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The sterility test has been utilized for many years to formally support the sterile release of products terminally sterilized by moist heat and other sterilization modalities. It is well-known that the sterility test is severely limited in providing a meaningful scientific and statistical basis to support the sterility of finished products. Accordingly, parametric release was developed as a sterility assurance release program that defines critical manufacturing process and sterilization control parameters that are essential for sterile product release, thereby eliminating the use of the sterility test. This article examines the limitations and shortcomings of the sterility test and reviews the findings from a case study to illustrate the risks associated with the overreliance on this test for the disposition of sterile products. The history of parametric release of products terminally sterilized with moist heat is presented with a summary of the current status, including a listing of global regulatory standards and guidances along with a discussion of applicable results from an industry survey of moist heat sterilization practice. This article is intended to be the initial step to establish a basic understanding of the parametric release concept and practices that can be leveraged as a baseline for future promotion and expanded implementation., (© PDA, Inc. 2022.)

Visible particulate matter contamination is responsible for the rejection or recall of numerous batches of injectable product each year. The result is wasted time, effort, money, product and the limited availability of medically necessary drug and biologic products. Recently published compendial standards have alleviated some of the confusion surrounding suitable test methods and acceptance criteria for visible particulates; however, the complexities of visual inspection methods across a wide range of injectable product types packaged in diverse and sometimes complex container systems has complicated the approach to visible particulate control in injectable products. The solution is a life-cycle approach to visible particulate contamination control that addresses the prevention, inspection, identification, and remediation of visible particulate contamination. More importantly, the life-cycle approach to visible particulate control is aligned with current United States Food and Drug Administration's good manufacturing practices and can serve as an effective tool for demonstrating regulatory compliance for inspections, audits, and regulatory submissions., (© PDA, Inc. 2020.)

Clinicians have had concerns about particulate matter contamination of injectable drug products since the development of the earliest intravenous therapeutics. All parenteral products contain particulate matter, and particulate matter contamination still has the potential to cause harm to patients. With tens of millions of doses of injectable drug products administered in the United States each year, it is critical to understand the types and sources of particulate matter that contaminate injectable drug products, the possible effects of injected particulate matter on patients, and the current state of regulations and standards related to particulate matter in injectable drug products. Today, the goal of manufacturers, regulators, and standards-setting organizations should be to continue to minimize the risk of particle-induced sequelae, especially in high-risk patients, without trading unnecessary manufacturing burden for minimal safety gains., Lay Abstract: All injectable drug products are contaminated with some level of solid particulate matter, including, for example, fibers, dust, rubber, and silicone. These materials enter drug products primarily during the manufacturing process. The possible effects on patients of injectable drug products containing particulate matter depend on a number of factors. However, given the large number of patients receiving injectable drug products each year in the United States and the potential for particulate matter to cause harm to patients, it is critical to continue to minimize particulate matter contamination in injectable drug products. Manufacturing standards and regulations have helped improve manufacturing quality. Nevertheless, manufacturers, regulators, and standards-setting organizations must continue to work toward improving manufacturing quality and minimizing the risk of harm from particle contamination, especially in high-risk patients.

This article summarizes a recently completed research assignment conducted on behalf of the Food and Drug Administration's (FDA) Office of Pharmaceutical Science. The assignment addressed the issue of changing sterile drug manufacturing sites from the point of view of both synthetic and biotech drug products. The article is intended to provide readers with an overview of the assignment and to summarize a recommended approach whereby the use of a comparability protocol can satisfy the FDA's review expectations on the one hand, and facilitate streamlined compliance and faster product to market by manufacturers on the other.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

Select groups of bacteria, including prothescate species, have an unusual capacity to sequester gold and bioconcentrate it to very high levels. Hyphomonas adhaerens MHS-3 (MHS-3) is one such species, as demonstrated by Energy Dispersive Spectroscopy. Transmission electron microscopy revealed that the binding site was specific on the polar polysaccharide capsule. A capsuleless mutant and periodate-treated wild type did not sequester gold. The gold may interact with the same sites in the capsule that naturally adhere MHS-3 to surfaces in the marine environment.

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.

The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.

Hyphomonas strain VP-6 is a prosthecate bacterium isolated from the Guayamas vent region and is a member of a genus of primary and common colonizers of marine surfaces. It adheres to solid substrata as a first step in biofilm formation. Fine-structure microscopy and the use of specific stains and lectins reveal that it synthesizes two different extracellular polymeric substances (EPS). One is a temporally synthesized, polar holdfast EPS, and the other is a capsular EPS that is present during the complete life cycle and surrounds the entire cell, including the prosthecum. The timing and location of Hyphomonas strain VP-6 EPS elaboration correlate with adhesion to surfaces, suggesting that the EPS serves not only as the biofilm matrix but also as a primary adhesin. The temporality and polarity of VP-6 EPS expression substantially differ from those properties of Hyphomonas strain MHS-3 EPS expression.