to Figure 4F). mass shifts observed in the spectra. In Table 5 some of the Identified species based on the annotated mass spectra observed ions are listed and compared to the expected are consistent with the main species expected in a PS m/z based on calculated masses. 80 sample, namely POE sorbitan oleic acid esters, and e-PO E e-POE

6.0e8 e-PO E Sorbitan-di-oleat

5.0e8 Sorbitan-oleat e-POE e-POE

4.0e8 e-PO E Sorbitan-tri-oleat -oleat et ra 3.0e8 Isosorbide-oleat Counts Sorbitan-t

2.0e8 Isosorbide-di-oleat POE, sorbitan-POE, isosorbide-POE 1.0e8

0 5 10 15 20 25 30 35 40 45 Minutes Figure 4A. TIC of PS 80 0.5 mg/mL (SA1). Conditions are described in Table 2 and Table 4.

1.2e6 3+ [Sorbitan-oleate-POEn + 2(NH )]2+ [Sorbitan-oleate-POEn + 3(NH4)] 4 Table1.1e6 5. Examples of detected ions with tentative identity assignment 826.8

1.0e6 Theoretical 782.8 Theoretical Theoretical Δ m/z m/z based on m/z based on870.9 m/z based (observed – 8.8e5 Detected most abundant760.7 monoisotopic on average Observed theoretical 849.2 Component Formula ion isotope mass 805.3 mass mass m/z average) 645.3 Sorbitan-oleate-POE7.5e5 C H O [M+2NH ]2+ 826.5 826.5 827.1 826.8 -0.3 27 78 152 33 630.8 660.0 4 738.7 3+ Sorbitan-oleate-POE6.3e5 33 C90H176O39 [M+3NH4] 645.1 645.1 893.2645.5 645.3 -0.2 616.0 660.4 914.9 Sorbitan-di-oleate-POE C H O [M+3NH ]3+ 733.5 733.2 733.6 733.5 -0.1 5.0e5 33 108 208 40 4

Sorbitan-di-oleate-POECounts C H O [M+2NH ]2+ 9 37.2 936.7 9 37.3 9 37.0 -0.3 26 94 180601.233 4 689.4 717.1 3.8e5 936.9 + Isosorbide-oleate-POE12 C48H90O17 [M+NH4] 704.1 956.7 956.7 9 57.3 956.7 -0.6 586.6 2.5e5 2+ 959.0 Isosorbide-oleate-POE16 C56H106O21 [M+2NH4] 575.4 575.4 575.8 575.6 -0.2

+ Isosorbide-di-oleate-POE1.3e5 12 C66H122O18 [M+NH4] 1220.9 1220.9 1221.7 1221.1 -0.6 Isosorbide-di-oleate-POE C H O [M+2NH ]2+ 685.5 685.5 686.0 685.7 -0.3 0 15 72 134 21 4 2+ Sorbitan-tri-oleate-POE28 C116H220O36 [M+2NH4] 1113.3 1112.8 1113.5 1113.6 +0.1

2+ Sorbitan-tetra-oleate-POE27 C132H248O36 [M+2NH4] 1223.4 1222.9 1223.7 1223.7 +0.0 450 500 600 700 800 900 1,000 1,100 6 m/z 6.0e8 Sorbitan-di-oleate-POE

5.0e8 Sorbitan-oleate-POE Since six ethylene oxide repeat units are isobaric to one Comparing the TIC in Figure 4A to the CAD chromatograms, oleate unit, it should be noted that the spectra alone could it is clear that the peaks were clustered in groups based on 4.0e8 not provide any information on the degree of esterification. degree of esterification. Sorbitan-tri-oleate-POE All groups indeed include species -oleate-POE However, it was assumed that the hydrophobicity within with the same degree of esterification, apart from Group 4, 3.0e8 a class of component increases with oleic acid content. Isosorbide-oleate-POE which combines tri and tetra esters. TIC, CAD with standard Based on this assumption, higher order esters would elute gradient, and CAD with inverse gradient are all suitable for Counts ra Sorbitan-tet later in the chromatogram2.0e8 relative to lower order ones. profiling PS 80. Isosorbide-di-oleate-POE When the CAD detection is considered, POE, sorbitan-POE, the substantial difference between standard and inverse isosorbide-POE In Group 1, free1.0e8 POE, POE sorbitan, and POE isosorbide gradient is the fact that the quantitation with the latter are detected; the identity assignment for most of the peaks provides the real mass-balance across all components of Group 1 is straightforward since most of them represent within each sample. When the standard gradient is applied, 0 single components, and consequently the spectra are easy the profiling is valuable for distinguishing one sample from to interpret with confidence (data not shown). another, but the method overestimates the amount of late eluting di-, tri-, and tetra-esters while underestimating 0 5 10 15 20 25 30 35 40 45 Minutesnon-esterified components.

1.2e6 3+ [Sorbitan-oleate-POEn + 2(NH )]2+ [Sorbitan-oleate-POEn + 3(NH4)] 4 1.1e6 826.8

1.0e6 782.8 870.9

8.8e5 760.7 849.2 805.3 7.5e5 645.3 630.8 660.0 738.7 6.3e5 893.2 616.0 660.4 914.9 5.0e5 Counts 601.2 689.4 717.1 3.8e5 936.9 704.1

586.6 2.5e5 959.0

1.3e5

450 500 600 700 800 900 1,000 1,100 m/z Figure 4B. Mass spectrum representing the averaged spectra across the retention time window 17.3–19.1 min, capturing the peak with apex at 17.9 min (Figure 4A). The signal ions are inferred as sorbitan-oleate-POEn doubly and triply ammoniated adducts. For doubly charged adducts polymer distribution, the detected species contain number of oxyethylene units n between 22 and 35; for the triple charged adducts polymer distribution n is between 29 and 35.

7 [Isosorbide-oleate-POEn + 2(NH )]2+ [Isosorbide-oleate-POEn + NH ]+ 1.2e6 4 4

1.1e6 553.7 575.6

1.0e6 531.6 9.0e5 597.7 [Isosorbide-oleate-POEn + 2(NH )]2+ [Isosorbide-oleate-POEn + NH ]+ 1.2e6 4 4 8.0e5 1.1e6 553.7 575.6 7.0e5 1.0e6 6.0e5 531.6 619.7 9.0e5 509.8 597.7 956.7 5.0e5 1000.8

Counts 8.0e5 4.0e5 1044.8 7.0e5 3.0e5 641.6 1089.0 6.0e5 619.7 2.0e5 1132.9 509.8 956.7 5.0e5 912.6 1000.8 1.0e5 Counts 4.0e5 1044.8 0 3.0e5 641.6 1089.0

2.0e5 1132.9 912.6 1.0e5450 500 600 700 800 900 1,000 1,100 1,200 1,300 m/z Figure0 4C. Mass spectrum representing the averaged spectra across the retention time window 19.4–20.3 min, capturing the peak [Sorbitan-di-oleate-POEn + 3(NH )]3+ [Sorbitan-di-oleate-POEn + 2(NH )]2+ with1.6e6 apex at 20.0 min (Figure 4A). The signal ions are inferred as 4isosorbide-oleate-POEn single and double ammoniated4 adducts. For singly charged adducts polymer distribution, the detected species contain a number of oxyethylene units n between 11 and 17; for the doubly charged adducts polymer distribution n is between 13 and 19. 937.0 959.9 1.4e6450 500 600 700 800 900 1,000 1,100 1,200 1,300 m/z 892.9 980.9 1.2e6 [Sorbitan-di-oleate-POEn + 3(NH )]3+ [Sorbitan-di-oleate-POEn + 2(NH )]2+ 1.6e6 4 1003.2 4

1.0e6 871.3 1.4e6 937.0 959.9 1025.1 733.5 8.0e5 719.1 748.2 892.9 980.9 1.2e6 1047.0 1003.2 762.9

Counts 6.0e5 704.1 1.0e6 871.3 848.9 1025.1 1069.6 777.5 4.0e5 733.5 8.0e5 689.5719.1 748.2 1091.5 1047.0 2.0e5 762.9

Counts 6.0e5 704.1 848.9 1069.6 0 777.5 4.0e5 689.5 1091.5

2.0e5450 500 600 700 800 900 1,000 1,100 1,200 1,300 m/z

450 500 600 700 800 900 1,000 1,100 1,200 1,300 m/z Figure 4D. Mass spectrum representing the averaged spectra across the retention time window 26.9–28.3 min, capturing the peak with apex at 27.2 min (Figure 4A). The signal ions are inferred as sorbitan-di-oleate-POEn double and triple ammoniated adducts. For doubly charged adducts polymer distribution, the detected species contain number of oxyethylene units n between 23 and 36; for the triply charged adducts polymer distribution n is between 27 and 37.

8 4.0e5 2+ + [Isosorbide-di-oleate-POEn + 2(NH4)] [Isosorbide-di-oleate-POEn + NH4]

3.5e5 685.7 663.8 707.8 3.0e5

2.5e5 730.1 643.7 2.0e5 1221.1 751.7 1265.2 Counts 1.5e5 960.0 1310.3 893.9 1.0e5 1004.0 849.9 1354.1 5.0e4

400 600 800 1,000 1,200 1,400 1,600 1,800 m/z Figure 4E. Mass spectrum representing the averaged spectra across the retention time window 29.0–30.4 min. The signal ions are inferred as isosorbide-di-oleate-POEn single and double ammoniated adducts. For single charged adducts polymer distribution, the detected species contain number of oxyethylene units n between 12 and 17; for the double charged adducts polymer distribution n is between 13 and 18.

[Sorbitan-tri-oleate-POEn + 2NH ]2+ 1.0e6 4

9.0e5 1092.1 1113.6 1069.6 8.0e5 1047.0 7.0e5 1025.1 1135.7

1003.5 6.0e5

5.0e5

981.2 1157.7

Counts 4.0e5

959.0 3.0e5

937.0 2.0e5

1.0e5

700 800 900 1,000 1,100 1,200 1,300 1,400 m/z Figure 4F. Mass spectrum representing the averaged spectra across the retention time window 33.9–35.0 min. The signal ions are inferred as sorbitan-tri-oleate-POEn doubly ammoniated adducts. The detected species contain a number of oxyethylene units n between 20 and 31.

9 2+ 4.0e5 [Sorbitan-tetra-oleate-POEn + 2NH4]

3.5e5 1223.7

1267.7 3.0e5 1201.8 1245.8 1179.7 2.5e5

1157.7 2.0e5 1289.7

Counts 1.5e5 1135.6 1113.6

1.0e5 1091.2 1312.3

1069.2 5.0e4

1,000 1,050 1,100 1,150 1,200 1,250 1,300 1,350 1,400 m/z Figure 4G. Mass spectrum representing the averaged spectra across the retention time window 36.9–37.7 minutes. The signal ions are inferred as sorbitan-tetra-oleate-POEn doubly ammoniated adducts. The detected species contain a number of oxyethylene units n between 20 and 31.

The sum of the area of all peaks measured in inverse highest main peaks in Group 2 and Group 3. Combining gradient varies with the sample (Table 6). Since the this insight with the information obtained by the mass detector response is independent of the species, and the detection, we can now conclude that those peaks contain concentration of PS used for the test is the same, this the POE sorbitan mono-esters and POE sorbitan di-esters. observation points to different purities of the PS standards. Thus, for the samples analyzed in this work, it appears that When comparing relative areas of the peak groups for the amount of the polyols of Group 1 inversely correlates inverse gradient analysis (Table 6), it is observed that SA1 with the amount of oleic acid esters of sorbitan. On the and SA2 contain the least amount of non-ester species other hand, the correlation with isosorbide esters is not (Group 1). Interestingly, these samples are those with the evident.

Table 6. Overview of the relative areas of the peak groups (standard and inverse gradient) and sum of all peak areas (inverse gradient). CAD detection: 1 mg/mL samples, and ISQ EM detection: 0.5 mg/mL. The data is the average of two injections.

Relative area (%) Gradient type Peak group SA1 (with MS detection) SA1 SA2 SA3 SB4 G1 7.8 9 1.58 1.66 2.31 2.16

G2 26.04 20.09 20.46 26.04 21.34 Standard G3 36.50 38.77 38.60 36.58 39.84

G4 29.58 39.59 39.30 29.59 36.67

G1 8.72 8.40 11.35 10.89

G2 33.69 34.09 34.95 33.87 n.a. G3 35.09 34.90 33.98 34.80 Inverse G4 22.51 22.62 19.73 20.45

Area (pA × min)

Sum area all groups 10.23 10.44 9.71 9.43

10 As shown in Table 7 and Figure 5, peak group area Table 7. Results of linear regression for the plots in Figure 5. measured for the sample concentration range between (0.5–2.5 mg/mL with 3 injections for each level)

0.5 and 2.5 mg/mL showed good linearity, for both Rel. standard and inverse gradient analyses. This observation, Std. Dev. Coeff. C0 C1 (%) of Det. (Intercept) (Slope) proves that the assessment of the groups’ relative abundance is consistent across the investigated Group 1 2.30 0.9985 -0.1895 0.8083 concentration range. The sum of the area of the four Group 2 0.94 0.9997 -1.6252 9.4848 Standard groups also correlates linearly with the amount of injected Group 3 1.30 0.9995 -3.7318 18.8525 gradient sample (Table 7). Utilizing the separation of groups Group 4 0.82 0.9998 -3.9703 19.2355 based on the level of esterification, quantitation can aid in All groups 0.92 0.9998 -9.5167 48.3811 monitoring trends in ester and polyol population. This is Group 1 2.40 0.9982 -0.1907 1.1080 useful for stability studies of PS-based drug formulations. Group 2 1.16 0.9996 -0.6553 4.1464 Inverse Group 3 1.66 0.9992 -0.8064 4.4371 gradient (A) Group 4 1.77 0.9991 -0.5396 2.8892 50 All groups 1.36 0.9994 -2.1920 12.5809 45 Group 4 Group 3 40 35 Conclusion 30 • An HPLC-CAD method for fingerprinting of PS 80 raw 25 material capable of resolving the components based on Group 2 20 the degree of esterification was developed. Area (pA × min) 15 • The method enables monitoring of different productions, 10 thereby providing a simple, albeit reliable, tool to ensure 5 the consistency of PS 80 raw materials and contribute to Group 1 0 00.5 11.5 22.5 3 consistent drug formulation production. mg/mL (B) • Thanks to the uniform response, CAD with inverse 12 gradient provides the real mass balance between Group 3 10 species with different degrees of esterification. Group 2 • Confirmation of the identity of the main components 8 of PS 80, namely POE, sorbitan POE, isosorbide POE, Group 4 6 isosorbide and sorbitan POE esters of oleic acid, is easily achieved by LC-MS with the ISQ EM mass detector.

Area (pA × min) 4 • The total ion chromatogram based on Full MS scans is a Group 1 2 viable alternative to CAD for PS 80 fingerprinting.

0 00.5 11.5 22.5 3 mg/mL Figure 5. Group area measured at different PS 80 concentration in SA1 (0.5–2.5 mg/mL with 3 injections for each level). Standard gradient (A) and inverse gradient (B).

11 References 5. Li, Y.; Hewitt, D.; Lentz Y. K.; Ji, J. A.; Zhang, T. Y.; Zhang, K. Characterization and 1. Tomlinson, A.; Zarraga, I. E.; and Demeule, B, Characterization of polysorbate ester stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by fractions and implications on protein drug product stability, Molecular Pharmaceutics, multidimensional ultrahigh-performance liquid chromatography–charged aerosol 2020, 17 (7), 2345–2353. detection–mass spectrometry, Analytical Chemistry, 2014, 86 (10), 5150–5157. 2. Doshi N.; Martin J., and Tomlinson A. Improving prediction of free fatty acid particle 6. Thermo Fisher Scientific Booklet, HPLC-Charged aerosol detector; surfactants and formation in biopharmaceutical drug products: incorporating ester distribution during emulsifiers applications notebook. https://assets.thermofisher.com/TFS-Assets/CMD/ polysorbate 20 degradation, Molecular Pharmaceutics, 2020, 17 (11), 4354–4363. Application-Notes/ai-71101-hplc-cad-surfactants-emulsifiers-ai71101-en.pdf 3. Lobback, C.; Backensfeld, T.; Funke, A.; Weitschies, W. Quantitative determination of 7. Thermo Fisher Scientific Technical Note 73449, Why use charged aerosol detection nonionic surfactants with CAD, Chromatography Techniques, 2007, 10, 18–20. with inverse gradient? https://assets.thermofisher.com/TFS-Assets/CMD/Technical- Notes/tn-73449-cad-inverse-gradient-tn73449-en.pdf 4. Fekete, S.; Ganzler, K.; Fekete, J. Fast and sensitive determination of Polysorbate 80 in solutions containing proteins, Journal of Pharmaceutical and Biomedical Analysis, 2010, 52(5), 672–679.

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