Characterization of a Novel Particle in a Pharmaceutical Drug Product.

A previously unreported particle type was observed during routine visual vial inspection of a liquid drug product and suspected to be the result of vial delamination. Delamination is the corrosive attack on the interior surface of a glass container resulting in the release of thin flake-like glass particles, lamellae, into solution. It is a major concern for pharmaceutical companies, especially for parenteral solutions, and drug programs with a high risk for delamination are typically monitored for lamellae formation through long-term stability studies. Although these particles observed resembled lamellae (i.e., thin, reflecting light, buoyant) they were not the result of glass delamination. In this study, the authors describe a previously unreported particle type and provide a detailed comparison with known lamellae exposed to the same drug formulation. The chemical, elemental, and morphological characteristics of the particles and respective vials are described in detail. Overall, the particles' high organic and low silica elemental signature, along with no signs of delamination on the glass vial inner surface demonstrate that this lamellae-like observation is a novel particle form that can be distinguished from lamellae formed from vial glass delamination.

Observation and Mitigation of Lamellar Silica Particles Formed in Pharmaceutical Products Packaged in Glass Vials.

A new type of lamellae-like particles was observed in protein based liquid therapeutic protein drug product (DP) packaged in standard (STD) and delamination controlled (DC) Type IB glass vials stored at 2-8°C as early as two weeks after manufacture. These particles were determined to be remarkably different from lamellae in not only in their chemical composition, but in the mechanism by which these are formed. The lamellae-like particles were an ultra-thin (< 200 nm) film, appeared curled, sheet-like, folded with no defined edges identified as lamellar silica composed of silica and polysorbate 80 (PS 80). It was also observed that the lamellar silica particles, when formed in a given drug product lot, not only were observed in a small percentage of vials, but also remained at low (≤ 5) numbers in affected vials, often decreasing in number over time. This is in contrast to the large number of commonly reported glass lamellae (hundreds per vial) observed in vials prone to delamination with a glass vial interior showing a delaminated inner surface. In this case study, evidence from low Si leachable levels in solution and various surface analytical techniques supported the conclusion that there was neither delamination nor early signs of glass delamination like reaction zones occurring in those impacted vials, regardless. A mechanism for particle formation was hypothesized and experimentally confirmed. Lamellar silica particles are composed of an admixture of condensed silica and PS 80 deposited on the interior walls of glass vials, which form and may be released into solution over time. The root cause was determined to be conditions present during preparation of the vials for drug product filling, specifically the vial washing and depyrogenation steps. These conditions are known to make glass vials prone to delamination; in this case study, they resulted in interactions between the glass and PS 80 present in the formulation. Incomplete drying of the glass vials during depyrogenation in closed ovens was confirmed as the contributing factors that led to lamellar silica particle formation via the studies of silicate spiked into the DC Type IB glass vials filled with the mAb DP in which lamellar silica particles were observed. Prevention of lamellar silica particles formation was successfully achieved through optimization of the duration and pressure of air blow during the vial washing and drying process in a depyrogenation oven. This was evidenced by the lack of appearance of the lamellar silica particles over 48 months for the DP lots filled post optimization. Additionally, the formation of lamellar silica was also mitigated by changing the vial washing process from a closed oven process to a tunnel process, which allowed for improved air flow and hence better drying of the vial primary container.

Influence of chain length on the diffusion and electrophoresis of DNA adsorbed on heterogeneous supported lipid bilayers.

This manuscript describes the influence of chain length on the diffusion and electrophoresis of single stranded DNA (ssDNA) adsorbed on heterogeneous cationic supported lipid bilayers. These studies are motivated by the increasing interest in developing novel strategies for the separation of DNA. We studied ssDNA molecules with the number of bases (N) varying from 21 to 84. Fluorescence recovery after photobleaching (FRAP) studies revealed that the diffusivity (D) of adsorbed ssDNA varied with N as D approximately N(-1), similar to a trend previously observed for the diffusion of double stranded DNA on homogeneous supported lipid bilayers. In contrast, the electrophoretic mobility of the adsorbed ssDNA in the presence of an applied tangential electric field was independent of N. Our studies indicated that the motion of ssDNA in the presence of an applied electric field was primarily due to electrophoresis and was not influenced significantly by electro-osmotic flow. Our results also suggest that the use of asymmetric diffusion barriers or other tunable obstacles may assist DNA separation on supported lipid bilayers.

Controlling DNA adsorption and diffusion on lipid bilayers by the formation of lipid domains.

We describe the influence of membrane heterogeneity on the adsorption and diffusion of DNA. Cellular membranes are believed to contain domains (lipid rafts) that influence processes ranging from signal transduction to the diffusion of membrane components. By analogy, we demonstrate that the formation of raft-like domains in supported lipid bilayers provides control over the adsorption and diffusion of DNA. The formation of bilayers from a mixture of the gel phase zwitterionic lipid 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) and the fluid phase cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) yielded coexisting DSPC-enriched and DOTAP-enriched phases. We demonstrated the ability to pattern the adsorption of DNA on the heterogeneous bilayers, with the adsorption being restricted to the DOTAP-enriched phase. We further demonstrated that the DSPC-enriched domains acted as obstacles to the lateral diffusion of adsorbed DNA. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the diffusivity of the adsorbed DNA tracked that of the underlying lipid, although the lipid diffusivity changed by an order of magnitude with changes in bilayer composition. Fundamental insight into the adsorption and diffusion of DNA on heterogeneous surfaces may be useful for the design of novel techniques for the size-based separation of DNA.

Enhancing protein stability by adsorption onto raftlike lipid domains.

We demonstrate that the stability of adsorbed proteins can be enhanced by controlling the heterogeneity of the surfaceby creating raftlike domains in a soft liposomal membrane. Recent work has shown that enzymes adsorbed onto highly curved nanoscale supports can be more stable than those adsorbed on flat surfaces with nominally the same chemical structure. This effect has been attributed to a decrease in lateral interenzyme interactions on a curved surface. Exploiting this idea, we asked if adsorbing enzymes onto "patchy" surfaces composed of adsorbing and nonadsorbing regions can be used to reduce lateral interactions even on relatively flat surfaces. We demonstrate that creating domains on which an enzyme can adsorb enhances the stability of that enzyme under denaturing conditions. Furthermore, we demonstrate that the size of these domains has a considerable effect on the degree of stability imparted by adsorption. Such biomimetic raft-inspired systems may find use in applications ranging from biorecognition to the design of novel strategies for the separation of biomolecules and controlling the interaction of multicomponent membrane-bound enzymes.

Statistical pattern matching facilitates the design of polyvalent inhibitors of anthrax and cholera toxins.

Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.

Microfluidic separation of DNA.

DNA separation is important for numerous applications in biotechnology and medicine. Efforts to improve DNA separation in microdevices have led to advances in capillary electrophoresis and the development of novel separation strategies. Current research on microcapillary electrophoresis materials is focused on the development of separation matrices with low injection viscosities and wall-coating capabilities. Microcapillary injector geometries are being designed to allow increased control of sample plug volumes. Novel separation strategies using entropic traps, arrays of pillars and Brownian ratchets are also being developed.

Impact of Uncontrolled vs Controlled Rate Freeze-Thaw Technologies on Process Performance and Product Quality.

Most biomolecules, owing to their marginal stability in liquid state, susceptibility to microbial growth, and tendency to foam upon storage/shipment in the liquid state, often require an alternate method of long-term storage. Cryopreservation is preferred, as it addresses most of these issues associated with liquid storage. However, the stability of the protein in the frozen state depends on the methodology of freezing/thawing and physico-chemical characteristics of the protein. A systematic study was undertaken to understand and evaluate the impact of freezing/thawing method on the process performance and product quality attributes using two freezing methods-conventional freezing in walk-in freezers and thawing in cold rooms using carboys as an uncontrolled rate method, and Celsius/CryoFin™ technologies as a controlled rate method. To assess the impact of freeze-thaw cycles on product quality, two types of proteins, a fusion protein and a peptibody (peptide fused to the Fc portion of the antibody), were used, employing appropriate stability-indicating assays. The results demonstrate superior process performance by the controlled rate freeze-thaw technology, both in terms of process times and cryoconcentration, compared to uncontrolled rate freeze thaw technology. Product impact studies indicate that the peptibody is sensitive to the method of freeze-thaw while the fusion protein is not and those that are sensitive to uncontrolled rate freeze-thaw processes can be effectively protected by controlled rate freeze-thaw technologies such as Celsius.

Lipid mobility controls the diffusion of small biopolymer adsorbates.

Recent studies on the diffusion of adsorbed polymers such as DNA on supported lipid bilayers have suggested that such strongly adsorbed polymers can be treated similarly to a polymer "in" a 2D fluid, but this conjecture has not been experimentally verified. To test this hypothesis and also to gain a better understanding of polymer dynamics in two dimensions, we designed an experimental protocol-the lateral transport of a short, single-stranded DNA oligonucleotide adsorbed on a supported cationic lipid bilayer. Fluorescence recovery after photobleaching (FRAP) analysis reveals that the diffusivity of the adsorbed DNA quantitatively tracks that of the underlying lipid, even though the bilayer mobility changes by 2 orders of magnitude with changes in temperature. Interestingly, our results for short, extended, adsorbed biopolymers quantitatively track those for globular proteins in lipid bilayers. We thus conclude that short macromolecules that are strongly adsorbed on lipid bilayers can be treated similarly to macromolecules in the bilayer.