identification and quantification of polysorbate ... · biopharmaceuticals using advanced mass...

Michael Zorn

Boehringer Ingelheim Pharma GmbH & Co. KG

Innovation Unit

Analytical Development Biologicals

Identification and Quantification of Polysorbate-degrading Host Cell Protein Impurities in Biopharmaceuticals using Advanced Mass Spectrometry-based Techniques

Outline

14 March 2019 AT Europe 2019, Dublin 2

• Introduction to process-related protein impurities

Bioprocess and host cell proteins (HCPs)

Polysorbate-degradation through lipolytic enzyme activity

• Advanced sample preparation strategies for sensitive identification and quantification of HCPs

in bulk drug substance via LC-MS

I. Quantification of target lipases in polysorbate-free drug substance

II. Hexapeptide-based HCP-enrichment and isotope dilution mass spectrometry for

relative quantification of lipases

III. Establishment of an activity-based probes (ABP) proteomic workflow for lipase profiling

• Summary

Process-related Protein Impurities

3

Bioreactor Primary recovery centrifugation

Affinity chromatography

Polishing I chromatography

Polishing II chromatography

DS/DP

Y

Y

Y Y

Y Y

Y

Y Y

HCP-

Level

~106 ppm ~103 ppm ~102 ppm <10 ppm

Upstream processing Downstream processing

Walker et al., 'A modular and adaptive mass spectrometry-based platform for support of bioprocess development toward optimal host cell protein clearance‚‚ mAbs Volume 9, 2017

14 March 2019

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwjFsouFnPnZAhWDY98KHTYODPUQjRx6BAgAEAU&url=http://altheacmo.com/drug-product-manufacturing/overview/&psig=AOvVaw2yIxRbPTVcyLbBaU3b7DVH&ust=1521577410838090

4

Impact of Process-related Protein Impurities

Potential to impact product quality, efficacy and patient safety

• May potentially induce immune response in patients

• Impact of API potency by blockage of mAb paratope

• Undesired API variants due to presence of catalytic activity of proteases and disulfide

reductases

• Polysorbate degradation by lipolytic enzymes

AT Europe 2019, Dublin 14 March 2019

Impact of Process-related Protein Impurities on Excipients Stability

• Polysorbates commonly used to improve

stability of API

• Present in majority of biopharmaceuticals

• Prone to degradation by hydrolysis and

auto-oxidation

• Susceptible to enzymatic cleavage of ester

bond because of structural similarity to

triglycerides

Polysorbate

Hydrolysis

FFA

+

mAb without PS20 mAb with PS20

5 14 March 2019

Siska et al., J. Pharm. Sci., 104, 447 (2015)

AT Europe 2019, Dublin

Strategy for Identification and Quantification of Process-related Protein Impurities

6

Protein A affinity chromatography

HCP Level

Dynamic Range

Downstream in-process control samples

Product pools Wash pools

Polishing chromatographies

Product pools Column regeneration pools

Bulk drug substance

Harvested cell culture fluid Target lipase profiling Application of untargeted LC-MS

methods

Identification & quantification of target lipases Single- or multi-analyte ELISA Targeted LC-MS methods


Targeted method development guided by theoretical and experimental input

14 March 2019

Outline

AT Europe 2019, Dublin 7










• Summary

14 March 2019

Quantification of Lipases in Polysorbate-free Drug Substance

8

untargeted LC-MS-analysis : Harvested cell culture fluid

Drug substance (polysorbate-free)

Assessment of analytical limits: LOD/LOQ

Targeted LC-MS/MS-analysis and quantification

HCP profile analysis

Development of targeted LC-MS/MS methods for the quantification of PLBL2, LPL and LPLA2

in polysorbate-free drug substance

Lipase quantification

14 March 2019

9

LOD and LOQ Assessment for LPL, PLBL2, and LPLA2

Sample preparation:

recombinant PLBL2, LPLA2 and LPL proteins were spiked

into polysorbate-free drug substance at 0, 0.1, 0.2, 0.5, 1,

2, 5, 10, and 20 ppm

Native digestion* and removal of incompletely digested

mAb by heating and centrifugation

LC-MS instrumental setup and data processing

Waters Acquity Arc HPLC (85 min gradient-run) coupled to

Thermo Q Exactive Plus

Parallel reaction monitoring data analysis software:

Skyline 4.1

*Huang, et. Al. Anal. Chem., 2017, 89 (10), pp 5436–5444

9

+

Peptide mixture

Spiking of recombinant lipases

14 March 2019

10

0

25

50

75

100

125

0 ppm 0.1 ppm 0.2 ppm 0.5 ppm 1 ppm 2 ppm 5 ppm 10 ppm 20 ppm

%C

V

Spiked-in Concentration

LPL precision for each peptide

LVAALYK

VFHYQVK

y = 389076x - 14284 R² = 0.9771

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

8.00E+06

9.00E+06

0 2 4 6 8 10 12 14 16 18 20

Tota

l Pea

k A

rea

mixed-in ppm

Calibration curve

LVAALYK

Limit of Quantification Assessment for LPL


11

Limit of Quantification Assessment for LPL


LPL can be confidently detected at 5 ppm spiking level. The recovery was determined by comparing the theoretical spiking level and the readout from the standard curve by peptide LVAALYK. The LOQ for LPL is determined as 10 ppm.

14 March 2019

Readout from standard curve (ppm)

LVAALYK - - - - 0.2 1.2 5.8 11.7 19.1

Spiked-in value 0.0 0.1 0.2 0.5 1.0 2.0 5.0 10.0 20.0

Spiked-in recovery [%]

- - - - 23.1 60.3 115.7 117.1 95.3

Limit of Detection and Limit of Quantification for PLBL2, LPL and LPLA2

12

Lipase Limit of detection Limit of quantification

PLBL2* <2 ppm <2 ppm

LPLA2 1 ppm 1 ppm

LPL 5 ppm 10 ppm

*PLBL2 exists in the respective drug substance.

LOD determination criteria: The MS2 detectible level

LOQ determination criteria: Spiked-in recovery= 75-125%, and precision CV% for each peptide measurement ≤25%


Outline











• Summary

14 March 2019

Mechanism of HCP Enrichment by ProteoMinerTM Kit


Elution of HCPs with MS- compatible solvents

LC-MS analysis

Load

Eluate

Combinatorial hexapeptide library – highly complex and a binding partner should exist for every protein

Majority of highly abundant API is depleted while low abundant HCPs are retained

14 March 2019


Relative Quantification of Lipase Level in Drug Substance via Isotope Dilution Mass Spectrometry

15

Untargeted LC-MS-analysis of harvested cell culture fluid

Hexapeptide-based enrichment of HCPs from bulk drug substance

Tryptic digestion and spiking of stable isotope labeled synthetic (SIS)-peptides

LC-MS/MS-analysis

Investigation of HCP profile

Targeted LC-MS/MS- analysis

Relative quantification of lipases

+



16

Bulk drug substance batch I II

Lipase I level 100% 98%

Lipase II level 100% 467%

Observation of PS20 degradation - +

Relative Quantification of Lipase Level in Drug Substance via Isotope Dilution Mass Spectrometry

Relative quantification of lipase levels in BDS indicates Lipase II as

the potential lipolytic enzyme causing PS20-degradation


Outline




Polysorbate-degradation upon lipolytic enzyme activity


via LC-MS in bulk drug substance





• Summary

14 March 2019

Activity-based Probes for Profiling lipolytic Enzymes

18

Product with lipolytic activity

Addition of activity- based probe (ABP)

Residual lipase activity testing

Affinity capture of enzyme-inhibitor complexes

Proteolytic digestion

LC-MS-analysis

m/z

Rel

. In

ten

sity

[%]

Protein identification

Incubation


Bioorg Med Chem. 2000 Mar;8(3):507-13. 19


Biotin - affinity tag Reductive cleavage site 4-nitrophenyl activated phosphonate

14 March 2019

https://www.ncbi.nlm.nih.gov/pubmed/?term=A+novel+biotinylated+suicide+inhibitor+for+directed+molecular+evolution+of+lipolytic+enzymes


• PS20 stability testing in DS/DP material in

presence of lipase-inhibitor

• Incubation for several days at 25°C

• LC-MS-monitoring of intact Polysorbate-esters

e. g. isosorbide C12

• LC-MS-monitoring of free fatty acids

e. g. lauric acid

Screening for effective Lipase-Inhibitors

+ ABP

DS/DP material

LC-MS-detection of POE and FFA

Hydrolysis

FFA

+

POE

Inhibited lipases

14 March 2019





0

20

40

60

80

100

120

rela

tive

In

ten

sity

[%

]

Incubation time [days]

Isosorbide C12 Placebo

0

20

40

60

80

100

120

rela

tive

In

ten

sity

[%

]


Isosorbide C12 Drug Substance

0

1000

2000

3000

4000

5000

6000

Co

nce

ntr

atio

n [

ng/

mL]


Lauric Acid Placebo

0

1000

2000

3000

4000

5000

6000

Co

nce

ntr

atio

n [

ng/

mL]


Lauric Acid Drug Substance

0

20

40

60

80

100

120

rela

tive

In

ten

sity

[%

]


Isosorbide C12 Inhibitor I

0

20

40

60

80

100

120

rela

tive

In

ten

sity

[%

]


Isosorbide C12 Inhibitor II

0

1000

2000

3000

4000

5000

6000

Co

nce

ntr

atio

n [

ng/

mL]


Lauric Acid Inhibitor I

0

1000

2000

3000

4000

5000

6000

Co

nce

ntr

atio

n [

ng/

mL]


Lauric Acid Inhibitor II

LC-MS-analysis of FFA content

LC-MS-analysis of POE content

14 March 2019

22

Summary


Strategy for the identification & quantififcation of polysorbate-degrading enzymes

in biopharmaceutical products

Native digestion and LC-MS-analysis of polysorbate-free drug substance allows

quantification of LPL, PLBL2 and LPLA2 ≤ 10 ppm

Hexapeptide-based HCP-enrichment and isotope-dilution mass spectrometry are

advantageous for relative lipase quantification in bulk drug substance material

Establishment of activity-based probes for profiling of lipolytically active enzymes

14 March 2019

23

Acknowledgement



DEV ADB & RES MedChem, Biberach

Bernd Reisinger

Andreas Feigler

Corinna Ruedi

Kathrin Fischer

Marianne Scheffold

Florian Binder

BP BPAD, Fremont

Yuan Gao

Guifeng Jiang

Sara Wright

Min Zhu

14 March 2019

Thank you for your Attention

24 AT Europe 2019, Dublin


DEV ADB & RES MedChem, Biberach

Bernd Reisinger

Andreas Feigler

Corinna Ruedi

Kathrin Fischer

Marianne Scheffold

Florian Binder

BP BPAD, Fremont

Yuan Gao

Guifeng Jiang

Sara Wright

Min Zhu

14 March 2019

identification and quantification of polysorbate ... · biopharmaceuticals using advanced mass...

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