Michael Borys (Bristol- Myers Squibb) Incorporation of QbD elements into the development and characterization of a second generation process

QbD principles are readily incorporated into mammalian cell processes to streamline process development and characterization. A key enabler of the implementation of these principles has been widespread adoption of platform technologies by the industry. This allows easy and efficient navigation of the QbD roadmap laid out in the A-Mab case study over the course of the development lifecycle of a product. Here we examine the case of a 2nd generation process for a legacy product that was originally developed and approved using the traditional approach to process development and characterization. The goal of the 2nd generation process was to achieve several fold increases to productivity while achieving similar process performance across scales. Furthermore, comparability profiles of quality attributes must be maintained to ensure treatment efficacy and patient safety, and to streamline the regulatory approval process. To meet these constraints, it was necessary to make significant deviations from the platform process. This presentation outlines some of the challenges encountered during process development, tech transfer, and process characterization and how QbD principles were incorporated at each of the stages. Specifically, advanced metabolomics and proteomics methods were used to understand and eliminate differences in process performance after tech transfer to manufacturing scale and small scale bioreactor operations were optimized to ensure an appropriate scale down model. Risk assessments were used to guide process characterization efforts and custom DOE approaches were used to minimize bioreactor experiments. The experimental data were then fit to models to understand the design space and used to establish quantitative criteria to guide parameter classification. The models were verified through additional experiments and raw material variability was accounted for to improve robustness. The examples provided here demonstrate the advantages of incorporating QbD principles into the development cycle of biologics processes even in situations of compressed timelines and off-platform processes

An upper limit to polarized submillimetre emission in Arp 220

We report the results of pointed observations of the prototypical ultraluminous infrared galaxy (ULIRG) Arp 220 at 850 μm using the polarimeter on the Submillimetre Common User Bolometer Array instrument on the James Clerk Maxwell Telescope. We find a Bayesian 99 per cent confidence upper limit on the polarized emission for Arp 220 of 1.54 per cent, averaged over the 15-arcsec beam-size. Arp 220 can serve as a proxy for other, more distant such galaxies. This upper limit constrains the magnetic field geometry in Arp 220 and also provides evidence that polarized ULIRGs will not be a major contaminant for next-generation cosmic microwave background polarization measurements

Metabolomics approach for increasing CHO cell specific productivity

Chinese hamster ovary cells are the most commonly used expression system in the production of monoclonal antibody therapeutic drugs. The biomanufacturing industry has made significant advances in increasing protein titers of these cell cultures by over 100-fold since the 1980s to gram-per-liter ranges, and much of this progress has been made via increasing cell density and viability. However, even next generation processes are approaching the limits of how high cell densities can be reached with available technologies. On the other hand, the specific productivity (qP) of the cell lines, though much higher now than at the advent of biologics production, has not been improved to the same degree, and advances on this front are needed to attain higher titers in shorter times. In this work, a library of twelve cell lines, having a wide range of qPs but all derived from the same parental cell line and expressing one of two different antibodies, was investigated using an untargeted metabolomics approach. Spent medium samples were collected from each fed-batch culture at two time points. BioCAn (Biologically Consistent Annotation), a recently developed automated annotation tool, was used to determine the most likely identities of features detected in LC-MS data from these cell lines. A correlation analysis was then performed to find annotated features that were significantly associated with either cell growth (37 features), qP (32 features), or both (56 features). Interestingly, all features associated with cell growth showed a negative correlation, while all features associated with qP showed a positive correlation. To investigate whether metabolites positively correlated with qP reflect endogenous metabolic activity beneficial for productivity, several metabolites were added to the culture medium at varying concentrations. We found that supplementing the medium with one or more select metabolites could improve qP without negatively impacting cell growth. We next evaluated whether these metabolites could be used as biomarkers to identify clones with potential for high productivity, as current screening methods can falsely eliminate clones due to sub-optimal culture media or process conditions. Together, these studies demonstrate opportunities for using untargeted metabolomics to achieve higher titer in biologics production processes. Further, the identification of biomarkers has potential to shorten cell line development timelines, which is on the critical path to biologics manufacturing

A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors

Achieving adequate CO2 stripping rates in large scale bioreactors is an important consideration during the scale up of animal cell cultures to large scale bioreactors due to the use of relatively low power input and gas sparging rates. It has previously been reported that cell growth, productivity, and product quality attributes such as glycosylation can be significantly impacted when cells are exposed to high CO2 environments. CO2 stripping models that depend on the CO2 mass transfer coefficient have been applied to simulate CO2 profiles in cell cultures using varied sparger types, reagents for pH adjustment, gas flow rates, and agitation speeds. These models were reported as being validated for a cell culture after cell exponential growth phase. However, in recent years, cell culture processes have been improved to enhance productivity in part through a longer exponential growth phase to achieve higher viable cell densities, making those models less relevant. The current CO2 stripping models were tested in several improved cell culture processes and resulted in predicted CO2 profiles not fitting the measured CO2 profiles. A modified CO2 stripping model was then developed, of which CO2 stripping is independent of the CO2 mass transfer coefficient. Instead, CO2 stripping is a function of gas flow rates, the residence time of bubbles in the liquid, the time of bubbles being saturated with CO2, and CO2 concentrations. The model was validated with two CHO cell culture processes that achieved different peak viable cell density (approximately 7 × 106 cells/mL and 12 × 106 cells/mL) in 25,000-L and 5,000-L manufacturing bioreactors, respectively. The CO2 stripping model was also applied to optimize cell culture conditions to reduce CO2 level in cell cultures in the manufacturing scale bioreactors

METABOLISM OF SKIN DERIVED PRECURSOR (SKP) CELLS IN STIRRED SUSPENSION BIOREACTORS

INTRODUCTION More than six million people suffer from burn injuries every year. These injuries can result in psychological trauma, disabilities and permanent disfigurement. A common treatment for burn victims is an autologous graft surgery in which skin is transplanted from a healthy part of the body to the injury site (i.e. split thickness skin graft). This graft, however, does not contain functional dermal tissue, hair follicles or glands, often causing graft contraction, chronic irritation, and unnatural in appearance. We have hypothesized that skin-derived precursor (SKP) cells, a multipotent dermal stem cell that resides within skin hair follicles, can be utilized in conjunction with split thickness skin grafts to improve their function and minimize irritation. SKPs have a high proliferative potential and need to be expanded in a well-controlled, standardized culture environment before they can be utilized in clinical treatments [1]. It is essential to optimize the expansion of SKP cells in order to generate a bioprocess capable of producing enough cells for a clinical setting. METHODS -qNUTR = ∆[NUTR]/Int(Xv)dtThe specific uptake and production rates were calculated for cells cultured in static T-Flask environments and stirred suspension bioreactors run at 40, 60, 80, and 100 rpm. Equation 1 was used to calculate specific rates (qNUTR). The integral change in viable cells (Int(Xv)dt) was calculated using a numerical trapezoid approximation, and the change in nutrient concentration (∆[NUTR]) was measured using the Nova Bioprofile 100+ analyzer. SKP cells were taken from a 68 year old female. RESULTS The specific uptake rates of glucose and glutamine and specific production rates of lactate and ammonia have been determined under altered cell culture environments for SKP cells (Figure1). This provides details into nutrient limitations and cell metabolic behaviours needed to access parameters to guide our bioprocess design and development of robust expansion protocols. DISCUSSION AND CONCLUSIONS Low levels of oxygen and nutrients result in significant changes to cell growth rates [2].  We were able to conclude that SKP cell growth is not limited by the glucose or glutamine concentrations in the media, and lactate and ammonia do not reach toxic levels. It is interesting to note that the stirred suspension environment does appear to have an effect on the specific consumption rates of glucose and glutamine. According to these initial results, we are predicting that under shear stress environments, SKP cells are changing their metabolic behaviour to allow more glucose to convert to pyruvate and enter the TCA cycle. The specific production rates of lactate and ammonia, however, do not follow the same patterns. Further validation and reasons behind these differences need to be investigated

The Phospha-Bora-Wittig Reaction

[Image: see text] We report the phospha-bora-Wittig reaction for the direct preparation of phosphaalkenes from aldehydes, ketones, esters, or amides. The transient phosphaborene Mes*P=B–NR(2) reacts with carbonyl compounds to form 1,2,3-phosphaboraoxetanes, analogues of oxaphosphetane intermediates in the classical Wittig reaction. 1,2,3-Phosphaboraoxetanes undergo thermal or Lewis acid-promoted cycloreversion, yielding phosphaalkenes. Experimental and density functional theory studies reveal far-reaching similarities between classical and phospha-bora-Wittig reactions

Protocol development to overcome bioprocess bottlenecks in the large-scale expansion of high quality hIPSC aggregates in vertical-wheel bioreactors

Human-induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self-renewal and trilineage differentiation. hiPSCs are cultured as adherent colonies at small scale, which is sufficient to generate cells for experimental purposes but impractical to achieve large quantities for clinical applications. Bioreactor-based processes are the method of choice for efficient expansion and differentiation of cells. Current protocols for the expansion of hiPSCs, however, utilize horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell-culture starting populations and achieve only moderate cell fold increases within the bioreactor. We have recently demonstrated that the vertical-wheel bioreactor produces a unique fluid flow pattern that results in a homogeneous distribution of hydrodynamic forces, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. Please click Additional Files below to see the full abstract

Computational fluid dynamics (CFD) modeling of single-use, vertical-wheel bioreactors as a predictive scale-up tool for large scale stem cell culture

Hydrodynamic variables in bioreactors such as velocity, shear rate, and energy dissipation rate have been shown to affect stem cell properties including: aggregate size, growth, plenotype, and differentiation potential. Unlike traditional bioreactor scale-up equations, CFD modeling allows the user to customize geometry so that scale-up equations can be derived between reactors of any given shape and size. We have recently published data that suggests maintaining the volume average energy dissipation rate, derived from CFD simulations, provides a robust method for scale-up of aggregate culture in stirred suspension bioreactors. Turbulent flow consists of eddies formed when kinetic energy is transferred. Energy dissipation rate is the parameter that determines the amount of energy lost by viscous forces in the flow, and interactions with turbulent eddies influence aggregate size. Aggregates in the culture that are smaller than eddies are engulfed and aggregates that are larger are sheared apart. Please click Additional Files below to see the full abstract

The spatial clustering of ultraluminous infrared galaxies over 1.5 < z < 3

We present measurements of the spatial clustering of galaxies with stellar masses 1011 M, infrared luminosities 1012 L, and star formation rates 200 M yr-1 in two redshift intervals: 1.5 &lt; z &lt; 2.0 and 2 &lt; z &lt; 3. Both samples cluster moderately strongly, with spatial correlation lengths of r0 = 6.14 B1 0.84 h-1 Mpc for the 2 &lt; z &lt; 3 sample and r0 = 5.36 B1 1.28 h-1 Mpc for the 1.5 &lt; z &lt; 2.0 sample. These clustering amplitudes are consistent with both populations residing in dark matter halos with masses of 7 C 1012 M, which is comparable to that seen for optical QSOs at the same epochs. We infer that a minimum dark matter halo mass is an important factor for all forms of luminous, obscured activity in galaxies at z &gt; 1, both starbursts and active galactic nuclei. Adopting plausible models for the growth of dark matter halos with redshift, the halos hosting the 2 &lt; z &lt; 3 sample will likely host poor to rich clusters of galaxies at z = 0, whereas the halos hosting the 1.5 &lt; z &lt; 2.0 sample will likely host L* elliptical galaxies or poor clusters at z = 0. We conclude that ultraluminous infrared galaxies (ULIRGs) at z 2.5 likely signpost stellar buildup in galaxies that will reside in clusters at z = 0 and that ULIRGs at z 1.7 signpost stellar buildup in sources that will either become L* elliptical galaxies or reside in poor clusters at z = 0

Computational fluid dynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture

Innovations in engineering and bioprocess development have accelerated the transition of induced pluripotent stem cell (iPSC) cultivation and use from the bench-top to large-scale clinical manufacturing. Owing to their potency, proliferation capabilities, and ability to overcome the challenges associated with traditional sources of pluripotent stem cells (PSCs), iPSCs have generated significant interest in the field of regenerative medicine for more than a decade. However, traditional bench scale methods to expand iPSCs, including petri dishes and T-flasks, are insufficient to achieve clinically relevant numbers. For iPSC treatments, cell dosages will range from 109 – 1012 cells per patient depending on the therapeutic target. To achieve the required number of cells in an effective and scalable manner, bioreactors will need to be used. Induced pluripotent stem cells (iPSCs) have proven to be extremely sensitive to the bioreactor hydrodynamic environment, making the use of suspension bioreactors to produce quality-assured cells at clinical and commercial scales very challenging. The PBS vertical-wheel (VW) bioreactor combines radial and axial flow components to produce uniform hydrodynamic force distributions, making it a promising platform to overcome the scale-up challenges associated with iPSCs. Please click Additional Files below to see the full abstract