Quantification of polysorbate 80 and polysorbate 20 in therapeutic protein formulations by mixed mode chromatography

Maryam Ghasemi,1,* Mahdi khodadadian,2 Hossein behrouz,3

1. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
2. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
3. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran

Abstract


Introduction

Polysorbates (pss) as an important class of amphiphilic, nonionic surfactants are widely used in protein pharmaceuticals to stabilize the proteins against interface-induced aggregation and to minimize surface adsorption of proteins. commonly applied processes and conditions involving interfacial interactions, such as filtration, pumping, agitation (e.g., shaking or stirring), freeze/thawing, and lyophilization may lead to aggregation. polysorbates are ubiquitous to protein formulations because of their effectiveness in protecting many proteins. in fact, specifically for monoclonal antibodies (mabs), more than 70% of the marketed formulations contain either ps20 or ps80. in addition, it is known that pss are prone to degradation by autoxidation and hydrolysis by the time, which are important parameters in protein stability. for all these reasons, it was necessary to quantify polysorbates in protein formulations. an evaporative light scattering detector (elsd) based hplc method that requires minimal sample preparation has been developed to quantitate polysorbate 80 and polysorbate 20 in therapeutic proteins.

Methods

The method utilizes a mixed-mode chromatography column (oasis max 30µm, 2.1ₓ 20 mm) and a 10 minutes step gradient of 0.02% formic acid in water and acetonitrile to separate ps from proteins and formulation excipients. . the elsd was set on 40 ºc and 75 ºc for nebulization and evaporation temperature, respectively. a flow rate of 1.0 ml/min and an injection volume of 25 µl was used in this method and the column temperature was set on 30 ºc. a 1.0 mg/ml ps80 stock solution was prepared by taking about 100 µl of pss (based on density) and dissolving in 100 ml of upw. the solution was vortexed until fully dissolved. the stock solution was further diluted in upw to prepare calibration standards of 0.05 mg/ml, 0.1 mg/ml, 0.15 mg/ml and 0.2 mg/ml ps80 and ps20 concentrations. a 400 µl of each prepared sample was taken into hplc vials and 25 µl was injected onto the column. the response from the elsd detector was plotted to prepare a calibration curve for 0.05 - 0.2 mg/ml ps80/ and ps20 . five different formulation buffers (ps80: aryoseven , aryoseven rt , zytux and ps20: aryotrust , stivant ) were evaluated for matrix effect on the method performance . protein samples in about 1-5 mg/ml were injected directly and proteins in more than 1.0 mg/ml were diluted to at least 5.0 mg/ml. the absolute areas of peaks at each sample were compared against the ps80 and ps20 calibration curve to estimate the ps concentration.

Results

In the employed method which is involving a mixed-mode stationary phase (mixed-mode anion-exchange and reversed-phase sorbent), proteins are typically positive charged and are not retained because of electrostatic repulsions from the quaternary amine in the mixed-mode resin. other formulation components elute in void volume because of their hydrophilicity. hydrophobic polylobate is retained, eluted with a step gradient and quantified as a single peak using an evaporative light scattering detector. as we expected, about 0.4 mg/ml for aryotrust, 0.08 mg/ml for stivant of ps 20 and 0.7 mg/ml for zytux and 0.07 mg/ml for aryoseven and aryoseven rt of ps 80 were obtained. the results have about ±0.02 mg/ml variation from labeled claim which is generally within their defined limit.

Conclusion

A new mixed –mode-hplc-elsd method has been developed to quantitate polysorbate 80 and polysorbate 20 in aryogen pharmed therapeutic proteins formulations .the method utilizes a step gradient of water and acetonitrile and a mixed-mode chromatography column which is containing a hydrophobic polymer backbone with positively charged quaternary amines to separate polysorbate from proteins and hydrophilic excipients. this method accurately measures polysorbate in at least 5 different protein solutions spanning a wide range of formulations with linearity (r2=0.99) and repeatability (0.7% rsd). the time reducing and solvent saving characteristics of the fast separation in this method is very advantageous, compared to the most widely used conventional hplc technique. also simplicity and low maintenance of elsd system are other useful features that were helpful in monitoring polysorbate concentrations in multiple protein samples during our formulation and also in stability studies.

Keywords

Polysorbate, evaporative light scattering detection, mixed-mode chromatography, monoclonal antibody,