Qualification of LDH Assay for Assessing Cell Lysis During Various Separation Methods of a Perfusion Cell Culture

Benedict Kang, Terése Joseph, Todd Sanderson, Michael Collins, David Sokolowski & Dana Pentia

INTRODUCTION

To assess potential cell damage in the separation of cells from media during perfusion cell culture using acoustic wave separator and depth filtration

Detection of lactate dehydrogenase (LDH) as a biomarker for cell damage
Change in LDH activity levels may indicate damages of cell membranes which are highly associated with cell lysis

LDH concentration was constant in all different % perfusion media
dotted lines show workable dilution range for the samples from the upstream bioprocess
1.5% and 0.8% are equivalent to 1:64 and 1:128 dilutions, respectively

●: extra points beyond the ranges of the standards that the kit provides
Linear range: 1.25 nmol/well to 15 nmol/well
The amount of NADH produced in the reaction (B value) was calculated using the equation of the curve

LDH activity was in linear relationship with the diluted positive control LDH in the perfusion media
As long as the samples are diluted down to the dilution range determined by the matrix effect test, back-calculated LDH activity levels will stay the same even though each assay reaction contains different % perfusion media

24 sample repeats of cell culture harvest show it is repeatable (8 samples x 3)
Repeatable in both dilutions: 1:64 and 1:128
Both dilutions (128-fold and 64-fold) agree with each other with coefficient of variation less than 7%

D19 and D26 were chosen to monitor the changes in LDH activity levels over the 7 days
No change in LDH activity level was observed

The Xpansion bioreactor plates were successfully coated with a proprietary protein critical to cell attachment and proliferation
Because every bioprocessing step has different buffer systems, LDH assay must be qualified to find the adequate windows of dilution that would allow to circumvent matrixed effects while detecting signals
Sequentially, matrix effect must be determined prior to the other tests such as linearity and range test, linearity of dilution test, and repeatability test
Unit definition of the LDH should be redefined when working with different buffer systems:
- Our unit definition of LDH for the samples in perfusion media: the amount of the LDH that generates 1 μmol NADH per minute at 37 °C in the diluted perfusion media (12% ~ 0.375%)