Approaches to Quality Risk Management When Using Single-Use Systems in the Manufacture of Biologics.

Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.

Therapeutic angiogenesis by implantation of a capillary structure constituted of human adipose tissue microvascular endothelial cells.

OBJECTIVE: We previously reported a novel technology for the engineering of a capillary network using an optical lithographic technique. To apply this technology to the therapy of ischemic diseases, we tested human omental microvascular endothelial cells (HOMECs) as an autologous cell source and decellularized human amniotic membranes (DC-AMs) as a pathogen-free and low immunogenic transplantation scaffold. METHODS AND RESULTS: Human umbilical vein endothelial cells were aligned on a patterned glass substrate and formed a capillary structure when transferred onto an amniotic membrane (AM). In contrast, HOMECs were scattered and did not form a capillary structure on AMs. Treatment of HOMECs with sphingosine 1-phosphate (S1P) inhibited HOMEC migration and enabled HOMEC formation of a capillary structure on AMs. Using quantitative RT-PCR and Western blot analyses, we demonstrated that the main S1P receptor in HOMECs is S1P(2), which is lacking in human umbilical vein endothelial cells, and that inhibition of cell migration by S1P is mediated through an S1P(2)-Rho-Rho-associated kinase signaling pathway. Implantation of capillaries engineered on DC-AMs into a hindlimb ischemic nude mouse model significantly increased blood perfusion compared with controls. CONCLUSIONS: A capillary network consisting of HOMECs on DC-AMs can be engineered ex vivo using printing technology and S1P treatment. This method for regeneration of a capillary network may have therapeutic potential for ischemic diseases.

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

An automated multiplex specific IgE assay system using a photoimmobilized microarray.

An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 µL of serum within a period of 20 min.

Implantation of capillary structure engineered by optical lithography improves hind limb ischemia in mice.

We previously reported a novel optical lithographic technique for the construction of a capillary network consisting of endothelial cells. To investigate the feasibility of clinical application in the treatment of ischemic diseases, capillary structures were formed on scaffolds made from amniotic membrane (AM) and implanted into mice. The capillary network remained in place for at least 5 days and blood perfusion through the implanted capillaries was histologically detected in an ear flap model. Moreover, blood was observed flowing through the capillary network implanted in abdominal subcutaneous tissue of mice at 5 days after insertion. Implantation of the AM capillary structure into the ischemic hind limbs of mice significantly increased reperfusion compared with controls (AM only). Blood flow was restored in the ischemic limbs to the level of corresponding nonischemic limbs as early as 9 days after surgical implantation. The treatment reversed ischemic symptoms, and ambulatory impairment was significantly improved. Thus, the implantation of a capillary network engineered ex vivo could have therapeutic potential for ischemic diseases.

Encouraging effect of cadherin-mediated cell-cell junctions on transfer printing of micropatterned vascular endothelial cells.

We made micropatterned vascular endothelial cells, which have a regular capillary tube-like structure, on a bioactive hydrogel matrix. We applied a stamping method to transfer micropatterned bovine aortic endothelial cells to a growth factor-reduced basement membrane matrix (Matrigel) and type I collagen gel. In this study, we addressed the issues of how to accelerate cell transfer and the effective factors in doing so. We focused on the effects of the cell-substratum and cell-cell adhesiveness prior to applying cultured endothelial cells to a hydrogel matrix on cellular behavior under transfer printing. We found that individual cells cultured sparsely on substrata with different cell adhesivity transferred to Matrigel up to 40%, whereas cells cultured on patterned substrata having lines of 60 mum in width, which involved cell-cell contacts, could transfer homogeneously to Matrigel within a few hours. The morphology of such cells changed from a tape-like monolayer into a thinner, tube-like structure. The speed and the ratio of transfer of micropatterned cells to Matrigel were affected by the period of cell culture on micropatterned substrata. We also found that the intensity of vascular endothelial cadherin staining at cell-cell junctions of micropatterned cells was correlated with cellular behavior when applying them to Matrigel, on which cells formed a tube-like structure or to which cells migrated individually. Furthermore, micropatterned cells made regular tube-like structures when applied to type I collagen gel. Such tube-like endothelial cells had good viability. These findings may be useful for creating in vitro angiogenesis assays and tissue-like constructs that include capillary-like networks of vascular endothelial cells.

On-chip single embryo coculture with microporous-membrane-supported endometrial cells.

In vitro culture (IVC) of the mammalian embryo is an essential technique in reproductive technology and other related life science disciplines. Although embryos are usually cultured in groups, a single embryo culture has been highly desired for IVC to investigate developmental processes. In this study, we proposed and developed the first single embryo coculture device, which allows making an array of a single embryo coculture with endometrial cells by controlling the culture environment in a microfluidic device. To realize this concept, we investigated three key issues: selection of a culture medium for the embryo coculture with endometrial cells using a mouse embryo and endometrial cells, evaluation of an on-microporous-membrane coculture of endometrial cells and an embryo to control the polarization of endometrial cells on the membrane, and evaluation of the coculture of endometrial cells and the embryo in the microfluidic device. We successfully obtained an array of a single coculture of embryo with endometrial cells in a microfluidic device. This concept will open and enhance the management of an individual embryo for assisted reproductive technology, livestock breeding, and fundamental stage research by further development.

In vitro formation of capillary networks using optical lithographic techniques.

Tissue engineering approaches have been developed for vascular grafts, but success has been limited to arterial replacements of large-caliber vessels. We have developed an innovative technique to transplant engineered capillary networks by printing techniques. Endothelial cells were cultured on a patterned substrate, in which network patterns were generated by prior optical lithography. Subsequently, the patterned cells were transferred to extracellular matrix and tissue at which point they changed their morphologies and formed tubular structures. Microinjection of dye showed that the micrometer-scale tubular structure had in vitro flow potential. When capillary-like networks engineered on amnion membranes were transplanted into mice, we found blood cells inside of the lumen of the transplanted capillary-like structure. This is the first report of the in vitro formation of capillary networks using cell transfer technique, and this novel technique may open the way for development of rapid and effective blood perfusion systems in regenerative medicine.