Analysis of leachable Bis Di-tert-butyl Phenyl Phosphate (bdtbpp) in bioprocessing films

The analysis of extractable and leachable compounds from medical grade plastics is a complex issue that is compounded by the presence of many chemical species that are either direct or indirect reaction and/or degradation products of additives, process aids and polymer agents. These compounds may be present at various levels and the potential for adverse effects on cell growth remains to be determined and warrants further investigation. Since many of these films undergo aggressive processing steps, such as, thermal extrusion and gamma irradiation there is the potential for many unknown degradation products to be formed at each step of the processing. In addition, the absolute identification of many of these chemical compounds remains unknown. Within the various applications of single-use disposable bioprocessing, the presence of bis di-tert butyl phenyl phosphate (bDtBPP), a common gamma irradiation degradation product of tris (2,4-ditert-butylphenyl) phosphite (TBPP), has been shown to have a profoundly negative impact on cell growth for certain lines. The presence of bDtBPP even at low levels (on the order of 10ppb) has been shown to inhibit cell growth performance percentages in some lines by as much as 30-50% [1]. The quantitative analysis of this compound becomes increasingly difficult at lower levels due to either; 1) irreversible binding of strongly charged phosphate groups to glassware and other labware used in processing samples, or 2) hydrolytic degradation in aqueous solutions, or 3) any combination of the two. Losses from either of these conditions has been show to give rise to variations in quantitative analysis results as high as 50% when testing ranges are set between 5-100 parts per billion (ppb) in water. To this end, we have investigated the fate of this compound at part per billion levels to gain insight into possible mechanisms associated the variations observed. The hydrolytic degradation as well as irreversible binding to various substrates such as HPLC vials, pipette tips, etc. has been investigated extensively. We propose mitigation strategies which allow for low level quantitative analysis of this compound to achieve a coefficient of variation (CV) within the range of 10-20%, for bDtBPP in water within concentration ranges of 5-100 ppb. References Lindskog, Eva., Blank, Eva., Ullsten, Sara., Yi, Shujian., Ganguli, Pokon., Carter, Jeffrey., Parma, Hernan. Implementation of Raw Material Control Strategies in the Manufacture of Single-Use Bioprocessing Containers; BioPharm International, Volume 28, Issue 1, January 1, 2015

Flexural fatigue behavior of rocking bioreactor films

The fields of biopharmaceutical processing and cell therapy are adopting single-use, closed systems throughout their workflows to enhance sterility, minimize waste wash effluent and enable manufacturing flexibility compared to traditional stainless steel bioreactors. One of the key single-use technologies in use is the rocking bioreactor, comprising a polymer film bag (outfitted with ports and sensors) mounted to a tray capable of mixing the contents of the bag and a control system (controlling temperature, agitation, and potentially media perfusion). One of the challenges encountered in rocking bioreactor bags is the fact that upon inflation/filling with media, the originally flat bioreactor bags often develop folds and dimples due to their inflated geometry. These deformations tend to be inconsequential at small volumes and low agitation rates/times, but can lead to flex fatigue failures such as whitening, delamination and through-cracking under more extreme conditions. In practice, these failures are dependent on a number of factors including bag material and volume, mounting geometry, rocking angle and rate, and the duration of culture, making a systematic study of the material properties controlling this behavior difficult and time-consuming. Several flex fatigue testing systems exist in the literature, including Gelbo and Sonntag-Universal, but none of these effectively model the unique geometry and stresses of the rocking bioreactor geometry. To this end, we have developed accelerated test methods to analyze the flexural fatigue behavior of multilayer rocking bioreactor films. These methods enable quality control testing of film lots, and have the potential to compare different film compositions with a rapid and reproducible test, thereby facilitating development of new films. Our test method models the local geometry surrounding the fold/dimple in a rocking bioreactor in a small sample of film, and cycles the sample to accelerate flexural fatigue at the dimple site. Initial results indicate the ability to accelerate film failure from tens of days on a rocking bioreactor platform (using a full bioreactor bag) to tens of hours using less than ten square inches of film. We will discuss the effects of various experimental parameters on film failure, optimization of test procedures and correlation with rocking bioreactor testing in the field

Nonequilibrium thermodynamics and energy efficiency in weight loss diets

Carbohydrate restriction as a strategy for control of obesity is based on two effects: a behavioral effect, spontaneous reduction in caloric intake and a metabolic effect, an apparent reduction in energy efficiency, greater weight loss per calorie consumed. Variable energy efficiency is established in many contexts (hormonal imbalance, weight regain and knock-out experiments in animal models), but in the area of the effect of macronutrient composition on weight loss, controversy remains. Resistance to the idea comes from a perception that variable weight loss on isocaloric diets would somehow violate the laws of thermodynamics, that is, only caloric intake is important ("a calorie is a calorie"). Previous explanations of how the phenomenon occurs, based on equilibrium thermodynamics, emphasized the inefficiencies introduced by substrate cycling and requirements for increased gluconeogenesis. Living systems, however, are maintained far from equilibrium, and metabolism is controlled by the regulation of the rates of enzymatic reactions. The principles of nonequilibrium thermodynamics which emphasize kinetic fluxes as well as thermodynamic forces should therefore also be considered

Test method development for next-generation bioprocessing applications

As single use disposable (SUD) bioprocessing systems become more commonplace, the range of applications and workflow steps served by single use continues to grow. Nearly all of the process steps and techniques currently used in bioprocessing were developed on stainless steel or glass vessels which exhibit thermal, chemical and mechanical properties which differ greatly from the polymer films used in SUD. While in many cases (e.g., cell culture) these differences have negligible effects on performance, as the industry pushes the limits of intensification and increases in the breadth of SUD applications (antibody-drug conjugation – ADC, microbial culture, etc.) single-use materials and systems face new challenges. This highlights the need for testing capabilities to qualify and develop new SUD materials, components and systems to ensure integrity, cleanliness and performance. In this presentation, we will comment on the current state-of-the-art in standardized testing as well as highlighting several small-scale test methods developed internally, specifically for testing materials for SUD bioprocessing. In order to assess film and component (e.g., port, tubing) capabilities for the full range of current and foreseeable process steps, we have developed/adapted/adopted test methodologies to model environments experienced both in conventional use (abrasion, flexure, pressurization), as well as challenging new use cases including high and low temperature extremes (freezing, pasteurization) and aggressive, non-aqueous environments used in bioconjugate chemistry (e.g., ADC, conjugate vaccines), chemical viral inactivation and non-traditional cell culture (e.g., microbial). For each of these applications, our methods were developed based on generating an understanding of the environment in which the chosen SUD system is to be used, identifying the primary failure modes that are (or could be) encountered in use and reducing these risks to their fundamental physics to generate small-scale tests that can be performed rapidly on small samples of material. These test methods allow for rapid screening of film and component materials to reduce risks in new applications prior to prototype development and assess product and process quality in the long ter