Polymer Analysis Melissa Ling Final Presentation Preview

2018 BMS PD Summer Internship Final Presentation

Development of a General Size-Exclusion Chromatography Method for Polymer Impurity Analysis Melissa Ling Mentors: Yun K. Ye, Xuejun Xu Date: 02 August 2018

Location: Building 50, Auditorium G10

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Outline

1. Problem Statement 2. Objective and Strategy 3. Principles of Size-Exclusion Chromatography (SEC) 4. Model Compound Selection 5. Stationary Phase Selection and Screening 6. Optimization of Mobile Phase and Temperature Conditions 7. Method Evaluation 8. Conclusion

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Problem Statement

❖ Potential polymerization in pharmaceutical chemical synthesis

❖ Mass balance issues ❖ Observe some final material missing ❖ No existing tools to confirm polymer existence

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Objective and Strategies

Objective ❑ To develop a general method to qualitatively screen polymer impurities

Strategy ▪

Cover all different types of polymers

▪

Select a robust SEC stationary phase and mobile phase conditions with diverse applications

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Model Polymer Standards Most Polar

Most Nonpolar

Polystyrene Sulfonate (PSS) • Ionic polymer • Soluble in Water, Methanol (MeOH) • MW 3K – 5000K

Polystyrene (PT) • Nonpolar polymer • Soluble in Tetrahydrofuran (THF) • MW 266 – 66K

Polyvinylpyrrolidone (PVP) • Dipolar polymer • Soluble in Water, MeOH, Acetonitrile (MeCN), THF • MW 10K – 1300K

Polysaccharide (PS) • Polar polymer • Soluble in Water • MW 342 – 800K

Different polymers have different physical and chemical properties! BMS Highly Confidential

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Objective and Strategies

Objective ❑ To develop a general method to qualitatively screen polymer impurities

Strategy ▪

Cover all different types of polymers

▪

Select a robust SEC stationary phase and mobile phase conditions with diverse applications

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Size-based Separation Principle Size Exclusion Chromatography (SEC) Polymers

Separation Mechanism • Separation based on size (hydrodynamic radius) • Stationary phase particles with pores • Larger analytes shorter path • Smaller analytes longer path

Monomers

Monomers Polymers

Late eluate Monomers

Early eluate Polymers BMS Highly Confidential

time 7

Column Interactions

Hydrophobic interactions

Strong organic solvents

Hydrophilic interactions

Aqueous solutions

Ionic interactions

Salt/Buffer solutions

Size Exclusion Separation

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Column Screening Experimental Data Using Selected Model Polymers

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Column Screening: PSS

• • •

(1)

MW 2350K

(1) (1)

MW 300K

(2) (2) (2) (3) (3) (3)

MW 2350K

Void

(1) TSKgel, MP 40:60

MW 41K

Mobile Phase: Detection: Sample Prep:

(2) XSelect C18, MP 90:10

MW 300K

MW 41K

(3) Jupiter C18, MP 90:10

MW 2350K

MW 300K MW 41K

MeOH:200 mM NH4Fc in Water UV, 220 nm PSS (1 mg/mL) in Water:MeOH (80:20)

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Column Screening: PVP

Void

(1) MW 1300K (1) MW 55K (1) MW 10K

(3)MW 1300K (3) MW 55K (3) MW 10K (4) MW 1300K (4) MW 55K (4) MW 10K

(2) MW 1300K (2) MW 55K (2) MW 10K (1) TSKgel, MP 40:60, Inj. Vol 20 µL (2) Aquagel, MP 40:60, Inj. Vol 30 µL • •

Mobile Phase: Sample Prep:

(3) XSelect C18 MP 90:10, Inj. Vol 2 µL (4) Jupiter C18 MP 90:10, Inj. Vol 2 µL

MeOH:200 mM NH4Fc in Water PVP (1 mg/mL) in MeCN:Water (50:50) BMS Highly Confidential

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SEC Column Selection C18 columns (SEC mode)

Conventional SEC columns

0

6

12

min

Pros ❖ Designed for size-based separation (relatively high peak resolution)

Cons o Large void volume o Low sensitivity o Long run time o Long equilibration time o May not be compatible with polar or nonpolar solvents o Not robust

0

4

min

Pros ❖ Small void volume ❖ High sensitivity ❖ Short run time ❖ Short equilibration time ❖ Compatible with a variety of polar and nonpolar solvents ❖ Robust ❖ Method transfer ❖ Commercially available

Cons o Need to eliminate column interaction

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Optimization of Conditions ❖ Mobile Phase conditions: ❖ THF, MeCN, MeOH, 200 mM Ammonium Formate (NH4Fc) in Water ❖ Temperature conditions

1% Polymer 99% Monomer

Monomers

Polymers time

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Mobile Phase Selection: 1% PSS PSS

Monomer

(1)

Mobile Phase Combinations

(2)

(1) MeCN:200 mM NH4Fc (90:10)

(3)

• • •

Column: Detection: Sample Prep:

(2) MeOH:200 mM NH4Fc (80:20)

(3) MeOH:200 mM NH4Fc (90:10)* XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm UV, 220 nm 1% PSS 7K (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50) BMS Highly Confidential

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Mobile Phase Selection: 1% PVP PVP

Monomer

(1) (2)

Mobile Phase Combinations MeOH:200 mM NH4Fc in Water

Low sensitivity and high baseline noise due to PVP low UV absorbance

(1) 70:30

(3)

(2) 80:20 (3) 90:10* • • •

Column: Detection: Sample Prep:

XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm UV, 220 nm 1% PVP 250K (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50) BMS Highly Confidential

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Temperature Variation

PSS

Monomer

80oC 60oC

40oC 20oC

• • •

Column: Detection: Sample Prep:

XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm UV, 220 nm 1% PSS 7K (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50) BMS Highly Confidential

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Method Evaluation: 1% Different MW Polymer Impurity Results Monomer MW 5000K

PSS

MW 300K

Monomer

MW 80K MW 41K MW 30K

MW 15K MW 7K MW 3K

• • • •

Mobile Phase: Column: Detection: Sample Prep:

MeOH: Water with 200 mM NH4Fc (90:10) XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm UV, 220 nm 1% PSS (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50) BMS Highly Confidential

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Method Evaluation: Sensitivity MW 266 MW 7K MW 2K

Monomer (Toluene)

PT

Toluene

MW 26K

3% 2% 1% 0.5% 0.2% 0.1%

• • • •

Mobile Phase: Column: Detection: Sample Prep:

THF XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm UV, 254 nm 1% PT (0.1 mg/mL)/99% Toluene (10 mg/mL) in THF • MW 266, 2K, 7K, 26K BMS Highly Confidential

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Final Method Conditions ❖ Column:

XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm

❖ Mobile Phase:

1. THF • For nonpolar, hydrophobic polymers 2. MeOH:200 mM NH4Fc in Water (90:10) • For mid-range polarity polymers 3. 200 mM NH4Fc in Water:MeOH (70:30) • For polar, hydrophilic polymers ❖ Inj. Volume:

20 µL

❖ Temperature:

60oC

❖ Flow Rate:

1 mL/min

❖ Detection:

UV (205, 220, 254 nm), CAD, and/or ELSD

❖ Sample Prep:

10 mg/mL in appropriate diluent BMS Highly Confidential

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Conclusions Experiment Summary ❖ Model compound selection: PT, PSS, PVP, PS ❖ Stationary phase selection: XSelect C18 ❖ Optimized solvent and temperature conditions Special Case Exceptions ❖ Solubility issues ❖ Use special case mobile phase with polymer ❖ Broad MW distribution ❖ Increase concentration in sample prep

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Acknowledgements

❖ Yun K. Ye ❖ Ziqing Lin ❖ Xuejun Xu ❖ CSD Department ❖ BMS 2018 PD Summer Internship Program

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