Polysorbate 80 Hydrolysis and

Considerations for Control Strategy

Development

Outline

1. Background

2. Considerations for control strategy development

• Development experience

• Impact to product quality

• Drug product testing

3. Conclusions

Evolving Understanding of Polysorbate

Degradation Pathways

• Kishore RK, et. al. 2011. Degradation of polysorbate 20 and 80: Studies on thermal autoxidation and hydrolysis. J Pharm Sci

• Kishore RK, et. al. 2011. The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics.

Pharm Res

• Labrenz SR. 2014. Ester hydrolysis of polysorbate 80 in mAb drug product: Evidence in support of the hypothesized risk after the

observation of visible particulate in mAb formulations. J Pharm Sci

• Cao X, et. al. 2015. Free fatty acid particles in protein formulations, Part 1: Microspectroscopic identification. J Pharm Sci

• Siska CC, et. al. 2015. Free fatty acid particles in protein formulations, Part 2: Contribution of polysorbate raw material. J Pharm

Sci

• Doshi N, et. al. 2015. Understanding particle formation: Solubility of free fatty acids as polysorbate 20 degradation byproducts in

therapeutic monoclonal antibody formulations. Mol Pharm

• Tomlinson A, et al. 2015. Polysorbate 20 degradation in biopharmaceutical formulations: Quantification of free fatty acids,

characterization of particulates, and insights into the degradation mechanism. Mol Pharm

• Dixit N, et. al. 2016. Residual host cell protein promotes polysorbate 20 degradation in a sulfatase drug product leading to free

fatty acid particles. J Pharm Sci

• Hall T. et al. 2016. Polysorbate 20 and 80 degradation by group XV lysosomal phospholipase A2 isomer X1 in monoclonal

antibody formulations. J Pharm Sci

Acknowledgements

Surface Science GroupXia DongCraig Kemp

NMR SpectroscopyScott BradleyLisa Zollars

AnalyticalAndy CarrMichael DeFelippisMelody GossageLihua HuangRick MeyerDawn NorrisCarissa SusallaTewelde TesfaiWilliam WeissShou Song Zhang

FormulationShermeen AbbasPatrick DonovanJose FloresNagarajan Thyagarajapuram

PurificationBill HolmesBrian Hotovec

StatisticsEric Adamec

Background

• Polysorbate 80 heterogeneity as a raw material

• Monoclonal antibody > 50 mg/mL

• Changing API storage from frozen in polycarbonate containers to

refrigerated in LDPE bags

Sorbitan headgroup

Oleic

Acid

PS80 Analytical Method

• PS80 lacks a chromophore for

standard HPLC-UV detection

• High protein concentrations

causes chromatographic

interference and/or

incompatibility with analysis of

PS80

• Selected the hydrolysis method

for detection of oleic acid relative

to PS80

MW = 1310

Polysorbate 80 Content Results for API

Stored in PC Bottles and LDPE Bags

Temperature Dependent Loss of PS80 (oleic acid) in LDPE Bags

Container Material of Construction

Stability Study

25ºC

Polysorbate 80 loss dependent on temperature and container material of construction

NMR Spectra of the Solutions

Sorbitan Headgroup Signal Signal for the CH2‘s in oleic acid

NMR data suggests a loss of the oleic acid portion, and not a decrease in the sorbitan headgroup

PS80 Standard

PC Bottle

(no loss)

LDPE Bag

(80% loss)

PETG Bottle

(no loss)

HDPE Bottle

(75% loss)

ToF-SIMS of Bag Interior Surfaces

Negative Ion Spectra

m/z 281Free oleic acid anion

240 260 280 300 320 340

Mass [m/z]

0

5

10

15

Co

un

ts

110131-0053-BN-176-01-20110329.TDC - Ions 400µm 776003 cts LA426 unused LLDPE bag rinsed with DI dried with N2

Unused Bag

273270243240339325263257251247

267

240 260 280 300 320 340

Mass [m/z]

0

5

10

15

20

25

Co

un

ts

110131-0053-BN-177-14-20110329.TDC - Ions 400µm 2099926 cts LA426 placebo bag 5C 7mo rinsed with DI water dried w N2

Bag Exposed to Placebo normal PS80 assay

311283275247243 279251 297 336267

237

261

240 260 280 300 320 340

Mass [m/z]

050

100150200250300350

Co

un

ts

110131-0053-BN-182-43-20110329.TDC - Ions 400µm 4094708 cts Lot 110124-0030-2 API bag No PS80 detected

Bag Exposed to Product low PS80 assay

251 284255 337

281

ToF-SIMS indicates oleic acid sorption to

the surface of the bag.

Further Evidence that Material of

Construction Influences Hydrolysis

Monoester Diester Triester

Polysorbate 80 control

Prefilled syringe 9 months 5C

Prefilled syringe 9 months 25C

LDPE bag with polysorbate 80 depleted

PS80 Monoester depleted with higher substituted PS80 remaining in prefilled

syringe but depleted in LDPE based on LC/MS

(Tetraester not shown)

Hydrolysis Assay

Oleic Acid

Sorbitan

Headgroup

Can we differentiate between

oleic acid free in solution prior

to sample preparation and the

oleic acid freed during the

base hydrolysis?

Internal standard: Cis-10-Nonadecenoic acid

Ole

ic A

cid

Inte

rna

l S

tan

da

rd

LDPE with hydrolysis

LDPE without hydrolysis

PC with hydrolysis

PC without hydrolysis

Hydrolysis Assay(with & without base hydrolysis sample preparation)

Cleavage of PS80 occurs in all containers

Sorption of oleic acid is dependent on container material of construction

Influence of Container Material of

Construction on Hydrolysis

Containers that reduce oleic acid by sorption accelerate the rate and extent of PS80 loss

• Surface area to volume ratio influences sorption (stability simulators)

PC Bottle vs LDPE Bag Based on TOAGlass Syringe Based on TOA and FOA

Three Lots of Drug Product at 5 and 25C

TOA: Total Oleic Acid (after sample hydrolysis)

FOA: Free Oleic Acid (NO sample hydrolysis)

Impact of Polysorbate 80 Hydrolysis

on Product Quality

• Analytical Control Strategy: In this case Hydrolysis Method

TOA – FOA = Polysorbate 80 Level

Three Lots of Drug Product Syringes at 5 & 25C

Impact of Polysorbate 80 Hydrolysis

on Product Quality (continued)• Determine the Minimum Effective Polysorbate 80 Level

Studies that contribute to setting minimum effective level:

• Freeze thaw, agitation, shipping, stability (include aged material)

• Include a safety margin to address product and method variability

Agitation Study Assessing PS80 Level Three Lots of Syringes at 5 & 25C

Impact of Polysorbate 80 Hydrolysis

on Product Quality (continued)• Product Quality: Three Lots of Drug Product Syringes at 5 & 25C

Studies that contribute to justifying product quality: Freeze thaw, agitation, shipping, stability

Impact of Polysorbate 80 Hydrolysis

on Product Quality (continued)3. Product Quality: Three Lots of Drug Product Syringes at 5 & 25C

Studies that contribute to justifying product quality: Freeze thaw, agitation, shipping, stability

Drug Product Testing

Minimum Effective

PS80 Level

Potential timing of importation testing

Timing of assay

versus

degradation rate

Drug Product Testing (continued)

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Poly

sorb

ate

80,

%

0 6 12 18 24 30 36

Adjusted TImepoint, Month

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

Batch

Number

Modeling of data indicates that at

least 0.020% PS80 is needed at

release to ensure PS80 remains

above lower effective level

Conclusions

1. The reduction of PS80 resulted from hydrolysis beyond the rate

expected from hydrolytic reactions

2. Container material of construction can influence the rate and extent of

hydrolysis

• Selection of stability simulator

• Drug substance storage at -70C essentially elements hydrolysis

3. The PS80 remaining in the drug product maintained product quality over

the shelf-life

4. Considerations for developing the drug product testing strategy

• Rate of degradation

• In-process assay versus release

• End of shelf life relative to minimum level of PS80