Critical process parameter identification using the ambr15(tm) for process characterization

Process characterization is a critical phase in the development of a commercial process for biotherapeutic production. Knowing the critical quality attributes of your molecule prior to beginning process development and/or characterization is imperative when using a quality by design (QbD) approach. Here we use a (QbD) approach for the characterization of a fed-batch process using an NSO cell line to express an IgG. For this molecule, the glycosylation profile, and in particular, the total fucosylation was identified as a critical quality attribute. After performing a primary hazard analysis, several process inputs were determined to potentially have an impact on this critical quality attribute. These parameters were then studied in a screening DoE using the ambr15™ to model the first and second order effects for each parameter on both the critical quality attributes and process performance. Of the 9 parameters studied, 5 were determined to have a statistically significant effect on the fucosylation of the molecule. In addition, 6 parameters were identified to have a significant impact on process performance. Through process modeling using JMP, a design space was determined for further studies to determine the proven acceptable range (PAR) for each parameter using the 10L, qualified scale down model. An example of the predicted PAR for pH and the timing of the temp shift can be seen in figure 1. Following the 10L studies, a PAR was determined for each parameter and compared with the predicted PAR from ambr. Here we demonstrate the feasibility to use the ambr15™ as a tool for key and even critical process parameter identification to reduce timelines for process characterization

Cell Attachment and Spreading on Carbon Nanotubes Is Facilitated by Integrin Binding

Owing to their exceptional physical, chemical, and mechanical properties, carbon nanotubes (CNTs) have been extensively studied for their effect on cellular behaviors. However, little is known about the process by which cells attach and spread on CNTs and the process for cell attachment and spreading on individual single-walled CNTs has not been studied. Cell adhesion and spreading is essential for cell communication and regulation and the mechanical interaction between cells and the underlying substrate can influence and control cell behavior and function. A limited number of studies have described different adhesion mechanisms, such as cellular process entanglements with multi-walled CNT aggregates or adhesion due to adsorption of serum proteins onto the nanotubes. Here, we hypothesized that cell attachment and spreading to both individual single-walled CNTs and multi-walled CNT aggregates is governed by the same mechanism. Specifically, we suggest that cell attachment and spreading on nanotubes is integrin-dependent and is facilitated by the adsorption of serum and cell-secreted adhesive proteins to the nanotubes

Modulation of functional pendant chains within poly(ethylene glycol) hydrogels for refined control of protein release

Hydrogels are highly attractive delivery vehicles for therapeutic proteins. Their innate biocompatibility, hydrophilicity and aqueous permeability allow stable encapsulation and release of proteins. The release rates also can be controlled simply by altering the crosslinking density of the polymeric network. However, the crosslinking density also influences the mechanical properties of hydrogels, generally opposite to the permeability. In addition, the release of larger proteins may be hindered below critically diminished porosity determined by the crosslinking density. Herein, the physical properties of the hydrogels are tuned by presenting functional pendant chains, independent of crosslinking density. Heterobifunctional poly(ethylene glycol) monomethacrylate (PEGMA) with various end functional groups is synthesized and copolymerized with PEG dimethacrylate (PEGDA) to engineer PEG hydrogels with pendant PEG chains. The pendant chains of the PEG hydrogels consisting of sulfonate, trimethylammonium chloride, and phenyl groups are utilized to provide negative charge, positive charge and hydrophobicity, respectively, to the hydrogels. The release rates of proteins with different isoelectric points are controlled in a wide range by the type and the density of functional pendant chains via electrostatic and hydrophobic interactions

The significance of peroxisomes in secondary metabolite biosynthesis in filamentous fungi

Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e.g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells

Star poly(ethylene glycol) as a tunable scaffold for neural tissue engineering

The primary focus of this work was to develop a novel synthetic hydrogel scaffold as an in vitro model to enable future detailed studies of how neurons grow in environments with controllable diffusion profiles of soluble cues and tunable neuron-matrix interactions. The development of in vitro models that enable elucidation of the mechanisms of system performance is a recently emerging goal of tissue engineering. The design of three-dimensional (3D) scaffolds in particular, is motivated by the need to develop model systems that better mimic native tissue as compared to conventional two-dimensional (2D) cell culture substrates. An ideal scaffold is degradable, porous, biocompatible, with mechanical properties to match those of the tissues of interest and with a suitable surface chemistry for cell attachment, proliferation, and differentiation. Although naturally derived materials are more versatile in providing complex biological cues, synthetic polymers are preferable for the design of in vitro models as they provide wider range of properties, controllable degradation rates, and easier processing. Most importantly, their mechanical properties can be decoupled from their biological properties, a crucial issue in interpreting cell responses. The synthetic material provides the structural backbone of the scaffold while biochemical function is added via incorporation of ligands or proteins aimed at triggering specific cell behaviors. As presented in this dissertation, we have developed and characterized a new synthetic 3D hydrogel scaffold from cross-linked poly(ethylene glycol) (PEG). PEG was selected because it is hydrophilic, non-toxic, biocompatible, and inert to protein adhesion. The chosen cross-linking chemistry was a highly specific reaction that occurred under physiological conditions so that cells could be embedded within the gel prior to cross-linking. Controllable degradability was imparted via series of hydrolytically degradable PEG cross-linkers. Thorough analysis demonstrated the independent tuning of the mechanical, biochemical and biological properties of the developed hydrogel. Because soluble cues such as neurotrophic factors are an effective means for promoting nerve regeneration, the diffusion of biomolecules through the PEG hydrogel were also explored in depth via two methods: fluorescence correlation spectroscopy (FCS) and bulk diffusion experiments. This is the first demonstration of FCS to delineate protein diffusivity within a cross-linked synthetic hydrogel and describe local and dynamic protein-polymer interactions that occur within these systems. Further, since PEG is inert, short ligands such as RGD were used to promote cell adhesion and new insights into how these ligands impact hydrogel mechanical and transport properties were established. Finally, to test the utility of the developed material as an in vitro model, neuronal cell-matrix interactions were studied by tuning hydrogel properties and assessing cell viability and neurite outgrowth. We believe that this work is major step in building an in vitro model for gaining an understanding of the key parameters that guide nerve regeneration and have the potential to lead to the development of better strategies to treat peripheral nerve injuries

Star poly(ethylene glycol) as a tunable scaffold for neural tissue engineering

The primary focus of this work was to develop a novel synthetic hydrogel scaffold as an in vitro model to enable future detailed studies of how neurons grow in environments with controllable diffusion profiles of soluble cues and tunable neuron-matrix interactions. The development of in vitro models that enable elucidation of the mechanisms of system performance is a recently emerging goal of tissue engineering. The design of three-dimensional (3D) scaffolds in particular, is motivated by the need to develop model systems that better mimic native tissue as compared to conventional two-dimensional (2D) cell culture substrates. An ideal scaffold is degradable, porous, biocompatible, with mechanical properties to match those of the tissues of interest and with a suitable surface chemistry for cell attachment, proliferation, and differentiation. Although naturally derived materials are more versatile in providing complex biological cues, synthetic polymers are preferable for the design of in vitro models as they provide wider range of properties, controllable degradation rates, and easier processing. Most importantly, their mechanical properties can be decoupled from their biological properties, a crucial issue in interpreting cell responses. The synthetic material provides the structural backbone of the scaffold while biochemical function is added via incorporation of ligands or proteins aimed at triggering specific cell behaviors. As presented in this dissertation, we have developed and characterized a new synthetic 3D hydrogel scaffold from cross-linked poly(ethylene glycol) (PEG). PEG was selected because it is hydrophilic, non-toxic, biocompatible, and inert to protein adhesion. The chosen cross-linking chemistry was a highly specific reaction that occurred under physiological conditions so that cells could be embedded within the gel prior to cross-linking. Controllable degradability was imparted via series of hydrolytically degradable PEG cross-linkers. Thorough analysis demonstrated the independent tuning of the mechanical, biochemical and biological properties of the developed hydrogel. Because soluble cues such as neurotrophic factors are an effective means for promoting nerve regeneration, the diffusion of biomolecules through the PEG hydrogel were also explored in depth via two methods: fluorescence correlation spectroscopy (FCS) and bulk diffusion experiments. This is the first demonstration of FCS to delineate protein diffusivity within a cross-linked synthetic hydrogel and describe local and dynamic protein-polymer interactions that occur within these systems. Further, since PEG is inert, short ligands such as RGD were used to promote cell adhesion and new insights into how these ligands impact hydrogel mechanical and transport properties were established. Finally, to test the utility of the developed material as an in vitro model, neuronal cell-matrix interactions were studied by tuning hydrogel properties and assessing cell viability and neurite outgrowth. We believe that this work is major step in building an in vitro model for gaining an understanding of the key parameters that guide nerve regeneration and have the potential to lead to the development of better strategies to treat peripheral nerve injuries

Adsorption and Sustained Delivery of Small Molecules from Nanosilicate Hydrogel Composites

Two-dimensional nanosilicate particles (NS) have shown promise for the prolonged release of small-molecule therapeutics while minimizing burst release. When incorporated in a hydrogel, the high surface area and charge of NS enable electrostatic adsorption and/or intercalation of therapeutics, providing a lever to localize and control release. However, little is known about the physio-chemical interplay between the hydrogel, NS, and encapsulated small molecules. Here, we fabricated polyethylene glycol (PEG)-NS hydrogels for the release of model small molecules such as acridine orange (AO). We then elucidated the effect of NS concentration, NS/AO incubation time, and the ability of NS to freely associate with AO on hydrogel properties and AO release profiles. Overall, NS incorporation increased the hydrogel stiffness and decreased swelling and mesh size. When individual NS particles were embedded within the hydrogel, a 70-fold decrease in AO release was observed compared to PEG-only hydrogels, due to adsorption of AO onto NS surfaces. When NS was pre-incubated and complexed with AO prior to hydrogel encapsulation, a >9000-fold decrease in AO release was observed due to intercalation of AO between NS layers. Similar results were observed for other small molecules. Our results show the potential for use of these nanocomposite hydrogels for the tunable, long-term release of small molecules