A cytotoxic leachable compound from single‐use bioprocess equipment that causes poor cell growth performance

The biopharmaceutical industry has invested considerably in the implementation of single-use disposable bioreactors in place of or in addition to their stainless steel-counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene-film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035-0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label-free LC-MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547-1558, 2016.

Section: Resultsmentioning

confidence: 99%

Process‐relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics

Kelly

McSweeney

Coleman

et al. 2016

“…Raw material analysis in cell culture media is one of the key areas in bioprocessing that requires more attention and characterization. Unknown or untested impurities within raw materials, such as trace metal contaminants, polymer impurities, or leachable elements in single‐use films are known to have an impact on cell growth, protein expression, or glycosylation . For example, the historical spinner flask and standard scale down bioreactor operation failed to detect the inefficiency of suspicious poloxamer 188 lots, which later caused a problem in the manufacturing scale.…”

Section: Discussionmentioning

confidence: 99%

Development of small scale cell culture models for screening poloxamer 188 lot‐to‐lot variation

Peng

Hall

Clayton

et al. 2014

Shear protectants such as poloxamer 188 play a critical role in protecting cells during cell culture bioprocessing. Lot-to-lot variation of poloxamer 188 was experienced during a routine technology transfer across sites of similar scale and equipment. Cell culture medium containing a specific poloxamer 188 lot resulted in an unusual drop in cell growth, viability, and titer during manufacturing runs. After switching poloxamer lots, culture performance returned to the expected level. In order to control the quality of poloxamer 188 and thus maintain better consistency in manufacturing, multiple small scale screening models were developed. Initially, a 5L bioreactor model was established to evaluate cell damage by high sparge rates with different poloxamer 188 lots. Subsequently, a more robust, simple, and efficient baffled shake flask model was developed. The baffled shake flask model can be performed in a high throughput manner to investigate the cell damage in a bubbling environment. The main cause of the poor performance was the loss of protection, rather than toxicity. It was also suggested that suspicious lots can be identified using different cell line and media. The screening methods provide easy, yet remarkable models for understanding and controlling cell damage due to raw material lot variation as well as studying the interaction between poloxamer 188 and cells.

“…Plastic materials can leach out of chemical compounds which may be toxic for microbial and cell cultures . As it is not possible to predict the toxicity of the leached compounds for individual cultures, a biocompatibility test should be conducted before each new combination of culture and plastic material.…”

Section: Resultsmentioning

confidence: 99%

“…As it is not possible to predict the toxicity of the leached compounds for individual cultures, a biocompatibility test should be conducted before each new combination of culture and plastic material. Comparison of growth behaviors of culture in the presence and absence of plastic materials is a common biocompatibility test setup . The plastic materials used in this study, namely the polyethersulfone membrane, heat shrink tubing, and silicon tubing were added separately to a culture of CHO DG44 in T75 flasks.…”

Section: Resultsmentioning

confidence: 99%

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Meier

Djeljadini

Regestein

et al. 2014

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse-flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse-flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non-feeding at a dilution rate of 0.2 h(-1) results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short-term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi-continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h(-1) and 0.5 h(-1) , respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h(-1) - 0.5 h(-1) . Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time.