Use of Monte Carlo simulations for improved facility fit planning in downstream biomanufacturing and technology transfer

Biologics manufacturing is capital and consumable intensive with need for advanced inventory planning to account for supply chain constraints. Early-stage process design and technology transfer are often challenging due to limited information on process variability regarding bioreactor titer, process yield, and product quality. Monte Carlo (MC) methods offer a stochastic modeling approach for process optimization where probabilities of occurrence for process inputs are incorporated into a deterministic model to simulate more likely scenarios for process outputs. In this study, we explore MC simulation-based design of a monoclonal antibody downstream manufacturing process. We demonstrate that this probabilistic approach offers more representative outcomes over the conventional worst-case approach where the theoretical minimum and maximum values of each process parameter are used without consideration for their probability of occurrence. Our work demonstrates case studies on more practically sizing unit operations to improve consumable utilization, thereby reducing manufacturing costs. We also used MC simulations to minimize process cadence by constraining the number of cycles per unit operation to fit facility preferences. By factoring in process uncertainty, we have implemented MC simulation-based facility fit analyses to efficiently plan for inventory when accounting for process constraints during technology transfer from lab-scale to clinical or commercial manufacturing.     

Different epithelia in the distal human male urethra

Summary The distal segment of the human male urethra, in particular the fossa navicularis, was studied with light- and electron microscopy as well as by means of histochemical and immunocytochemical methods. The fossa navicularis of the urethra contains a circumscribed zone of extremely thick, non-keratinized stratified squamous epithelium composed of cells containing a large amount of glycogen. These cells lack acid phosphatase activity and lysozyme-like immunoreactivity, both of which can be demonstrated to varying extents in the other zones of the distal male urethra. These glycogen-rich cells are considered to be the substrate for an endogenous flora of lactobacteria, whereas the acid-phosphatase activity and the lysozyme-like immunoreactivity indicate the presence of macrophages and the secretion of bactericidal agents at the epithelial surface. These observations suggest that the different zones with heterogeneous properties in the distal male urethra probably represent a defense system against the invasion of pathogenic microorganisms. Moreover, the glycogen-rich zone, which resembles the glycogen-rich epithelium of the vagina, is estrogen-dependent. This is demonstrated in cases of sex reversal in which after long-lasting estrogen treatment the glycogen-rich zone becomes extremely extended by displacement of the neighbouring epithelium.     

Cell cycle length, cell size, and proliferation rate in hydra stem cells

We have analyzed the cell cycle parameters of interstitial cells in Hydra oligactis. Three subpopulations of cells with short, medium, and long cell cycles were identified. Short-cycle cells are stem cells; medium-cycle cells are precursors to nematocyte differentiation; long-cycle cells are precursors to gamete differentiation. We have also determined the effect of different cell densities on the population doubling time, cell cycle length, and cell size of interstitial cells. Our results indicate that decreasing the interstitial cell density from 0.35 to 0.1 interstitial cells/epithelial cell (1) shortens the population doubling time from 4 to 1.8 days, (2) increases the [3H]thymidine labeling index from 0.5 to 0.75 and shifts the nuclear DNA distribution from G2 to S phase cells, and (3) decreases the length of G2 in stem cells from 6 to 3 hr. The shortened cell cycle is correlated with a significant decrease in the size of interstitial stem cells. Coincident with the shortened cell cycle and increased growth rate there is an increase in stem cell self-renewal and a decrease in stem cell differentiation.     

Purinergic receptor stimulation reduces cytotoxic edema and brain infarcts in mouse induced by photothrombosis by energizing glial mitochondria

Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5' diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y(1)R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP(3))-dependent Ca(2+) release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.     

Permeation of ions across the potassium channel: brownian dynamics studies

The physical mechanisms underlying the transport of ions across a model potassium channel are described. The shape of the model channel corresponds closely to that deduced from crystallography. From electrostatic calculations, we show that an ion permeating the channel, in the absence of any residual charges, encounters an insurmountable energy barrier arising from induced surface charges. Carbonyl groups along the selectivity filter, helix dipoles near the oval chamber, and mouth dipoles near the channel entrances together transform the energy barrier into a deep energy well. Two ions are attracted to this well, and their presence in the channel permits ions to diffuse across it under the influence of an electric field. Using Brownian dynamics simulations, we determine the magnitude of currents flowing across the channel under various conditions. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength causes a steady decrease in the channel conductance. The current also decreases systematically when the effective dielectric constant of the channel is lowered. The conductance with the optimal choice of dipoles reproduces the experimental value when the dielectric constant of the channel is assumed to be 60. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates from Ohm's law at a higher applied potential. The reversal potentials obtained with asymmetrical solutions are in agreement with those predicted by the Nernst equation. The conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings for the transport of ions across the potassium channels and membrane channels in general.     

Targeted disruption of the Insl3 gene causes bilateral cryptorchidism.

The sexual dimorphic position of the gonads in mammals is dependent on differential development of two ligaments, the cranial suspensory ligament (CSL) and the gubernaculum. During male embryogenesis, outgrowth of the gubernaculum and regression of the CSL result in transabdominal descent of the testes, whereas in the female, development of the CSL in conjunction with failure of the gubernaculum development holds the ovaries in a position lateral to the kidneys. Several lines of evidence suggest that regression of the CSL and induction of gubernaculum development are mediated by testosterone and a yet unidentified testicular factor, respectively. The Insl3 gene (originally designated Ley I-L), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the postnatal ovary. Here we show that male mice homozygous for a targeted deletion of the Insl3 locus exhibit bilateral cryptorchidism with free moving testes and genital ducts. These malformations are due to failure of gubernaculum development during embryogenesis. In double-mutant male mice for Insl3 and androgen receptor genes, testes are positioned adjacent to the kidneys and steadied in the abdomen by the CSL. These findings demonstrate, that the Insl3 induces gubernaculum development in an androgen-independent way, while androgen-mediated regression of the CSL occurs independently from Insl3.     

Boundary cells of endodermal origin define the mouth of Hydra vulgaris (Cnidaria)

We investigated morphology, dynamics and origin of cells surrounding the mouth of Hydra vulgaris using the monoclonal antibody L96. This antibody recognises a one cell-thick ring of endodermal epithelial cells exactly at the boundary between endoderm (gastrodermis) and ectoderm (epidermis). L96⁺ cells can stretch considerably without any cell rupture during mouth opening. Thus, our data prove the existence of a distinct cell population defining hydra's mouth. A model for mouth opening is proposed and the significance of L96⁺ cells for boundary formation between ectoderm and endoderm is discussed.