Impact of cell lysis on the description of cell growth and death in cell culture

Abstract

The primary task of process development is a process design that guarantees product quality and maximizes product quantity. One part of the process development is the identification of critical process parameters. Especially in cell culture processes, unwanted cell damage as critical process parameter is still challenging in stirred tank reactors and needs therefore to be considered. Nevertheless, this topic and its effects on process performance are currently not well discussed and not verified in literature until now. The process of cell damage or lysis can be defined as the loss of integrity of the cells. For the investigation of this phenomenon, a model‐based designed fed‐batch cultivation with Chinese hamster ovary cells was performed. Besides measurements of viable and dead cell concentration, lysed cell count was determined by DNA measurements. Based on these analytics, different hypotheses, characterizing the cell death, were compared. From this, four main statements could be derived: (i) consideration of lysis in cell culture processes is of great importance for the description of the living biomass population in terms of growth and dying; (ii) a higher effort in process monitoring facilitates significantly model development; (iii) in contradiction to existing models from literature, our verification approach demonstrated a direct correlation of lysis with viable cell concentration and therefore with productivity; and (iv) lysis could be effectively described by only one model parameter, the specific lysis rate from viable cells to lysed cells. All these statements could be accurately proven by statistical methods. Our results enable future detailed investigations of the causes of cell damage and the influences of cell lysis on product quality and quantity. Potential application fields are scale‐up issues, process optimization, and impurity screening.

Soft sensor for monitoring biomass subpopulations in mammalian cell culture processes

“…Target values of the soft sensor are the three biomass states X VCC , X DCC and X LCC . In a previously published study, it was shown that the investigated biomass subpopulations can be described by three parameters (Kroll et al 2016 ), which are the specific growth rate (µ), the specific death rate (k D ) and the specific lysis rate (k DL ). In order to determine these three parameters, and consequently the three biomass subpopulations, at least three independent relationships must be established.…”

“…In this section the last necessary relation for X LCC is established based on a previously published growth model considering a similar process (Kroll et al 2016 ). This ordinary differential equation (ODE) contains the specific lysis rate k DL as parameter that can be assumed to be constant (Kroll et al 2016 ). It is a system-describing parameter which depends on lysis influencing parameters such as aeration, power input and reactor geometry.…”

“…Thus, for a fed-batch process X LCC can be described by Eq. ( 6 ) (Kroll et al 2016 ). For this purpose, it is necessary to measure feed rates F in and the reactor volume V R .…”

“…Frequently, biomass is considered to be homogeneous in its composition. In mammalian cell culture processes, however, it was shown previously that segregation of biomass in subpopulations of viable cells, dead cells and lysed cells provides useful information that can be used for process understanding, data evaluation and modeling (Klein et al 2015 ; Kroll et al 2016 ). With regard to viable and dead cells, several methods are available basing on dielectric and Raman spectroscopy using data driven models (Abu-Absi et al 2011 ; Patel and Markx 2008 ).…”

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

“…Therefore, lysed cells cannot be directly detected via cell counting methods. Thus, they are usually indirectly measured through the detection of the released internal constituents, such as DNA, lactate dehydrogenase, or via the detection of cell debris . The change in rheological characteristics represents an additional opportunity to account for cell lysis; however, this is only applicable for microbial systems or high cell density cell culture systems.…”