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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

 

 

acoustic wave separator and depth filter

 

Scope

 

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

 

 

 

Procedure

 

  • 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

 

 

 

MATERIALS AND METHODS

 

  • 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 activity per ml

 

 

RESULTS AND DISCUSSION

 

 

matrix effect test

 

 

  • 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

 

 

linearity and range of the standard curve of nadh

 

 

  • ●: 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 

 

 

linearity of dilution test

 

 

  • 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

 

 

repeatability test

 

  • 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%
ldh activity levels for acoustic wave separator upstream bioprocessing instrument

 

 

 

 

 

 

  • 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

 

 

 

CONCLUSION

 

  • 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%)