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

BMS Highly Confidential

1
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

BMS Highly Confidential

2
Problem Statement

❖ Potential polymerization in pharmaceutical chemical synthesis

❖ Mass balance issues

❖ Observe some final material missing

❖ No existing tools to confirm polymer existence

BMS Highly Confidential

3
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

BMS Highly Confidential

4
Model Polymer Standards

Most Nonpolar Most Polar

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

Polystyrene (PT)
• Nonpolar polymer
• Soluble in Polyvinylpyrrolidone Polysaccharide (PS)
Tetrahydrofuran (PVP) • Polar polymer
(THF) • Dipolar polymer • Soluble in Water
• MW 266 – 66K • Soluble in Water, • MW 342 – 800K
MeOH, Acetonitrile
(MeCN), THF
• MW 10K – 1300K

Different polymers have different physical and chemical properties!

BMS Highly Confidential
5
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

BMS Highly Confidential

6
Size-based Separation Principle

Size Exclusion Chromatography (SEC)

Polymers

Separation Mechanism

• Separation based on size

Monomers
(hydrodynamic radius)
• Stationary phase particles
with pores
• Larger analytes shorter path
• Smaller analytes longer path

Monomers

Polymers
Late eluate Early eluate
Monomers Polymers
time
BMS Highly Confidential
7
Column Interactions

Hydrophobic interactions Strong organic solvents

Hydrophilic interactions Aqueous solutions

Ionic interactions Salt/Buffer solutions

Size Exclusion Separation

BMS Highly Confidential

8
Column Screening Experimental Data
Using Selected Model Polymers

BMS Highly Confidential

9
Column Screening: PSS

Void
(1) MW 2350K
(1) MW 300K (1) TSKgel,
(1) MW 41K MP 40:60
(2) MW 2350K (2) XSelect C18,
(2) MW 300K MP 90:10
(2) MW 41K
(3) Jupiter C18,
(3) MW 2350K MP 90:10
(3) MW 300K
(3) MW 41K

• Mobile Phase: MeOH:200 mM NH4Fc in Water

• Detection: UV, 220 nm
• Sample Prep: PSS (1 mg/mL) in Water:MeOH (80:20)

BMS Highly Confidential

10
Column Screening: PVP

Void
(1) MW 1300K
(3)MW 1300K
(1) MW 55K
(3) MW 55K
(1) MW 10K
(3) MW 10K
(4) MW 1300K
(2) MW 1300K
(2) MW 55K (4) MW 55K
(2) MW 10K (4) MW 10K

(1) TSKgel, (3) XSelect C18

MP 40:60, Inj. Vol 20 µL MP 90:10, Inj. Vol 2 µL
(2) Aquagel, (4) Jupiter C18
MP 40:60, Inj. Vol 30 µL MP 90:10, Inj. Vol 2 µL
• Mobile Phase: MeOH:200 mM NH4Fc in Water
• Sample Prep: PVP (1 mg/mL) in MeCN:Water (50:50)

BMS Highly Confidential

11
SEC Column Selection

Conventional SEC columns C18 columns (SEC mode)

0 6 12 min
0 4 min
Pros Pros
❖ Designed for size-based separation ❖ Small void volume
(relatively high peak resolution) ❖ High sensitivity
❖ Short run time
Cons ❖ Short equilibration time
o Large void volume ❖ Compatible with a variety of polar
o Low sensitivity and nonpolar solvents
o Long run time ❖ Robust
o Long equilibration time ❖ Method transfer
o May not be compatible with polar or ❖ Commercially available
nonpolar solvents
o Not robust Cons
o Need to eliminate column interaction
BMS Highly Confidential
12
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

BMS Highly Confidential

13
Mobile Phase Selection: 1% PSS

PSS Monomer

(1)

Mobile Phase Combinations

(2)
(1) MeCN:200 mM NH4Fc
(90:10)
(3) (2) MeOH:200 mM NH4Fc
(80:20)

(3) MeOH:200 mM NH4Fc

(90:10)*
• Column: XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm
• Detection: UV, 220 nm
• Sample Prep: 1% PSS 7K (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic
acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50)
BMS Highly Confidential
14
Mobile Phase Selection: 1% PVP

PVP Monomer

(1)

(2)
Low sensitivity and high Mobile Phase Combinations
baseline noise due to MeOH:200 mM NH4Fc in Water
PVP low UV
absorbance (1) 70:30
(3)
(2) 80:20

(3) 90:10*

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

• Detection: UV, 220 nm
• Sample Prep: 1% PVP 250K (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic
acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50)

BMS Highly Confidential

15
Temperature Variation

PSS Monomer

80oC

60oC

40oC

20oC

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

• Detection: UV, 220 nm
• Sample Prep: 1% PSS 7K (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic
acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50)

BMS Highly Confidential

16
Method Evaluation: 1% Different MW Polymer Impurity Results

Monomer
MW 5000K
PSS Monomer
MW 300K

MW 80K
MW 41K
MW 30K

MW 15K

MW 7K

MW 3K

• Mobile Phase: MeOH: Water with 200 mM NH4Fc (90:10)

• Column: XSelect CSH C18, 4.6 x 250 mm, 130 Å, 5 µm
• Detection: UV, 220 nm
• Sample Prep: 1% PSS (0.1 mg/mL)/99% 1,5-naphthalenedisulfonic
acid tetrahydrate (10 mg/mL) in MeCN:Water (50:50)
BMS Highly Confidential
17
Method Evaluation: Sensitivity
MW 266
PT Toluene
Monomer
MW 7K (Toluene)
MW 2K
MW 26K

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

• Mobile Phase: THF

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

BMS Highly Confidential

18
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

19
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

BMS Highly Confidential

20
Acknowledgements

❖ Yun K. Ye

❖ Ziqing Lin

❖ Xuejun Xu

❖ CSD Department

❖ BMS 2018 PD Summer Internship Program

BMS Highly Confidential

21