Investigation of low viability in sparged bioreactor CHO cell cultures points to variability in the Pluronic F-68 shear protecting component of cell culture media

Apostolidis, Pani A.; Tseng, Alice; Koziol, Marie-Eve; Betenbaugh, Michael J.; Chiang, Barbara

doi:10.1016/j.bej.2015.01.013

Cited by 15 publications

(10 citation statements)

References 34 publications

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“…Moreover, supplier's certificate of analysis (CoA), which is largely compendial based, have been inadequate to assess raw material variance and its biomanufacturing impact. Poloxamer P188, a raw material in mammalian cell culture, is a prime example where traditional test methods and published pharmaceutical monographs were inadequate to detect the now well‐known lot‐to‐lot contaminant impurity variance that significantly impacts cell growth …”

Section: Introductionmentioning

confidence: 99%

Development of a rapid and reliable analytical method for screening poloxamer 188 for use in cell culture process

Bareford

Peng

Ali

et al. 2019

Biotechnology Progress

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Poloxamer P188 is a common nonionic surfactant additive used in cell culture media as a cellular protectant from the hydrodynamic forces and shear stress during bioprocessing. Presence of a hydrophobic high molecular weight impurity contaminant has been shown to compromise its protective properties and lead to batch failure. In this work we present, a reliable, sensitive, and rapid analytical method to detect and quantify the contaminant impurity in poloxamer 188. This method replaces a laborious and time-consuming functional test in the form of a shake flask assay. The method is based upon reversed-phase liquid chromatography with charged aerosol detection, simple mobile phase compositions, and a three-step gradient. The method was optimized to resolve the impurity from the main P188 fraction in less than 10 min. Analytical method qualification and functional test comparison demonstrate equivalent or better high throughput impurity screening performance. Attempts to identify the impurity and establish suitable method positive control standards are also discussed. K E Y W O R D S charged aerosol detector, impurity, mammalian cell culture, poloxamer 188, surfactant

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Section: Introductionmentioning

confidence: 99%

Development of a rapid and reliable analytical method for screening poloxamer 188 for use in cell culture process

Bareford

Peng

Ali

et al. 2019

Biotechnology Progress

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“…Cell lines used for the production of recombinant monoclonal antibodies for therapeutic use have shifted over the years, starting from SP2/0 and NS0 mouse myeloma to the currently favored CHO cell line and human cell lines such as HEK293 [3]. Expression platforms have also improved significantly since the use of dihydrofolate reductase (DHFR) and moved towards site directed integration, making platforms more robust albeit still susceptible to raw material variability [4][5][6]. The use of chemically defined media is the most straightforward way to ensure protection from animal derived viruses that could potentially arise from components such as fetal bovine serum (FBS) [7].…”

Section: Introductionmentioning

confidence: 99%

“…Poloxamers support viability in batch and fed batch processes in stirred tank reactors through protection against mechanical stress, such as agitation and bubble bursting at the surface layer [13][14][15]. Poloxamer lowers surface tension, stabilizes foam and reduces cell-bubble interactions [5,13]. Biologically, they have been shown to incorporate into the plasma membrane, increasing the membrane fluidity, which also can aid in protection against shear stress [10,16,17].…”

Section: Introductionmentioning

confidence: 99%

“…Poloxamer P188, manufactured primarily for industrial use is also widely used in cell culture media for recombinant biopharmaceuticals. Incidents of P188 variability which significantly affect mammalian cell culture processes have been published and are a known problem in the industry [1,5]. Analytical research showed contaminants and product carryover present in P188 (referred to as poloxamer "X"), which was confirmed upon the release of a pharmaceutical grade version of poloxamer X, manufactured with more strict controls, ensuring less contaminants, carryover and ultimately less variability, referred to as poloxamer "X-MOD" [1].…”

Section: Introductionmentioning

confidence: 99%

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Uncovering new poloxamer attributes significantly impacting mammalian cell growth performance

Safta

Case

Brandt

et al. 2022

Preprint

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A biopharmaceutical grade poloxamer designed with less hydrophobic contaminants and shown to resolve growth issues for mammalian cell lines was implemented in a commercial biopharmaceutical process and expected to improve protection of cells from hydrodynamic stress [[1]](#ref-0001). However, contrary to expected results, the cell growth and resultant titer further declined. This was not consistent with prior reports in literature [[2]](#ref-0002), which demonstrated that these hydrophobic contaminants present in P188 poloxamer contributed to low performance due to inefficiency in protecting cells from shear stress. An analytical screening tool as well as a novel, high shear stress bioreactor model was developed and used to determine that an overall higher poloxamer average molecular weight was actually the root cause of poor growth in this SP2/0 production cell line. This demonstrates an increased SP2/0 sensitivity to subtle differences in poloxamer molecular weight distribution.

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“…For this reason, bioreactors and control strategies for cell culture processes are constructed in such a way that low local energy dissipation rates are achieved . In addition, it is common practice to supplement additives such as Pluronic F‐68 to the media to keep the inflicted cell damage in an acceptable range .…”

Section: Introductionmentioning

confidence: 99%

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

Kröll

Eilers

Fricke

et al. 2016

Engineering in Life Sciences

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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.

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Investigation of low viability in sparged bioreactor CHO cell cultures points to variability in the Pluronic F-68 shear protecting component of cell culture media

Cited by 15 publications

References 34 publications

Development of a rapid and reliable analytical method for screening poloxamer 188 for use in cell culture process

Development of a rapid and reliable analytical method for screening poloxamer 188 for use in cell culture process

Uncovering new poloxamer attributes significantly impacting mammalian cell growth performance

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

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