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Processing stresses on therapeutic proteins may cause formation of subvisible particles. Different stress mechanisms generate particle populations with characteristic morphological "fingerprints," and machine learning techniques like convolutional neural networks (CNNs) allow classification of microscopy images of these particles according to known stresses at their root cause. Using CNNs to classify novel particle types not included during network training may lead to inaccurate classification, however, using CNNs to monitor the presence of particulate matter not explicitly used in training could serve as a useful process analytical technology. We used CNNs to classify and identify the root cause of particles generated by subjecting three monoclonal antibodies (mAbs) to various common manufacturing stresses. We probed the generality of particles generated by stressing different mAbs in different formulations and showed that CNN analyses were sensitive not only to the applied stress, but also the buffer conditions and the particular mAb that generated particle populations. Thus, models trained on images of particles created with one mAb and buffer system may not provide accurate root cause analysis when applied to particles generated by other mAb and buffer systems. A lever-rule analysis of CNN-derived fingerprints was used to characterize the composition of mixtures of particle types. Finally, we monitored the temporal evolution of CNN-derived fingerprints when novel populations of particles, which were not included during training, were generated by pumping mAb solutions through a peristaltic pump.

Container choice can influence particle generation within protein formulations. Incompatibility between proteins and containers can manifest as increased particle concentrations, shifts in particle size distributions and changes in particle morphology distributions. In this study, flow imaging microscopy (FIM) combined with machine learning-based goodness-of-fit hypothesis testing algorithms were used in accelerated stability studies to investigate the impact of containers on particle formation. Containers in four major container categories subdivided into eleven container types were filled with monoclonal antibody formulations and agitated with and without headspace, producing subvisible particles. Digital images of the particles were recorded using flow imaging microscopy and analyzed with machine learning algorithms. Particle morphology distributions depended on container category and type, revealing differences that would not have been obvious by analysis of particle concentrations or container surface characteristics alone. Additionally, the algorithm was used to compare morphologies of particles generated in containers against those generated using isolated stresses at air-liquid and container-air-liquid interfaces. These comparisons showed that the morphology distributions of particles formed during agitation most closely resemble distributions that result from exposure of proteins to moving triple interface lines at points where container-air-liquid interfaces intersect. The approach described here can be used to identify dominant causes of particle generation due to protein-container interactions.

Currently licensed vaccines require a cold-chain to maintain efficacy. This cold-chain requirement reduces the availability of vaccines in resource-poor areas of the world. Commercially available human papillomavirus (HPV) vaccines protect against the most common HPV types related to cervical cancer; however, their impact is limited in many regions due to cold-chain requirements. The goal of this study was to test the thermostability of an adjuvanted, trivalent HPV L1 capsomere-based vaccine (containing HPV types 16, 18, and 31) that was formulated by using lyophilization to embed the antigens within a solid, glassy matrix. Thermal stabilities were determined by storing the vaccine formulations for 3 months at 50 °C, followed by immunization of BALB/c mice and measurement of antibody responses. Antibody responses to capsomere vaccines formulated with alum were unchanged after storage for 3 months at 50 °C. Neutralizing responses to these vaccines were unchanged by high-temperature storage, and were equivalent to those generated after administration of the commercially available liquid HPV vaccine Gardasil®9.

This work reports the ability of hydrogel coatings to protect therapeutic proteins from cavitation-induced aggregation caused by mechanical stress. Here, we show that polyacrylamide hydrogel coatings on container surfaces suppress mechanical shock-induced cavitation and the associated aggregation of intravenous immunoglobulin (IVIg). First, crosslinked polyacrylamide hydrogels were grown on the surfaces of borosilicate glass vials. Treatment with ultrasound showed that these hydrogel surfaces suppressed cavitation events to levels below those found for unfunctionalized borosilicate glass. Next, IVIg solutions were loaded into these vials and subjected to tumbling, horizontal shaking, and drop testing. Aggregation was quantified by bisANS fluorescence staining and particle counting by flow imaging microscopy (FIM). In all cases, the presence of polyacrylamide hydrogels on the vial surfaces reduced the amount of IVIg aggregation and the number of particulates. In addition, the polyacrylamide appeared to have a protective effect that prevented additional aggregates from forming at extended tumbling times. Finally, drop test studies showed that the polyacrylamide coatings suppressed detectable cavitation. This work reveals how even a simple hydrogel vial coating can have a profound effect on stabilizing protein therapeutics.

Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus are the filoviruses most commonly associated with human disease. Previously, we administered a three-dose regimen of trivalent vaccines comprising glycoprotein antigens from each virus in mice and non-human primates (NHPs). The vaccines, which contained a polysorbate 80-stabilized squalane-in-water emulsion adjuvant and were lyophilized from a solution containing trehalose, produced high antibody levels against all three filovirus antigens. Subsequently, single-vial formulations containing a higher concentration of adjuvant were generated for testing in NHPs, but these vaccines elicited lower neutralizing antibody titers in NHPs than previously tested formulations. In order to explain these results, in the current work we measured the size of adjuvant emulsion droplets and the peroxide levels present in the vaccines after lyophilization and reconstitution and tested the effects of these variables on the immune response in mice. Increases in squalane droplet sizes were observed when the ratio of adjuvant to trehalose was increased beyond a critical value, but antibody and neutralizing antibody titers in mice were independent of the droplet size. Higher levels of peroxides in the vaccines correlated with higher concentrations of adjuvant in the formulations, and higher peroxide levels were associated with increased levels of oxidative damage to glycoprotein antigens. Neutralizing titers in mice were inversely correlated with peroxide levels in the vaccines, but peroxide levels could be reduced by adding free methionine, resulting in retention of high neutralizing antibody titers. Overall, the results suggest that oxidation of glycoprotein antigens by peroxides in the polysorbate 80-stabilized squalane-in-water emulsion adjuvant, but not lyophilization-induced increases in adjuvant emulsion droplet size may have been responsible for the decreased neutralizing titers seen in formulations containing higher amounts of adjuvant.

Therapeutic proteins are exposed to numerous stresses during their manufacture, shipping, storage and administration to patients, causing them to aggregate and form particles through a variety of different mechanisms. These varied mechanisms generate particle populations with characteristic morphologies, creating “fingerprints” that are reflected in images recorded using flow imaging microscopy. Particle population fingerprints in test samples can be extracted and compared against those of particles produced under baseline conditions using an algorithm that combines machine learning tools such as convolutional neural networks with statistical tools such as nonparametric density estimation and Rosenblatt transform-based goodness-of-fit hypothesis testing. This analysis provides a quantitative method with user-specified Type 1 error rates to determine whether the mechanisms that produce particles in test samples differ from particle formation mechanisms operative under baseline conditions. As a demonstration, this algorithm was used to compare particles within intravenous immunoglobulin (IVIg) formulations that were exposed to freeze-thawing and shaking stresses within a variety of different containers. This analysis revealed that seemingly subtle differences in containers (e.g., glass vials from different manufacturers) generated distinguishable particle populations after the stresses were applied. This algorithm can be used to assess the impact of process and formulation changes on aggregation-related product instabilities.

Filling pump operation is an important cause of particle formation in therapeutic protein formulations. The goals of the present study were to investigate the impacts of peristaltic filling pump tubing type, pump operating parameters (acceleration and velocity), and formulation on both nanoparticle and microparticle formation in water, placebo, and a 120 mg/mL mAb drug formulation. Microparticles were quantified using flow imaging microscopy and light obscuration, and nanoparticles were counted with nanoparticle tracking analysis. Pumping of all solutions through Pharmed® tubing resulted in much higher particle levels than processing with Accusil™ or Masterflex® tubing. Pump acceleration did not measurably affect particle levels in pumped solutions, but in some cases, a relatively high pumping velocity of 400 rpm enhanced nanoparticle formation. The presence of surfactants reduced pumping-induced particle formation in the mAb solution, and the effects of 4 different surfactants tested were similar. Biophysical properties (secondary and tertiary structure, and thermal stability) of the protein in solution were not altered by pumping. Overall, this study demonstrates that investigations of pumping parameters and formulations using both nanoparticle and microparticle measurement methods are important for understanding pumping-induced particle formation and developing effective control strategies.

When administered intravenously, various particles and nanomedicines activate complement, potentially leading to infusion reactions and other adverse drug reactions. Particles form within formulations of therapeutic proteins due to stresses incurred during shipping, handling and administration to patients. In this study, IVIg solutions were stored in multiple types of vials and prefilled syringes and exposed to agitation and freeze thawing stresses to generate particles. The stressed samples were added to human serum to determine whether these particles activated complement. Sub-visible IVIg particles ranging in size between 2 and 10 microns activated complement in a fashion that was linearly dependent on the number of particles dosed, whereas little correlation was found between doses of larger particles (>10 microns) and complement activation. Activation of complement by sub-visible particles of IVIg followed the alternative pathway, as shown by the release of complement cascade factor Bb and the production of the anaphylatoxins C3a and C5a without generation of C4a. The number and the morphology of sub-visible particles formed depended on the applied stress, formulation and on the container material. But the capacity of the 2-10 micron-sized particles to activate complement in human serum appeared to depend only on particle concentration.

Reconstitution of lyophilized disaccharide formulations results in the formation of nano-sized air bubbles that persist in suspension for weeks. If proteins are present, interactions with nanobubbles may cause loss of monomeric protein and formation of sub-visible particles. The goals of this work are to determine the mechanism(s) by which nanobubbles form in reconstituted lyophilized formulations, and to develop strategies for reducing nanobubble generation. We hypothesize that nanobubbles are created from nano-sized gas pockets within lyophilized solids, which become bubbles when the surrounding matrix is dissolved away during reconstitution. Nano-sized voids may originate from small ice crystals formed within the concentrated liquid during freezing that subsequently sublime during drying. Nanobubble concentrations are correlated with the extent of mannitol crystallization during freezing. Nano-sized ice crystals, induced by the release of water during mannitol crystallization, were responsible for nanobubble formation. The presence of trehalose or sucrose, in formulations with low mannitol concentrations, inhibited excipient crystallization during lyophilization and reduced nanobubble levels following reconstitution. Our results show a correlation between nanobubble formation and concentrations of insoluble IL-1ra aggregates, suggesting that minimizing nanobubble generation may be an effective strategy for reducing protein aggregation following reconstitution.

Concerns regarding the impact of subvisible particulate impurities on the safety and efficacy of therapeutic protein products have led manufacturers to implement strategies to minimize protein aggregation and particle formation during manufacturing, storage, and shipping. However, once these products are released, manufacturers have limited control over product handling. In this work, we investigated the effect of di(2-ethylhexyl) phthalate (DEHP) nanodroplets generated in polyvinyl chloride (PVC) bags of intravenous (IV) saline on the stability and immunogenicity of IV immunoglobulin (IVIG) formulations. We showed that PVC IV bags containing saline can release DEHP droplets into the solution when agitated or transported using a pneumatic tube transportation system in a clinical setting. We next investigated the effects of emulsified DEHP nanodroplets on IVIG stability and immunogenicity. IVIG adsorbed strongly to DEHP nanodroplets, forming a monolayer. In addition, DEHP nanodroplets accelerated IVIG aggregation in agitated samples. The immunogenicity of DEHP nanodroplets and IVIG aggregates generated in these formulations were evaluated using an in vitro assay of complement activation in human serum. The results suggested DEHP nanodroplets shed from PVC IV bags could reduce protein stability and induce activation of the complement system, potentially contributing to adverse immune responses during the administration of therapeutic proteins.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a poly-disperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Especially in developing countries, the impact of vaccines can be limited by logistical obstacles associated with multiple dose regimens, pathogen variants, and challenges imposed by requirements for maintaining vaccines at low temperatures during shipping and storage. Thus, there is a need for vaccines that can be flexibly modified to address evolving pathogen landscapes, are stable outside of narrow "cold-chain" temperatures and require administration of only single doses. Here we demonstrate in proof-of-concept studies a vaccine platform that addresses these impediments to more widespread use of vaccines. The platform relies on bacteriophage-derived phage-like-particles (PLPs) that utilize a "plug-and-play" antigen delivery system that allows for fast, easy alteration of antigens on the surface of the PLPs. Thermostability of PLP-based vaccines can be achieved by embedding the PLPs within glassy particles produced by spray drying, and nanoscopic aluminum oxide layers applied using atomic layer deposition (ALD) can serve to control release of antigen in vivo, yielding vaccine formulations that elicit strong immune responses after administration of single doses. Bacteriophage λ was stabilized by spray drying to form powders that were incubated at 37 °C for up to a year without loss of infectious activity. PLPs derived from bacteriophage λ were expressed and purified from E. coli cultures, and an in vitro conjugation strategy was used to decorate specific PLP surface sites with T4-lysozyme, a model vaccine antigen. The resulting T4-lysozyme:PLP complexes (Lys-PLPs) were embedded in glassy dry powders formed by spray drying and coated with nanometer-thick layers of alumina deposited by ALD in a fluidized bed reactor. Alumina-coated Lys-PLP vaccines were stable for a least a month at 50 °C, and single doses of the alumina-coated vaccines elicited immune responses that were indistinguishable from responses generated by conventional two-dose, prime-and-boost dosing regimens of alum-adjuvanted Lys-PLP vaccines.

Digital microscopy is used to monitor particulates such as protein aggregates within biopharmaceutical products. The images that result encode a wealth of information that is underutilized in pharmaceutical process monitoring. For example, images of particles in protein drug products typically are analyzed only to obtain particle counts and size distributions, even though the images also reflect particle characteristics such as shape and refractive index. Multiple groups have demonstrated that convolutional neural networks (CNNs) can extract information from images of protein aggregates allowing assignment of the likely stress at the "root-cause" of aggregation. A practical limitation of previous CNN-based approaches is that the potential aggregation-inducing stresses must be known a priori, disallowing identification of particles produced by unknown stresses.We demonstrate an expanded CNN analysis of flow imaging microscopy (FIM) images incorporating judiciously chosen particle standards within a recently proposed "fingerprinting algorithm" (Biotechnol.Bioeng. (2020) 117:3322) that allows detection of particles formed by unknown root-causes. We focus on ethylene tetrafluoroethylene (ETFE) microparticles as standard surrogates for protein aggregates. We quantify the sensitivity of the new algorithm to experimental parameters such as microscope focus and solution refractive index changes, and explore how FIM sample noise affects statistical testing procedures.Applied to real-world microscopy images of protein aggregates, the algorithm reproducibly detects complex, distinguishing "textural features" of particles that are not easily described by standard morphological measurements. This offers promise for quality control applications and for detecting shifts in protein aggregate populations due to stresses resulting from unknown process upsets.

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), and Marburg marburgvirus (MARV) are the most prevalent and pathogenic species of filovirus. Previously, we showed that glycoprotein antigens from each virus could be lyophilized to create thermostable monovalent subunit vaccines. However, cross-protection is not expected from the monovalent vaccines and therefore developing a trivalent filovirus vaccine would be desirable. Subunit protein vaccines often require the addition of an adjuvant to sufficiently boost the immunogenicity. Typically, liquid suspensions or emulsions of adjuvants and lyophilized antigens are stored in separate vials to avoid destabilizing interactions and are only mixed immediately before administration. Herein, we describe the development and characterization of monovalent and trivalent filovirus vaccines that are co-lyophilized with a squalane-in-water emulsion adjuvant. We found that the single-vial presentation retained adjuvant particle diameter and zeta potential after lyophilization and reconstitution. Furthermore, the trivalent vaccines elicited high antibody levels against all three antigens in mice and non-human primates. These results advance the prospect of developing a single-vial trivalent filovirus vaccine, which would enable easier distribution and administration of the vaccine to resource-poor areas.

Cavitation can occur when liquids are exposed to pressure waves of sufficient amplitude, producing rapidly expanding and collapsing gas bubbles that generate localized regions of high energy dissipation. When vials containing insulin were subjected to mechanical shock or when ultrasound was applied to the vials, the resulting cavitation events induced formation of insulin amyloid fibril nuclei that were detected by transmission electron microscopy and quantified by fluorescence spectroscopy following staining with the amyloid-sensitive dye thioflavin-T. Dropping insulin solutions in glass vials produced only minute amounts of insulin fibril nuclei, which could be detected by allowing the nuclei to grow. Cavitation-induced formation of amyloid aggregates may be relevant for iatrogenic insulin deposition disease, where insulin fibrils formed in vitro prior to administration to patients could serve as nuclei for growing fibril deposits in vivo.

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra, even though in solution, PEG rhIL-1ra had a lower ΔG(unf) and exhibited a more perturbed tertiary structure at the interface. Net proteinprotein interactions in solution for rhIL-1ra were attractive, but increased steric repulsion due to PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were five times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.

Therapeutic proteins are among the most widely prescribed medications, with wide distribution and complex supply chains. Shipping exposes protein formulations to stresses that can trigger aggregation, although the exact mechanism(s) responsible for aggregation are unknown. To better understand how shipping causes aggregation, we compared populations of aggregates that were formed in a polyclonal antibody formulation during live shipping studies to populations observed in accelerated stability studies designed to mimic both the sporadic high g-force and continuous low g-force stresses encountered during shipping. Additionally, we compared the effects on aggregation levels generated in two types of secondary packaging, one of which was designed to mitigate the effects of large g-force stresses. Aggregation was quantified using fluorescence intensity of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) dye, size exclusion high performance liquid chromatography (SECHPLC), and flow imaging microscopy (FIM). FIM was also combined with machine learning methods to analyze particle morphology distributions. These comparisons revealed that the morphology distributions of aggregates formed during live shipping resemble distributions that result from low g-force events, but not those observed following high g-force events, suggesting that low g-force stresses play a predominant role in shipping-induced aggregation.

Aggregation of therapeutic proteins can result from a number of stress conditions encountered during their manufacture, transportation, and storage. This work shows the effects of two interrelated sources of protein aggregation: the chemistry and structure of the surface of the container in which the protein is stored, and mechanical shocks that may result from handling of the formulation. We investigated how different mechanical stress conditions (dropping, tumbling and agitation) and container surface passivation affected the stability of solutions of intravenous immunoglobulin (IVIG). Application of mechanical shock caused cavitation to occur in the protein solution, followed by bubble collapse and the formation of high-velocity fluid microjets that impinged on container surfaces, leading to particle formation. We also observed cavitation following dropping of vials from heights as low as 5 cm and that polyethylene glycol (PEG) grafting could provide temporary protection against drop-induced cavitation. PEG treatment of the vial surface reduced the formation of protein aggregates observed after repeated dropping events, most likely by reducing protein adsorption to container surfaces. These studies enable the development of new coatings and surface chemistries that can reduce the particulate formation induced by surface adsorption or/and mechanical shock.

Cold-chain requirements affect worldwide distribution of many vaccines. In addition, vaccines requiring multiple doses impose logistical and financial burdens, as well as patient compliance barriers. To address such limitations, we have developed new technologies to prepare thermostable, single-shot, prime-boost microparticle vaccines. Antigen/adjuvant formulations containing glass-forming polymers and trehalose first are spray-dried to form glassy microparticles that confer thermostability. Atomic layer deposition (ALD) reactions conducted in fluidized beds are then used to coat the microparticles with defined numbers of molecular layers of alumina that modulate the timed release of the internalized antigen and act as adjuvants. We have used a model HPV16 L1 capsomere antigen to evaluate the properties of these technologies. Thermostabilized powders containing HPV16 L1 capsomeres were prepared by spray-drying, coated by ALD with up to 500 molecular layers of alumina, and injected into mice. Antigen distribution was assessed by live-animal IR dye tracking of injected labeled antigen. Antibody responses were measured weekly by ELISA, and neutralizing antibodies were measured by pseudovirus neutralization assays at selected time points. Thermostability was evaluated by measuring antibody responses after incubating ALD-coated antigen powders for one month at 50 °C. Single doses of the ALD-coated vaccine formulations elicited a prime-boost immune response, and produced neutralizing responses and antibody titers that were equivalent or superior to conventional prime-boost doses of liquid formulations. Antibody titers were unaffected by month-long incubation of the formulations at 50 °C. Single-dose, thermostable antigen preparations may overcome current limitations in HPV vaccine delivery as well as being widely applicable to other antigens.

The filoviruses Zaire ebolavirus (EBOV), Marburg marburgvirus (MARV), and Sudan ebolavirus (SUDV) are some of the most lethal infectious agents known. To date, the Zaire ebolavirus vaccine (ERVEBO®) is the only United States Food and Drug Administration (FDA) approved vaccine available for any species of filovirus. However, the ERVEBO® vaccine requires cold-chain storage not to exceed −60 °C. Such cold-chain requirements are difficult to maintain in low- and middle-income countries where filovirus outbreaks originate. To improve the thermostability of filovirus vaccines in order to potentially relax or eliminate these cold-chain requirements, monovalent subunit vaccines consisting of glycoproteins from EBOV, MARV, and SUDV were stabilized within amorphous disaccharide glasses through lyophilization. Lyophilized formulations and liquid controls were incubated for up to 12 weeks at 50 °C to accelerate degradation. To identify a stability-indicating assay appropriate for monitoring protein degradation and immunogenicity loss during these accelerated stability studies, filovirus glycoprotein secondary, tertiary, and quaternary structures and vaccine immunogenicity were measured. Size-exclusion chromatography was the most sensitive indicator of glycoprotein stability in the various formulations for all three filovirus immunogens. Degradation of the test vaccines during accelerated stability studies was reflected in changes in quaternary structure, which were discernible with size-exclusion chromatography. Filovirus glycoproteins in glassy lyophilized formulations retained secondary, tertiary, and quaternary protein structure over the incubation period, whereas the proteins within liquid controls both aggregated to form higher molecular weight species and dissociated from their native quaternary structure to form a variety of structurally-perturbed lower molecular weight species.

Aggregates and particles may be generated by positive displacement piston pumps during fill-finishing operations for protein formulations. We investigated potential factors that might contribute to aggregation in intravenous IgG (IVIG) formulations during pumping, including electrostatic interactions between protein molecules and pump surfaces, cavitation, and aggregate nucleation from particles shed from pumps. Electrostatic interactions were investigated by modifying pump surface chemistry. Cavitation as a potential cause of particle formation was investigated by changing pumping speeds, and the possibility that particles shed from pump surfaces act to nucleate protein aggregation was explored by spiking prepumped buffer solutions into IVIG formulations. Neither cavitation nor particles shed from pump surfaces played dominant roles in generating particles. Per pump cycle, production of particles and protein aggregates was constant, and corresponded with the amount of protein expected to adsorb on pump surfaces at monolayer coverage. More subvisible particles and protein aggregates were generated in formulations containing higher concentrations of IVIG, but they reached a plateau at protein concentrations above 2 mg/mL, where adsorption isotherms saturated. Negatively charged pump surfaces interacted with the positively charged IVIG to produce more particles and aggregates than positively charged surfaces, an effect ascribed to electrostatic interactions that moderated rates of protein adsorption.

Stable formulations of inactivated polio vaccine (IPV) could reduce cold-chain requirements and increase distribution of the vaccine to developing countries. Recently, significant improvement in thermal stability of IPV vaccines has been achieved by including urea in lyophilized formulations. In the present study, we investigated the effects of urea on recovery of potency of IPV after lyophilization and storage at 37°C and the correlation of potency recovery with key biophysical properties of IPV. By dynamic light scattering and transmission light microscopy, we found that loss of potency appeared to be due to agglomeration of virus particles during lyophilization and that moderate concentrations (e.g., 0.4 M) of urea reduced agglomeration and improved potency recovery. In addition, the relative thermal stability of the viron proteins was assessed after rehydration with temperature-dependent intrinsic fluorescence. Lyophilization of formulations without urea and postdrying storage resulted in reduced apparent melting temperatures in rehydrated samples. In formulations with urea, the rehydrated samples had thermal transitions and melting temperatures that were similar to those observed in aqueous control samples. Overall, the results indicated that in IPV formulations, urea improved potency recovery by inhibiting viron particle agglomeration and reducing denaturation of viron proteins.

The safety and efficacy of protein pharmaceuticals depend not only on biological activity but also on purity levels. Impurities may be process related because of limitations in manufacturing or product related because of protein degradation occurring throughout the life history of a product. Although the pharmaceutical biotechnology industry has made great progress in improving bulk and drug product manufacturing as well as company-controlled storage and transportation conditions to minimize the level of degradation, there is less control over the many factors that may subsequently affect product quality after the protein pharmaceuticals are released and shipped by the manufacturer. Routine handling or unintentional mishandling of therapeutic protein products may cause protein degradation that remains unnoticed but can potentially compromise the clinical safety and efficacy of the product. In this commentary, we address some potential risks associated with (mis)handling of protein pharmaceuticals after release by the manufacturer. We summarize the environmental stress factors that have been shown to cause protein degradation and that may be encountered during typical handling procedures of protein pharmaceuticals in a hospital setting or during self-administration by patients. Moreover, we provide recommendations for improvements in product handling to help ensure the quality of protein pharmaceuticals during use.

The presence of subvisible particles in formulations of therapeutic proteins is a risk factor for adverse immune responses. Although the immunogenic potential of particulate contaminants likely depends on particle structural characteristics (e.g., composition, size, and shape), exact structure-immunogenicity relationships are unknown. Images recorded by flow imaging microscopy reflect information about particle morphology, but flow microscopy is typically used to determine only particle size distributions, neglecting information on particle morphological features that may be immunologically relevant. We recently developed computational techniques that utilize the Kullback-Leibler divergence and multidimensional scaling to compare the morphological properties of particles in sets of flow microscopy images. In the current work, we combined these techniques with expectation maximization cluster analyses and used them to compare flow imaging microscopy data sets that had been collected by the U.S. Food and Drug Administration after severe adverse drug reactions (including 7 fatalities) were observed in patients who had been administered some lots of peginesatide formulations. Flow microscopy images of particle populations found in the peginesatide lots associated with severe adverse reactions in patients were readily distinguishable from images of particles in lots where severe adverse reactions did not occur.

Flow-imaging microscopy (FIM) is commonly used to characterize subvisible particles in therapeutic protein formulations. Although pharmaceutical companies often collect large repositories of FIM images of protein therapeutic products, current state-of-the-art methods for analyzing these images rely on low-dimensional lists of "morphological features" to characterize particles that ignore much of the information encoded in the existing image databases. Deep convolutional neural networks (sometimes referred to as "CNNs or ConvNets") have demonstrated the ability to extract predictive information from raw macroscopic image data without requiring the selection or specification of "morphological features" in a variety of tasks. However, the inherent heterogeneity of protein therapeutics and optical phenomena associated with subvisible FIM particle measurements introduces new challenges regarding the application of ConvNets to FIM image analysis. We demonstrate a supervised learning technique leveraging ConvNets to extract information from raw images in order to predict the process conditions or stress states (freeze-thawing, mechanical shaking, etc.) that produced a variety of different protein particles. We demonstrate that our new classifier, in combination with a "data pooling" strategy, can nearly perfectly differentiate between protein formulations in a variety of scenarios of relevance to protein therapeutics quality control and process monitoring using as few as 20 particles imaged via FIM.

We investigated the effects of protein-protein interaction strength on interfacial viscoelastic properties and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) at silicone oil-water interfaces. Osmotic second virial coefficients determined by static light scattering were used to quantify protein-protein interactions in bulk solution. Attractive protein-protein interactions dominated at low ionic strengths and their magnitude decreased with increasing ionic strength, in contrast to repulsive interactions that would be expected based on uniformly charged sphere models. Interfacial shear rheometry was used to characterize rhIL-1ra interfacial layers. More attractive protein-protein interactions in bulk solution correlated with stronger interfacial gels. Thioflavin-T fluorescence measurements indicated that the intermolecular β-sheet content of rhIL-1ra incubated in the presence of silicone oil-water interfaces correlated with gel strength. Siliconized syringes were used to probe the effects of mechanical perturbation of the interfacial gel layers. When rhIL-1ra solutions in siliconized glass syringes were subjected to end-over-end rotation, monomeric rhIL-1ra was lost from solution, and particles containing aggregated protein were released into the bulk aqueous phase. The loss of monomeric rhIL-1ra in response to mechanical perturbation was highest under the conditions where the strongest gels were observed. Aggregation of rhIL-1ra was strictly interface-induced and growth of aggregates in the bulk solution was not observed, even in the presence of particles released from silicone oil-water interfaces.

The effect of an aqueous/organic interface on the folding and aggregation of amphipathic peptides is examined by applying discontinuous molecular dynamics (DMD) simulations combined with an intermediate resolution protein model, PRIME20, to a peptide/interface system. The systems contain 48 (KLLK)4 peptides in random coil or α-helical conformations interacting with both strong and weak interfaces. In the absence of an interface, most of the oligomers form helical bundles, a small fraction of which convert to β-sheets when the temperature is above the folding transition. Adding a weak interface decreases oligomer formation above the folding temperature and increases it below. Little monolayer formation is observed at the weak interface; instead reversible adsorption increases the local peptide concentration near the interface, promoting helical bundle formation in the aqueous phase below the folding temperature and β-sheet formation above the folding temperature. Introducing a strong interface lead...

Subvisible particles in therapeutic protein formulations are an increasing manufacturing and regulatory concern because of their potential to cause adverse immune responses. Flow imaging microscopy is used extensively to detect subvisible particles and investigate product deviations, typically by comparing imaging data using histograms of particle descriptors. Such an approach discards much information and requires effort to interpret differences, which is problematic when comparing many data sets. We propose to compare imaging data using the Kullback-Leibler divergence, an information theoretic measure of the difference of distributions (Kullback S, Leibler RA. 1951. Ann Math Stat. 22:79-86). We use the divergence to generate scatter plots representing the similarity between data sets and to classify new data into previously determined categories. Our approach is multidimensional, automated, and less biased than traditional techniques. We demonstrate the method with FlowCAM® imagery of protein aggregates acquired from monoclonal antibody samples subjected to different stresses. The method succeeds in classifying aggregated samples by stress condition and, once trained, is able to identify the stress that caused aggregate formation in new samples. In addition to potentially detecting subtle incipient manufacturing faults, the method may have applications to verification of product uniformity after manufacturing changes, identification of counterfeit products, and development of closely matching bio-similar products.

Mechanical shock may cause cavitation in vials containing liquid formulations of therapeutic proteins and generate protein aggregates and other particulates. To test whether common formulation components such as protein molecules, air bubbles, or polysorbate 20 (PS20) micelles might nucleate cavitation, a high-speed video camera was used to detect cavitation in vials containing antibody formulations after application of controlled mechanical shock using a shock test. Higher concentrations of subvisible particles were found in formulations where cavitation had occurred. Bubbles trapped on vial surfaces were a primary site for cavitation nucleation; other potential cavitation nuclei were ineffective. The incidence of cavitation events observed after application of mechanical shock was lower in type I glass vials than in cyclic olefin polymer vials or in SiOPlas™ cyclic olefin polymer vials and correlated with the surface roughness of the different vials. To reduce the incidence of cavitation and the adsorption of mAb on glass-water and silicone oil-water interfaces and thus minimize protein damage due to cavitation, PS20, a common nonionic surfactant, was added to formulations. Addition of PS20 to formulations in glass and silicone oil-coated glass vials significantly reduced both incidence of mechanical shock-induced cavitation and the particle formation that resulted from cavitation events.

No United States Food and Drug Administration-licensed vaccines protective against Ebola virus (EBOV) infections are currently available. EBOV vaccine candidates currently in development, as well as most currently licensed vaccines in general, require transport and storage under a continuous cold chain in order to prevent potential decreases in product efficacy. Cold chain requirements are particularly difficult to maintain in developing countries. To improve thermostability and reduce costly cold chain requirements, a subunit protein vaccine against EBOV was formulated as a glassy solid using lyophilization. Formulations of the key antigen, Ebola glycoprotein (EBOV-GP), adjuvanted with microparticulate aluminum hydroxide were prepared in liquid and lyophilized forms, and the vaccines were incubated at 40 °C for 12 weeks. Aggregation and degradation of EBOV-GP were observed in liquid formulations during the 12-week incubation period, whereas changes were minimal in lyophilized formulations. Antibody responses against EBOV-GP following three intramuscular immunizations in BALB/c mice were used to determine vaccine immunogenicity. EBOV-GP formulations were equally immunogenic in liquid and lyophilized forms. After lyophilization and reconstitution, adjuvanted vaccine formulations produced anti-EBOV-GP IgG antibody responses in mice similar to those generated against corresponding adjuvanted liquid vaccine formulations. More importantly, antibody responses in mice injected with reconstituted lyophilized vaccine formulations that had been incubated at 40 °C for 12 weeks prior to injection indicated that vaccine immunogenicity was fully retained after high-temperature storage, showing promise for future vaccine development efforts.

The generation of nanobubbles following reconstitution of lyophilized trehalose formulations has recently been reported. Here, we characterize particle formation and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in reconstituted formulations of lyophilized trehalose. Particle characterization methods including resonant mass measurement and nanoparticle tracking analysis were used to count and size particles generated upon reconstitution of lyophilized trehalose formulations. In addition, accelerated degradation studies were conducted to monitor rhIL-1ra aggregation in solutions containing various concentrations of suspended nanobubbles. Reconstitution of lyophilized trehalose formulations with solutions containing rhIL-1ra reduced nanobubble concentrations and generated negatively buoyant particles attributed to aggregated rhIL-1ra. Furthermore, levels of rhIL-1ra aggregation following incubation in aqueous solution correlated with concentrations of suspended nanobubbles. The results of this study suggest that nanobubbles may be a contributor to protein aggregation and particle formation in reconstituted, lyophilized therapeutic protein formulations.

In this study, we investigated the effect of ionic strength (1.25-231 mM) on viscoelastic interfacial gels formed by a monoclonal antibody at silicone oil-water interfaces, and the formation of subvisible particles due to rupture of these gels. Rates of gel formation and their elastic moduli did not vary significantly with ionic strength. Likewise, during gel rupture no significant effects of ionic strength were observed on particle formation and aggregation as detected by microflow imaging, resonance mass measurement, and size exclusion chromatography. Subvisible particles formed by mechanical rupturing of the gels agglomerated over time, even during quiescent incubation, due to the colloidal instability of the particles.

Human serum albumin (HSA) is an excipient present in formulations of several recombinant protein products that are approved for clinical use. We investigated the relative contributions of HSA and HSA particles to the generation of antibody responses against recombinant human erythropoietin (rhEPO) and the excipient HSA itself. Protein samples were characterized before injection for quantities of monomeric proteins, soluble protein aggregates, and nano- and micron-sized particles. rhEPO, containing various concentrations of HSA particles, were injected three times a week for 8 weeks into mice. Hematocrits and the production of anti-rhEPO and anti-HSA antibodies were determined at various time points. Levels of antibodies against rhEPO in mice injected with HSA-containing rhEPO were higher than those in mice treated with HSA-free rhEPO. Mice injected with formulations that contained particles of HSA produced strong anti-HSA antibody responses; whereas these responses were greatly reduced when particle-free formulations were administered. In contrast, anti-rhEPO antibody responses were not affected by the presence of particles.

Protein aggregation and particle formation have been observed when protein solutions contact hydrophobic interfaces, and it has been suggested that this undesirable phenomenon may be initiated by interfacial adsorption and subsequent gelation of the protein. The addition of surfactants, such as polysorbate 20, to protein formulations has been proposed as a way to reduce protein adsorption at silicone oil-water interfaces and mitigate the production of aggregates and particles. In an accelerated stability study, monoclonal antibody formulations containing varying concentrations of polysorbate 20 were incubated and agitated in pre-filled glass syringes (PFS), exposing the protein to silicone oil-water interfaces at the siliconized syringe walls, air-water interfaces, and agitation stress. Following agitation in siliconized syringes that contained an air bubble, lower particle concentrations were measured in the surfactant-containing antibody formulations than in surfactant-free formulations. Polysorbate 20 reduced particle formation when added at concentrations above or below the critical micelle concentration (CMC). The ability of polysorbate 20 to decrease particle generation in PFS corresponded with its ability to inhibit gelation of the adsorbed protein layer, which was assessed by measuring the interfacial diffusion of individual antibody molecules at the silicone oil-water interface using total internal reflectance fluorescence (TIRF) microscopy with single-molecule tracking.

The impact of drug conjugation on intra- and intermolecular interactions of trastuzumab (TmAb) was determined by comparing the conformational and colloidal stabilities of TmAb and trastuzumab emtansine (T-DM1). In low ionic strength formulations, drug conjugation to native lysine residues of TmAb significantly reduced the repulsive electrostatic interactions between T-DM1 molecules. When these electrostatic interactions were screened in solutions with high ionic strength, intermolecular interactions between T-DM1 molecules were found to be more attractive than those between TmAb molecules. Drug conjugation lowered the colloidal stability of T-DM1 compared to TmAb, making T-DM1 more susceptible to agitation-induced aggregation. The presence of polysorbate-20 in the formulations inhibited aggregation of TmAb and T-DM1 induced by the hydrophobic air-water interface. Furthermore, the effect of increased hydrophobic interactions between T-DM1 molecules was studied by monitoring aggregation in TmAb and T-DM1 solutions that were incubated at 4°C, 25°C, and 50°C. Conjugating DM1 to TmAb increased the hydrophobicity of the molecule, and faster aggregation of T-DM1 at 50°C could be attributed to a temperature-dependent increase in hydrophobic interactions between T-DM1 molecules.

Current technologies for monitoring the subvisible particles that may be generated during fill-finish operations for protein formulations are cumbersome. Measurement times are generally too long for real-time analysis, and the high protein concentrations that are characteristic of many antibody products interfere with common optical techniques for particle analysis. To rapidly monitor protein particle levels in high-concentration protein solutions, we developed a fluorescence-based method that uses extrinsic fluorescent dyes such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid that are sensitive to the presence of aggregated protein. To test the method, antibody formulations containing various concentrations of protein particles were generated by application of various mechanical and freeze-thaw stresses. After addition of fluorescent dyes, fluorescence intensities were measured and compared to fluorescence intensities in particle-free formulations. The differences in fluorescence intensities were linearly proportional to protein particle levels, which for calibration purposes were measured offline by fluid imaging microscopy and protein assays. Protein particle levels could be measured without requiring sample dilution, even in high-concentration (e.g., 40 mg/mL) antibody formulations.

High-throughput single-molecule total internal reflection fluorescence microscopy was used to investigate the effects of pH and ionic strength on bovine serum albumin (BSA) adsorption, desorption, and interfacial diffusion at the aqueous-fused silica interface. At high pH and low ionic strength, negatively charged BSA adsorbed slowly to the negatively charged fused silica surface. At low pH and low ionic strength, where BSA was positively charged, or in solutions at higher ionic strength, adsorption was approximately 1000 times faster. Interestingly, neither surface residence times nor the interfacial diffusion coefficients of BSA were influenced by pH or ionic strength. These findings suggested that adsorption kinetics were dominated by energy barriers associated with electrostatic interactions, but once adsorbed, protein-surface interactions were dominated by short-range nonelectrostatic interactions. These results highlight the ability of single-molecule techniques to isolate elementary processes (e.g., adsorption and desorption) under steady-state conditions, which would be impossible to measure using ensemble-averaging methods.

Using high throughput single-molecule total internal reflection fluorescence microscopy (TIRFM), we have acquired molecular trajectories of bovine serum albumin (BSA) and hen egg white lysozyme during protein layer formation at the silicone oil-water interface. These trajectories were analyzed to determine the distribution of molecular diffusion coefficients, and for signatures of molecular crowding/caging, including subdiffusive motion and temporal anticorrelation of the instantaneous velocity vector. The evolution of these properties with aging time of the interface was compared with dynamic interfacial tension measurements. For both lysozyme and BSA, we observed an overall slowing of protein objects, the onset of both subdiffusive and anticorrelated motion (associated with crowding), and a decrease in the interfacial tension with aging time. For lysozyme, all of these phenomena occurred virtually simultaneously, consistent with a homogeneous model of layer formation that involves gradual crowding of weakly interacting proteins. For BSA, however, the slowing occurred first, followed by the signatures of crowding/caging, followed by a decrease in interfacial tension, consistent with a heterogeneous model of layer formation involving the formation of protein clusters. The application of microrheological methods to single molecule trajectories described here provides an unprecedented level of mechanistic interpretation of interfacial events that occurred over a wide range of interfacial protein coverage.

Silicone oil is used as a lubricant in glass pre-filled syringes (PFS) but can contribute to the generation of particles within protein formulations in PFS. To mitigate the production of such particles, various silicone oil coating processes have been proposed. In this study, three siliconization methods (the "covalent" method, the "baked silicone oil" method, and the "liquid silicone oil" method) were used to coat glass syringes with silicone oil. Glide forces were determined for syringes coated by each method. Then, a monoclonal antibody formulation or a buffer solution were incubated in the coated syringes in either the presence or absence of an air bubble, and the syringes were rotated end-over-end to induce air bubble movement within the syringe. The particle concentrations were measured throughout the incubation period using flow microscopy. The coating method did not affect particle concentrations measured in buffer alone, nor did the coating method affect particle concentrations measured in antibody formulations in the absence of an air bubble. Particle concentrations were influenced by the syringe coating method in protein formulations agitated in the presence of an air bubble, with the most particles formed in syringes lubricated with liquid silicone oil. Fewer particles were produced in syringes lubricated with baked silicone oil, and the fewest particles were produced in syringes with covalently-attached silicone oil. However, the glide forces measured in syringes coated with silicone oil by each method are inversely correlated with the measured particle concentrations.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

In this study we investigated the potential roles of nanoparticles (< 100 nm) and submicron (100-1000 nm) particles in the formation of microparticles (> 1000 nm) in protein formulations under some pharmaceutically-relevant stress conditions. Exposure of intravenous immunoglobulin (IVIG) solutions to the interface-associated stresses of freeze-thawing or agitation resulted in relatively large increases in microparticle concentrations, which depended directly on the levels of preexisting nano- and submicron particles. Thus, agglomeration of nanoparticles and submicron particles appears to play a role in microparticle formation under these stresses. In contrast, increases in microparticle concentrations during quiescent incubation at elevated temperatures were independent of the initial nano- and submicron particle concentrations in solution.

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg-white lysozyme (Lyz) and its diPEGylated (5 kDa) analog as a result of typical handling stress conditions. Both proteins were subjected to mechanical stress in the absence and presence of silicone oil (SO), elevated temperatures, and freeze-thaw cycles. Flow imaging microscopy showed that PEGylated Lyz formed approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation. Surprisingly, the PEGylated Lyz adsorbed an order of magnitude faster onto SO, despite being much larger in size, as determined by small-angle X-ray scattering and dynamic light scattering measurements. Thus, PEGylation may significantly reduce, but not prevent, micron aggregate formation of a protein during typical handling stresses.

Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60 °C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements 〈 u 2 〉 −1 for hydrogen atoms (fast β relaxation), and the relaxation time τ β , which correlates with relaxation due to fast Johari–Goldstein motions in the glass (Xu et al., 2013) [1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer–Emmet–Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid–air interface. KGF-2 degradation rates were highest in formulations wherein the protein’s structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid–air interface.

The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures. Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated. In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures. This study shows that not all protein drug aggregates are equally immunogenic.

The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of protein molecules to silicone oil-water interfaces and air-water interfaces. In addition, agitation, such as that experienced during transportation, may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study, surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed to silicone oil-water interfaces (siliconized syringe walls), air-water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 μm diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil-water-air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil-water interfaces, air-water interfaces, and agitation in the generation of particles in protein formulations.

We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 °C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's CH2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the CH2 domains leads to aggregation.