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








  • 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 assay kit (BioVision, Milpitas, CA, USA)
  • Spectrophotometer (Molecular Devices, San Jose, CA, USA)
  • Incubator (Thermo Fisher Scientific, Waltham, MA, USA) u Clear 96-well plate









  • 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%)



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