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Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation

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Abstract

Purpose

Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause. The purpose of this study was to develop an assay to detect lipolytic activity in biopharmaceutical DS and DP formulations.

Methods

The assay is based on the hydrolysis of the lipase substrate 4-methylumbelliferyl oleate to yield the fluorescent product 4-methylumbelliferone.

Results

First, the assay components and their concentrations (buffer salts and pH, solvent and inhibitor Orlistat) were established and optimized using a model lipase (Porcine pancreatic lipase) and cell culture harvest fluid that exhibited lipolytic activity. The assay was then successfully applied and thereby qualified in protein formulations and at lipase concentrations possibly encountered in actual biopharmaceutical DS and DP formulations.

Conclusion

The lipase assay can be used to detect lipolytic activity in intermediate and final DS, for example during process optimization in downstream purification, to better and specifically reduce the level, or deplete, lipases from HCPs. The assay is also suitable to be applied during root cause investigations related to polysorbate degradation in biopharmaceutical DP.

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Abbreviations

API:

Active pharmaceutical ingredient

BIS-TRIS:

Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methan

CCHF :

Cell culture harvest fluid

CHO:

Chinese hamster ovary

DMSO:

Dimethyl sulfoxide

DP:

Drug product

DS:

Drug substance

ELISA :

Enzyme-linked immunosorbent assays

FMA:

Fluorescence micelle assay

HCMB:

High concentration matrix buffer

HCP:

Host cell protein

HPLC:

High performance liquid chromatography

IPA :

Iso-propanol

mAb:

Monoclonal antibody

MeOH:

Methanol

4Mu :

4-Methylumbelliferone

4MuO :

4-Methylumbelliferyl oleate

4 Np :

4-Nitrophenol

NPN :

N-Phenyl-1-naphtylamine

PLBL2:

Phospholipase B-like 2

PPL :

Porcine pancreatic lipase

NEH:

Non enzymatic hydrolysis

PS20:

Polysorbate 20

PS80:

Polysorbate 80

RT:

Room temperature

TRIS:

Tris(hydroxymethyl)aminomethane (commercial abbreviation: Trizma®)

w/v :

Weight to volume ratio

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Authors and Affiliations

  1. Lonza AG, Drug Product Services, 60 A, CH-4057, Basel, Switzerland

    Michael Jahn, Andreas Zerr, Filip Maciej Fedorowicz, Finn Brigger, Atanas Koulov & Hanns-Christian Mahler

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  1. Michael Jahn
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  2. Andreas Zerr
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  3. Filip Maciej Fedorowicz
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  4. Finn Brigger
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  6. Hanns-Christian Mahler
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Correspondence to Michael Jahn.

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Jahn, M., Zerr, A., Fedorowicz, F.M. et al. Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation. Pharm Res 37, 118 (2020). https://doi.org/10.1007/s11095-020-02812-0

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