Best Practices for Microbial Challenge In-use Studies to Evaluate the Microbial Growth Potential of Parenteral Biological Products; Industry and Regulatory Considerations.

Microbial challenge in-use studies are performed to evaluate the potential for microbial proliferation in preservative-free single dose biological products after first puncture and potential accidental contamination during dose preparation (e.g. reconstitution, dilution) and storage. These studies, in addition to physicochemical in-use stability assessments, are used as part of product registration to define in-use hold times in Prescribing Information and in the pharmacy manual in the case of clinical products. There are no formal guidance documents describing regulator expectations on how to conduct microbial challenge in-use studies and interpret microbial data to assign in-use storage hold-times. In lieu of guidance, US Food and Drug Administration (FDA) regulators have authored publications and presentations describing regulator expectations. Insufficient or unavailable microbial challenge data can result in shortened in-use hold times, thus microbial challenge data enables flexibility for health care providers (HCPs) and patients, while ensuring patient safety. A cross-industry/FDA in-use microbial working group was formed through the Innovation & Quality (IQ) Consortium to gain alignment among industry practice and regulator expectations. The working group assessed regulatory guidance, current industry practice via a blinded survey of IQ Consortium member companies, and scientific rationale to align on recommendations for experimental design, execution of microbial challenge in-use studies, and a decision tree for microbial data interpretation to assign in-use hold times. Besides the study execution and data interpretation, additional considerations are discussed including use of platform data for clinical stage products, closed system transfer devices (CSTDs), transport of dose solutions, long infusion times, and the use of USP <797> by HCPs for preparing sterile drugs for administration. The recommendations provided in this manuscript will help streamline biological product development, ensure consistency on assignment of in-use hold times in biological product labels across industry, and provide maximum allowable flexibility to HCPs and patients, while ensuring patient safety.

Viscosities and Protein Interactions of Bispecific Antibodies and Their Monospecific Mixtures.

Solution viscosities (η) and protein-protein interactions (PPI) of three monoclonal antibodies (mAb-A, mAb-B, mAb-C), two bispecific antibodies (BsAb-A/B, BsAb-A/C), and two 1:1 binary mixtures (mAb-A + mAb-B and mAb-A + mAb-C) were measured. mAb-A and mAb-C have similar isoelectric point (pI) values but significantly different η versus protein concentration ( c2) profiles. The viscosity of the mAb-A + mAb-C mixture followed an Arrhenius mixing rule and was identical to viscosity of the bispecific BsAb-A/C. In contrast, mAb-A and mAb-B had similar η versus c2 profiles, but the Arrhenius mixing rule failed to predict the higher viscosities of their mixtures. The viscosity of the bispecific BsAb-A/B followed the 1:1 mAb-A + mAb-B mixture at all concentrations. The nature of the interactions for mAb-A, mAb-B, the BsAb-A/B bispecific, and the 1:1 mAb-A + mAb-B mixture were characterized by static and dynamic light scattering (SLS and DLS). mAb-A and mAb-B exhibited net-attractive and -repulsive electrostatic interactions, respectively. The bispecific antibody (BsAb-A/B) had short-ranged attractive interactions, suggesting that the increase in viscosity for this molecule and the mAb-A + mAb-B mixture was due to cross-interactions between Fab regions. At high and low ionic strengths and protein concentrations, the Rayleigh scattering profile, the collective diffusion coefficient, and viscosity for the mixture closely followed that for the bispecific antibody. These results highlight the possible anomalous viscosity increases of bispecific antibodies constructed from relatively low-viscosity mAbs but demonstrates a potentially fruitful approach of using mAb mixtures to predict the viscosity of candidate bispecific constructs.

Monoclonal Antibody Interfaces: Dilatation Mechanics and Bubble Coalescence.

Monoclonal antibodies (mAbs) are proteins that uniquely identify targets within the body, making them well-suited for therapeutic applications. However, these amphiphilic molecules readily adsorb onto air-solution interfaces where they tend to aggregate. We investigated two mAbs with different propensities to aggregate at air-solution interfaces. The understanding of the interfacial rheological behavior of the two mAbs is crucial in determining their aggregation tendency. In this work, we performed interfacial stress relaxation studies under compressive step strain using a custom-built dilatational rheometer. The dilatational relaxation modulus was determined for these viscoelastic interfaces. The initial value and the equilibrated value of relaxation modulus were larger in magnitude for the mAb with a higher tendency to aggregate in response to interfacial stress. We also performed single-bubble coalescence experiments using a custom-built dynamic fluid-film interferometer (DFI). The bubble coalescence times also correlated to the mAbs aggregation propensity and interfacial viscoelasticity. To study the influence of surfactants in mAb formulations, polyethylene glycol (PEG) was chosen as a model surfactant. In the mixed mAb/PEG system, we observed that the higher aggregating mAb coadsorbed with PEG and formed domains at the interface. In contrast, for the other mAb, PEG entirely covered the interface at the concentrations studied. We studied the mobility of the interfaces, which was manifested by the presence or the lack of Marangoni stresses. These dynamics were strongly correlated with the interfacial viscoelasticity of the mAbs. The influence of competitive destabilization in affecting the bubble coalescence times for the mixed mAb/PEG systems was also studied.

A Method To Measure Protein Unfolding at an Air-Liquid Interface.

Proteins are surface-active molecules that have a propensity to adsorb to hydrophobic interfaces, such as the air-liquid interface. Surface flow can increase aggregation of adsorbed proteins, which may be an undesirable consequence depending on the application. As changes in protein conformation upon adsorption are thought to induce aggregation, the ability to measure the folded state of proteins at interfaces is of particular interest. However, few techniques currently exist to measure protein conformation at interfaces. Here we describe a technique capable of measuring the hydrophobicity, and therefore the conformation and folded state, of proteins at air-liquid interfaces by exploiting the environmentally sensitive fluorophore Nile red. Two monoclonal antibodies (mAbs) with high (mAb1) and low (mAb2) surface activity were used to highlight the technique. Both mAbs showed low background fluorescence of Nile red in the liquid subphase and at a glass-liquid interface. In contrast, at the air-liquid interface Nile red fluorescence for mAb1 increased immediately after protein adsorption, whereas the Nile red fluorescence of the mAb2 film evolved more slowly in time even though the adsorbed quantity of protein remained constant. The results demonstrate that hydrophobicity upon mAb adsorption to the air-liquid interface evolves in a time-dependent manner. Interfacial hydrophobicity may be indicative of protein conformation or folded state, where rapid unfolding of mAb1 upon adsorption would be consistent with increased protein aggregation compared to mAb2. The ability to measure protein hydrophobicity at interfaces using Nile red, combined with small sample requirements and minimal sample preparation, fills a gap in existing interfacial techniques.

Single-molecule enzymology based on the principle of the Millikan oil drop experiment.

The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.

Structural and rheological properties of meibomian lipid.

PURPOSE: We explore the unique rheological and structural properties of human and bovine meibomian lipids to provide insight into the physical behavior of the human tear-film lipid layer (TFLL). METHODS: Bulk rheological properties of pooled meibomian lipids were measured by a commercial stress-controlled rheometer; a home-built interfacial stress rheometer (ISR) probed the interfacial viscoelasticity of spread layers of meibomian lipids. Small- and wide-angle x-ray scattering detected the presence and melting of dispersed crystal structures. Microscope examination under cross polarizers provided confirmation of ordered crystals. A differential scanning calorimeter (DSC) analyzed phase transitions in bulk samples of bovine meibum. RESULTS: Bulk and interfacial rheology measurements show that meibum is extremely viscous and highly elastic. It is also a non-Newtonian, shear-thinning fluid. Small- and wide-angle x-ray diffraction (SAXS and WAXS), as well as differential scanning calorimetry (DSC) and polarizing microscopy, confirm the presence of suspended lamellar-crystal structures at physiologic temperature. CONCLUSIONS: We studied meibum architecture and its relation to bulk and interfacial rheology. Bovine and human meibomian lipids exhibit similar physical properties. From all structural probes utilized, we find a melt transition near eye temperature at which lamellar crystals liquefy. Our proposed structure for the tear-film lipid layer at physiologic temperature is a highly viscoelastic, shear-thinning liquid suspension consisting of lipid lamellar-crystallite particulates immersed in a continuous liquid phase with no long-range order. When spread over on-eye tear, the TFLL is a duplex film that exhibits bulk liquid properties and two separate interfaces, air/lipid and water/lipid, with aqueous protein and surfactantlike lipids adsorbed at the water/lipid surface.

3-Hydroxybutyric acid interacts with lipid monolayers at concentrations that impair consciousness.

3-Hydroxybutyric acid (also referred to as ß-hydroxybutyric acid or BHB), a small molecule metabolite whose concentration is elevated in type I diabetes and diabetic coma, was found to modulate the properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers when added to the subphase at clinical concentrations. This is a key piece of evidence supporting the hypothesis that the anesthetic actions of BHB are due to the metabolite's abilities to alter physical properties of cell membranes, leading to indirect effects on membrane protein function. Pressure-area isotherms show that BHB changes the compressibility of the monolayer and decrease the size of the two-phase coexistence region. Epi-fluorescent microscopy further reveals that the reduction of the coexistence region is due to the significant reduction in morphology of the liquid condensed domains in the two-phase coexistence region. These changes in monolayer morphology are associated with the diminished interfacial viscosity of the monolayers (measured using an interfacial stress rheometer), which gives insight as to how changes in phase and structure may contribute to membrane function.

Molecular structure of interfacial human meibum films.

Meibum is the primary component of the tear film lipid layer. Thought to play a role in tear film stabilization, understanding the physical properties of meibum and how they change with disease will be valuable in identifying dry eye treatment targets. Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at an air-water interface to identify molecular organization. At room temperature, interfacial meibum films formed two coexisting scattering phases with rectangular lattices and next-nearest neighbor tilts, similar to the Ov phase previously identified in fatty acids. The intensity of the diffraction peaks increased with compression, although the lattice spacing and molecular tilt angle remained constant. Reflectivity measurements at surface pressures of 18 mN/m and above revealed multilayers with d-spacings of 50 Å, suggesting that vertical organization rather than lateral was predominantly affected by meibum-film compression.

Temperature-induced transitions in the structure and interfacial rheology of human meibum.

Meibomian lipids are the primary component of the lipid layer of the tear film. Composed primarily of a mixture of lipids, meibum exhibits a range of melt temperatures. Compositional changes that occur with disease may alter the temperature at which meibum melts. Here we explore how the mechanical properties and structure of meibum from healthy subjects depend on temperature. Interfacial films of meibum were highly viscoelastic at 17°C, but as the films were heated to 30°C the surface moduli decreased by more than two orders of magnitude. Brewster angle microscopy revealed the presence of micron-scale inhomogeneities in meibum films at higher temperatures. Crystalline structure was probed by small angle x-ray scattering of bulk meibum, which showed evidence of a majority crystalline structure in all samples with lamellar spacing of 49 Å that melted at 34°C. A minority structure was observed in some samples with d-spacing at 110 Å that persisted up to 40°C. The melting of crystalline phases accompanied by a reduction in interfacial viscosity and elasticity has implications in meibum behavior in the tear film. If the melt temperature of meibum was altered significantly from disease-induced compositional changes, the resultant change in viscosity could alter secretion of lipids from meibomian glands, or tear-film stabilization properties of the lipid layer.

Insertion mechanism of a poly(ethylene oxide)-poly(butylene oxide) block copolymer into a DPPC monolayer.

Interactions between amphiphilic block copolymers and lipids are of medical interest for applications such as drug delivery and the restoration of damaged cell membranes. A series of monodisperse poly(ethylene oxide)-poly(butylene oxide) (EOBO) block copolymers were obtained with two ratios of hydrophilic/hydrophobic block lengths. We have explored the surface activity of EOBO at a clean interface and under 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers as a simple cell membrane model. At the same subphase concentration, EOBO achieved higher equilibrium surface pressures under DPPC compared to a bare interface, and the surface activity was improved with longer poly(butylene oxide) blocks. Further investigation of the DPPC/EOBO monolayers showed that combined films exhibited similar surface rheology compared to pure DPPC at the same surface pressures. DPPC/EOBO phase separation was observed in fluorescently doped monolayers, and within the liquid-expanded liquid-condensed coexistence region for DPPC, EOBO did not drastically alter the liquid-condensed domain shapes. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XRR) quantitatively confirmed that the lattice spacings and tilt of DPPC in lipid-rich regions of the monolayer were nearly equivalent to those of a pure DPPC monolayer at the same surface pressures.

The interfacial viscoelastic properties and structures of human and animal Meibomian lipids.

As the interface between the aqueous layer of the tear film and air, the lipid layer plays a large role in maintaining tear film stability. Meibomian lipids are the primary component of the lipid layer; therefore the physical properties of these materials may be particularly crucial to the functionality of the tear film. Surface pressure versus area isotherms, interfacial shear and extensional rheology, and Brewster angle microscopy (BAM) were used to characterize the Meibomian lipids from different species known to have different lipid compositions. The isotherms of humans, bovinae, wallabies, rabbits and kultarrs (a small desert marsupial) were qualitatively similar with little hysteresis between compression and expansion cycles. In contrast, several isocycles were necessary to achieve equilibrium behavior in the koala lipids. With the exception of kultarr lipids, the interfacial complex viscosity of all samples increased by one or two orders of magnitude between surface pressures of 5 mN/m and 20 mN/m and exhibited classic gel behavior at higher surface pressures. In contrast, the kultarr lipids were very fluid up to 22 mN/m; the behavior did not depend on surface pressure. Human lipids were very deformable in extensional flow and the BAM images revealed that the film became more homogeneous with compression as the elasticity of the film increased. The morphology of the kultarr lipids did not change with compression indicating a strong correlation between film structure and behavior. These results suggest that the lipid layer of the tear film forms a gel in vivo, which may aid in mechanically stabilization of the tear film.

A comparison of alternative 60-mer probe designs in an in-situ synthesized oligonucleotide microarray.

BACKGROUND: DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers) is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. RESULTS: We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, beta-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined) of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined) designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment) produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. CONCLUSION: Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that several different measures commonly utilized in probe design were not predictive of outlier probes.