Analysis of aroma components changes in Gannan navel orange at different growth stages by HS-SPME-GC-MS, OAV, and multivariate analysis.

The ripe Gannan navel oranges have an appealing aroma, but few studies have reported the changes of these aromatic substances during the growth of navel oranges. In this study, changes of aroma components in Gannan navel orange from 119 to 245 days after flowering were systematically studied using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) coupled with multivariate analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). A total of 43 and 54 aroma components were identified in pulp and peel of navel orange, respectively. The odor active value (OAV) results indicated that 14 substances were the key aroma components during the growth of navel orange. Among them, the contribution of linalool, ß-myrcene and limonene were the highest. The multivariate statistical analysis further confirmed that 14 and 18 compounds could be used as key markers to distinguish the pulp and peel at different growth stages, respectively. Results from this study contributed to a better understanding of the dynamic variation and retention of aroma compounds during navel orange growth, and have great potential for industrial application.

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

No ordinary proteins: Adsorption and molecular orientation of monoclonal antibodies.

The interaction of monoclonal antibodies (mAbs) with air/water interfaces plays a crucial role in their overall stability in solution. We aim to understand this behavior using pendant bubble measurements to track the dynamic tension reduction and x-ray reflectivity to obtain the electron density profiles (EDPs) at the surface. Native immunoglobulin G mAb is a rigid molecule with a flat, "Y" shape, and simulated EDPs are obtained by rotating a homology construct at the surface. Comparing simulations with experimental EDPs, we obtain surface orientation probability maps showing mAbs transition from flat-on Y-shape configurations to side-on or end-on configurations with increasing concentration. The modeling also shows the presence of ß sheets at the surface. Overall, the experiments and the homology modeling elucidate the orientational phase space during different stages of adsorption of mAbs at the air/water interface. These finding will help define new strategies for the manufacture and storage of antibody-based therapeutics.

Armoring the Interface with Surfactants to Prevent the Adsorption of Monoclonal Antibodies.

The pharmaceutical industry uses surface-active agents (excipients) in protein drug formulations to prevent the aggregation, denaturation, and unwanted immunological response of therapeutic drugs in solution as well as at the air/water interface. However, the mechanism of adsorption, desorption, and aggregation of proteins at the interface in the presence of excipients remains poorly understood. The objective of this work is to explore the molecular-scale competitive adsorption process between surfactant-based excipients and two monoclonal antibody (mAb) proteins, mAb-1 and mAb-2. We use pendant bubble tensiometry to measure the ensemble average adsorption dynamics of mAbs with and without the excipient. The surface tension measurements allow us to quantify the rate at which the molecules "race" to the interface in single-component and mixed systems. These results define the phase space, where coadsorption of both mAbs and excipients occurs onto the air/water interface. In parallel, we use X-ray reflectivity (XR) measurements to understand the molecular-scale dynamics of competitive adsorption, revealing the surface-adsorbed amounts of the antibody and excipient. XR has revealed that at a sufficiently high surface concentration of the excipient, mAb adsorption to the surface and subsurface domains was inhibited. In addition, despite the fact that both mAbs adsorb via a similar mechanistic pathway and with similar dynamics, a key finding is that the competition for the interface directly correlates with the surface activity of the two mAbs, resulting in a fivefold difference in the concentration of the excipient needed to displace the antibody.

Impact of electroviscous effect on viscosity in developing highly concentrated protein formulations: Lessons from non-protein charged colloids.

Subcutaneous delivery of highly concentrated protein formulations is paramount for reducing healthcare cost and improving patient compliance, where reducing the solution viscosity of formulations is critical for drug delivery. The objective of this paper is to provide some mechanistic understanding about the contribution of electrostatic repulsion to the viscosity of protein solutions at high concentrations, along with the effect of excipients such as salts on relative viscosity. Proteins are treated as charged colloids in this paper. At high concentrations, the electrical double layer starts to overlap, and secondary electroviscous effect becomes significant in addition to primary electroviscous effect. In other words, the hydrodynamic volume of proteins plays a great role in influencing their solution viscosity because of the excluded volume effect. Currently, it is hypothesized that the high viscosity of concentrated protein solutions is attributed to formation of clusters due to either electrostatic attraction or hydrophobic interactions, especially for monoclonal antibodies, in which anybody molecules in high concentration formulations may form networks. Consequently, viscosity reduction in the presence of inorganic or organic salts in these formulations is due to breaking up of these networks. In this review, authors hope to provide another point of view based on the effect of the electrostatic repulsion on the excluded volume-hydrodynamic volume. Finally, authors hope the proposed theoretical framework can be used to guide excipient selection in the product development of highly concentrated proteins.

Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective.

Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor-liquid, solid-liquid, and liquid-liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended.

Adsorption of polypropylene oxide-polyethylene oxide type surfactants at surfaces of pharmaceutical relevant materials: effect of surface energetics and surfactant structures.

Protein therapeutics are exposed to various surfaces during product development, where their adsorption possibly causes unfolding, denaturation, and aggregation. In this paper, we aim to characterize four types of typical surfaces used in the development of biologics: polycarbonate, polyethersulfone, borosilicate glass, and cellulose. Contact angles of these surfaces were measured using three probing liquids: water, formamide, and diidomethane, from which acid/base (AB) and Lifshitz-van der Waals (LW) interaction components were derived. To explore the interactions of surfactants of Pluronics/Poloxamers (PEO-PPO-PEO copolymers) with these surfaces, the adsorption of three Pluronics (F68, F127, and L44) at these surfaces was determined using a quartz crystal microbalance with dissipation technique (QCM-D). For hydrophobic surfaces without AB component (polycarbonate and polyethersulfone), these copolymers exhibited significant adsorption with a little dissipation at low concentrations. For hydrophilic surfaces with AB component (cellulose and borosilicate), the adsorption at low-surfactant concentration is low while dissipation is relatively high. Additionally, the chemical properties of Pluronics such as the ratio of PPO to PEO, along with the interaction of PPO with surfaces were observed to play a critical role in adsorption. Furthermore, the interfacial structure of the adsorbed layer was affected by both AB interaction and the presence of PEO block.

Real-time observation of protein aggregates in pharmaceutical formulations using liquid cell electron microscopy.

Understanding the properties of protein-based therapeutics is a common goal of biologists and physicians. Technical barriers in the direct observation of small proteins or therapeutic agents can limit our knowledge of how they function in solution and in the body. Electron microscopy (EM) imaging performed in a liquid environment permits us to peer into the active world of cells and molecules at the nanoscale. Here, we employ liquid cell EM to directly visualize a protein-based therapeutic in its native conformation and aggregate state in a time-resolved manner. In combination with quantitative analyses, information from this work contributes new molecular insights toward understanding the behaviours of immunotherapies in a solution state that mimics the human body.

Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species.

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B 22*, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.

Particle Characterization for a Protein Drug Product Stored in Pre-Filled Syringes Using Micro-Flow Imaging, Archimedes, and Quartz Crystal Microbalance with Dissipation.

Micro-flow imaging (MFI) has been used for formulation development for analyzing sub-visible particles. Archimedes, a novel technique for analyzing sub-micron particles, has been considered as an orthogonal method to currently existing techniques. This study utilized these two techniques to investigate the effectiveness of polysorbate (PS-80) in mitigating the particle formation of a therapeutic protein formulation stored in silicone oil-coated pre-filled syringes. The results indicated that PS-80 prevented the formation of both protein and silicone oil particles. In the case of protein particles, PS-80 might involve in the interactions with the hydrophobic patches of protein, air bubbles, and the stressed surfaces of silicone oil-coated pre-filled syringes. Such interactions played a role in mitigating the formation of protein particles. Subsequently, quartz crystal microbalance with dissipation (QCM-D) was utilized to characterize the interactions associated with silicone oil, protein, and PS-80 in the solutions. Based on QCM-D results, we proposed that PS-80 likely formed a layer on the interior surfaces of syringes. As a result, the adsorbed PS-80 might block the leakage of silicone oil from the surfaces to solution so that the silicone oil particles were mitigated at the presence of PS-80. Overall, this study demonstrated the necessary of utilizing these three techniques cooperatively in order to better understand the interfacial role of PS-80 in mitigating the formation of protein and silicone oil particles.

An Approach to Mitigate Particle Formation on the Dilution of a Monoclonal Antibody Drug Product in an IV Administration Fluid.

To support dose reduction, low dose of a monoclonal antibody (mAb) was required to be administered via IV infusion at a concentration of 0.1 mg/mL. To achieve the target protein concentration, the infusion solution was prepared by diluting the drug product containing 10-mg/mL mAb with normal saline, a 0.9% sodium chloride injection solution. However, particles were observed in the diluted solution. Particle formation must be avoided to administer the low dose using the existing drug product. To mitigate the particle formation, an unconventional compounding approach was used. With this approach, a stabilizing vehicle containing polysorbate-80 was added to saline before drug-product dilution to maintain suitable surfactant level to prevent precipitation of the mAb. In this way, use of the stabilizing vehicle to support low doses ensured suitable quality across a wider range of mAb concentrations, thereby allowing additional flexibility to the clinical trial. Such an approach may be useful for broader application in early-stage clinical trials where there is an uncertainty regarding doses or the need to revise to lower doses based on clinical observations or other drivers.

Sensitive fluorescence-based method for the rapid determination of polysorbate-80 content in therapeutic monoclonal antibody products.

A sensitive and effective method has been developed for the rapid determination of polysorbate-80 content in therapeutic monoclonal antibody (mAb) products. The method is based on the detection of the fluorescence emission of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) enhanced by the presence of polysorbate-80. The developed method includes two approaches. One requires removal of the mAb from solution prior to analysis, while the other requires only simple sample dilution. The limits of detection and quantitation, calculated from the calibration curve generated in the absence of mAb-A, were 1.5 and 4.7 parts per million, respectively. Given the comparable linear range and linearity of the linear line between the solutions, with or without mAb, the limit of detection and quantitation is assumed to be similar. The dilution method is not only fast and simple in terms of sample preparation, but it is also particularly useful for analyzing the level of polysorbate-80 contained in highly concentrated mAb products. However, given that this method does require availability of polysorbate-80-free materials of mAb for preparation of calibration standards, the protein removal method may be useful for the cases where appropriate protein materials for standard preparation are limited or unavailable.

A model membrane protein for binding volatile anesthetics.

Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4-3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism.

Amphiphilic 4-helix bundles designed for biomolecular materials applications.

Artificial peptides previously designed to possess alpha-helical bundle motifs have been either hydrophilic (i.e., soluble in polar media) or lipophilic (i.e., soluble in nonpolar media) in overall character. Realizations of these bioinspired bundles have succeeded in reproducing a variety of biomimetic functionality within the appropriate media. However, to translate their functionality into any biomolecular device applications at the macroscopic level, the bundles must be oriented in an ensemble, for example, at an interface. This goal has been realized in a new family of alpha-helical bundle peptides which are amphiphilic; namely, they assemble into 4-helix bundles with well-defined hydrophilic and hydrophobic domains. These peptides are capable of binding metalloporphyrin prosthetic groups at selected locations within these domains. We describe here the realization of one of the first members of this family, AP0, successfully designed for vectorial incorporation into soft interfaces between polar and nonpolar media.

Comparative structural studies of Vpu peptides in phospholipid monolayers by x-ray scattering.

Vpu is an 81-residue HIV-1 accessory protein, its transmembrane and cytoplasmic domains each responsible for one of its two functions. Langmuir monolayers of phospholipid incorporating a membrane protein with a unidirectional vectorial orientation, on a semiinfinite aqueous subphase, provide one "membranelike" environment for the protein. The cytoplasmic domain's interaction with the surface of the phospholipid monolayer in determining the tertiary structure of the peptide within the monolayer was investigated, employing a comparative structural study of Vpu with its submolecular fragments Tm and TmCy truncated to different extents in the cytoplasmic domain, via synchrotron x-ray scattering utilizing a new method of analysis. Localizations of the transmembrane and cytoplasmic domains within the monolayer profile structure were similar for all three proteins, the hydrophobic transmembrane helix within the hydrocarbon chain region tilted with respect to the monolayer plane and the helices of the cytoplasmic domains lying on the surface of the headgroups parallel to the monolayer plane. The thickness of the hydrocarbon chain region, determined by the tilt of the hydrocarbon chains and transmembrane domain with respect to the monolayer plane, was slightly different for Tm, TmCy, and Vpu systematically with protein/lipid mole ratio. Localization of the helices in the cytoplasmic domains of the three proteins relative to the headgroups depends on their extents and amphipathicities. Thus, the interaction of the cytoplasmic domain of Vpu on the surface may affect the tilt of the transmembrane helix within the hydrocarbon chain region in determining its tertiary structure in the membrane.