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Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution. Previous studies have suggested that they can potentially act as adjuvant resulting in potential safety risks for patients. In this paper we present several analytical case studies describing the impact of the presence of SiOPs in biotherapeutics on the analysis of the drug as well as clinical case studies examining the effect of SiOPs on patient safety. The analytical case studies demonstrate that orthogonal techniques, especially flow imaging, can help differentiate SiOPs from other types of particulate matter. The clinical case studies showed no difference in the observed patient safety profile across multiple drugs, patient populations, and routes of administration, indicating that the presence of SiOPs does not impact patient safety., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses., Methods and Results: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials., Conclusion: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP. The risk factors include ingress of hazardous materials such as oxidizing residuals from the sterilization process, delamination- or rubber stopper-derived particles, silicone oil droplets, and leachables into the formulation, as well as surface interactions between the protein and packaging materials, all of which may cause protein degradation. The type of primary packaging container used (such as vials and prefilled syringes) may substantially influence the impact of transportation and handling stresses on DP Critical Quality Attributes (CQAs). Mitigations via process development and robustness studies as well as control strategies for DP CQAs are discussed, along with current industry best practices for scale-down and in-use stability studies. We conclude that more research is needed on postproduction transportation and handling practices and their implications for protein DP quality., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Silicone oil is a lubricant for prefilled syringes (PFS), a common primary container for biotherapeutics. Silicone oil particles (SiOP) shed from PFS are a concern for patients due to their potential for increased immunogenicity and therefore also of regulatory concern. To address the safety concern in a context of manufacturing and distribution of drug product (DP), SiOP was increased (up to ∼25,000 particles/mL) in PFS filled with mAb1, a fully human antibody drug, by simulated handling of DP mimicked by drop shock. These samples are characterized in a companion report (Jiao N et al. J Pharm Sci. 2020). The risk of immunogenicity was then assessed using in vitro and in vivo immune model systems. The impact of a common DP excipient, polysorbate 80, on both the formation and biological consequences of SiOP was also tested. SiOP was found associated with (1) minimal cytokine secretion from human peripheral blood mononuclear cells, (2) no response in cell lines that report NF-κB/AP-1 signaling, and (3) no antidrug antibodies or significant cytokine production in transgenic Xeno-het mice, whether or not mAb1 or polysorbate 80 was present. These results suggest that SiOP in mAb1, representative of real-world DP in PFS, poses no increased risk of immunogenicity., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Subvisible particles (SbVPs) are a critical quality attribute for biotherapeutics. Particle content in prefilled syringes (PFSs) of a biotherapeutic can include protein particles and silicone oil particles (SiOP). Here, a real-world protein therapeutic PFS shows that although polysorbate is effective in preventing protein particle formation, it also leads to the formation of SiOP. PFSs of protein and buffer formulations in the presence and absence of polysorbate are subjected to a drop shock to generate SbVP and the effect of polysorbate and protein in generating SbVP is investigated. Particle characterization by light obscuration and flow imaging shows that polysorbate prevents protein particle formation as intended, but the presence of polysorbate substantially increases the formation of SiOP. The protein itself also acts as a surfactant and leads to increased SiOP, but to a lesser degree compared to polysorbate. In a separate companion study by Joh et al., the risk of immunogenicity was assessed using in vivo and in vitro models. Flow imaging distinguishes between SiOP and protein particles and enables risk assessment of the natures of different SbVP in PFSs., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Purpose: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO)., Methods: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins., Results: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified., Conclusion: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

The success of clinical and commercial therapeutic proteins is rapidly increasing, but their potential immunogenicity is an ongoing concern. Most of the studies that have been conducted over the past few years to examine the importance of various product-related attributes (in particular several types of aggregates and particles) and treatment regimen (such as dose, dosing schedule, and route of administration) in the development of unwanted immune responses have utilized one of a variety of mouse models. In this review, we discuss the utility and drawbacks of different mouse models that have been used for this purpose. Moreover, we summarize the lessons these models have taught us and some of the challenges they present. Finally, we provide recommendations for future research utilizing mouse models to improve our understanding of critical factors that may contribute to protein immunogenicity., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Therapeutic proteins have a propensity for aggregation during manufacturing, shipping, and storage. The presence of aggregates in protein drug products can induce adverse immune responses in patients that may affect safety and efficacy, and so it is of concern to both manufacturers and regulatory agencies. In this vein, there is a lack of understanding of the physicochemical determinants of immunological responses and a lack of standardized analytical methods to survey the molecular properties of aggregates associated with immune activation. In this review, we provide an overview of the basic immune mechanisms in the context of interactions with protein aggregates. We then critically examine the literature with emphasis on the underlying immune mechanisms as they relate to aggregate properties. Finally, we highlight the gaps in our current understanding of this issue and offer recommendations for future research., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5-10 μm in size displayed elevated cytokine release profiles compared with other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared with controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by microflow imaging, transmission electron microscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in vitro PBMC studies to rank-order the immunogenic potential of various types of mAb particles are discussed., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies. The mice were tested for their ability to break tolerance to self-tolerant monomeric antibodies. Aggregates made by mechanical stirring elicited an anti-drug antibody (ADA) response, but did not induce a robust and long-term memory B and T-cell response. Chemically modified antibodies made by oxidation were only weak and transient inducers of an immune response, as measured by a lack of both an ADA response and a B-cell antigen-specific response. Aggregate size was an important characteristic, as specific-sized protein-coated beads were able to elicit an immune response. We propose the use of this model to identify risk factors such as aggregation during manufacturing at early development for an increased potential immunogenicity risk., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1β, IL-6, IL-10, MCP-1, MIP-1α, MIP-1β, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2-10 μm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.

Comparison of protein aggregates/self-associated species between laboratories and across disciplines is complicated by the imprecise language presently used to describe them. In this commentary, we propose a standardized nomenclature and classification scheme that can be applied to describe all protein aggregates. Five categories are described under which a given aggregate may be independently classified: size, reversibility/dissociation, conformation, covalent modification, and morphology. Possible subclassifications within each category, several examples of applications of the nomenclature, and difficulties in making appropriate assignments will be discussed., (Copyright © 2011 Wiley Periodicals, Inc.)

Design of experiment and statistical analyses were applied to evaluate the effects of several formulation components on the thermal and colloidal stability for a series of monoclonal antibody (mAb) formulations. The high-throughput assessment of the protein stability was performed by measuring the temperature of hydrophobic exposure (T(h) , thermal stability) and the diffusion interaction parameter (k(D) , colloidal stability). To correlate the measured parameters with protein stability, the propensity to aggregate was tested by exposing the mAb samples to two types of stress: mechanical stress caused by shaking agitation and thermal stress. Mechanical stress led to increased formation of large particles, whereas temperature stress resulted in an increase in oligomers. The data obtained from the stress studies were used to determine the critical values for the stability parameters. The optimal formulation compositions were determined based on the statistical models and the predication tests. This approach of high-throughput thermal and colloidal stability screening can be applied to the characterization and prediction of protein formulation properties., (Copyright © 2011 Wiley-Liss, Inc.)

A host of diverse stress techniques was applied to a monoclonal antibody (IgG(2)) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q., Joubert, M. K., Stevenson, R., Narhi, L. O., and Wypych, J. (2011) J. Biol. Chem. 286, 25134-25144). Aggregates were categorized into seven discrete classes, based on the traits described. Several additional molecules (from the IgG(1) and IgG(2) subtypes as well as intravenous IgG) were stressed and found to be defined with the same classification system. The mechanism of protein aggregation and the type of aggregate formed depends on the nature of the stress applied. Different IgG molecules appear to aggregate by a similar mechanism under the same applied stress. Aggregates created by harsh mechanical stress showed the largest number of subvisible particles, and the class generated by thermal stress displayed the largest number of visible particles. Most classes showed a disruption of the higher order structure, with the degree of disorder depending on the stress process. Particles in all classes (except thermal stress) were at least partially reversible upon dilution in pH 5 buffer. High copper content was detected in isolated metal-catalyzed aggregates, a stress previously shown to produce immunogenic aggregates. In conclusion, protein aggregates can be a very heterogeneous population, whose qualities are the result of the type of stress that was experienced.

In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met(246), His(304), and His(427) in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxide-treated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.

The purpose of this study was to demonstrate the utility of combining a design of experiment (DOE) approach with high-throughput formulation screening to identify the main factors affecting protein thermostability and solution viscosity. The optimization of buffer compositions was guided by statistical analysis of the data to obtain the targeted combination of low viscosity and high thermostability. Different monoclonal antibody (mAb) formulation variables were evaluated in the study to achieve optimization of two parameters: (i) thermostability characterized by temperature of hydrophobic exposure and (ii) viscosity. High-throughput measurements were employed to characterize both parameters. The significance of each factor and the two-way interactions between them was studied by multivariable regression analysis. An experimental design was used to estimate the significance of all factors, including interaction effects. The range of optimal buffer compositions that maximized thermostability and minimized viscosity of a mAb formulation was determined. The described high-throughput methods are well suited for characterization of multiple protein formulation compositions with minimized resources such as time and material. The DOE approach can be successfully applied to the screening of mAb formulations early in the development lifecycle., (Copyright © 2010 Wiley-Liss, Inc.)

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles., ((c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Differential scanning fluorimetry (DSF) was employed to increase the throughput of the thermostability screening of monoclonal antibody (mAb) formulations. The method consists of measuring the fluorescence intensity of a polarity sensitive probe at gradually increasing temperatures, and obtaining the transition temperature of exposure of the hydrophobic regions of proteins (T(h)). The change in fluorescence intensity was directly related to protein unfolding levels and temperatures. The results from thermostability measurements were compared with the data acquired using differential scanning calorimetry (DSC), and a good correlation between T(h) and the temperature of protein unfolding or melting (T(m)) was observed. The method was applied to screen four mAb molecules in 84 different buffers. The studies revealed a good correlation of T(h) values with the known effects of pH and excipients on protein stability in solution. Specifically, the elevated aggregation levels induced by salt, low pH, and high protein concentrations could be successfully predicted by this thermal stability screening. This method is efficient, with high throughput capability, and could be widely applied in the biopharmaceutical industry for formulation and process development, and characterization., (2009 Wiley-Liss, Inc. and the American Pharmacists Association)

We propose a new method to measure the viscosity of concentrated protein solutions in a high-throughput format. This method measures the apparent hydrodynamic radius of polystyrene beads with known sizes using a dynamic light scattering (DLS) system with a microplate reader. Glycerol solution viscosities obtained by the DLS method were in good agreement with those reported in the literature. Viscosity of the solutions of two monoclonal antibody molecules was acquired using both DLS and cone-and-plate techniques, and the results were comparable. The DLS method described here has the potential to be used in many aspects of protein characterization., (Copyright 2009 Elsevier Inc. All rights reserved.)

Biologics represent a large and growing segment of the therapeutic medicinal market. Sub-visible particles present in these products are a product quality attribute and a potential patient safety concern yet to be fully explored. Early and consistent particle quantitation and control throughout the product life cycle of these drugs from development to commercial lot release is critical in mitigating any concerns. This requires appropriate analytical methods which can be applied to biopharmaceuticals across a large variety of protein concentrations and modes of administration. The compendial light obscuration method for quantitating sub-visible particles in small volume parenterals is not ideally suited for therapeutic biologics. Approaches to modify the current compendial method so that it is applicable to biologics, including appropriate sample preparation, reduced assay sample volume, increased sizing information, and development of an appropriate sampling plan, are presented in this article. Successful applications of a modified light obscuration method to therapeutic protein products are demonstrated, and a strategy to utilise complimentary methods and techniques at different phases of product development is discussed.

We studied the physical and biophysical affects of the nonionic surfactants polysorbate 20 and 80 and their mechanism of interaction using darbepoetin alfa, a 4-helix bundle protein, as the exemplary protein. Differences were observed between the abilities of the polysorbates to prevent surface loss/aggregation and correlated with each polysorbates initiation of micelle formation prior to the critical micelle concentration (CMC). The biophysical properties monitored by far-UV circular dichroism (CD) and tryptophan (Trp) fluorescence showed effects due to polysorbates, but were not correlated with their CMC. At a constant protein concentration PS-80 induced alpha-helix in the protein with a maximal effect at 15:1 molar ratio of PS-80/protein. PS-20 initially induced alpha-helix with a maximal effect at 1.5:1 ratio followed by a decrease in the alpha-helix content. PS-80 had no effect on near-UV CD but increased Trp fluorescence only at the 150:1 polysorbate/protein ratio. PS-20 decreased the near-UV CD and Trp fluorescence. Thermodynamic studies by isothermal titration calorimetry (ITC) demonstrated that the protein interacts with monomeric polysorbate, but not with polysorbate micelles. The data suggest that the polysorbates differentially interact with the protein and that the biophysical effects are dependent on the structure of the polysorbate and the polysorbate to protein ratio.

The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

Purpose: The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies., Methods: Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG(2) mAbs. The C(H)2 domain melting temperature (T (m1)) and reduced valence (z*) of the mAbs were measured., Results: Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F(-) < Cl(-) < Br(-) < I(-) < SCN(-) ~ ClO(4) (-)) and correlated with a decrease in T (m1) and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li(+), Na(+), K(+), Rb(+), or Cs(+)) had no effect on T (m1), z* or aggregation upon agitation., Conclusions: The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution.

Purpose: Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions., Methods: The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.7-5.0), salt concentration (0-400 mM), and ion identity using a series of monovalent, Hofmeister anions (F(-), Cl(-), Br(-), I(-), ClO(4) (-), SCN(-)) and cations (Li+, Na+, K+, Rb+, Cs+). The effects of salt on conformational stability and reduced valence were determined using Fourier-transform infrared spectroscopy, circular dichroism, and capillary electrophoresis/analytical ultracentrifugation., Results: PbA precipitation occurred upon salt addition and could be modulated with solution pH, salt identity & concentration. The precipitation was sensitive to anions, but not cations, and increased with anion size. A reverse Hofmeister effect (SCN(-) approximately ClO(4) (-)>I(-)>Cl(-)>Br(-)>F(-)) was observed with "salting-in" anions being the more effective precipitants. An increase in the precipitation rate below pH 4.3 indicated that protonation of aspartyl and glutamyl side-chains was also important for precipitation. The reversibility of precipitation was excellent (100%) at 4 degrees C but decreased upon storage at 25 degrees C and 37 degrees C; the loss in reversibility correlated with an increase in intermolecular beta-sheet content of the precipitate., Conclusion: Salts, employed as buffering, tonicifying, and viscosity modifying agents, may adversely affect the solubility of basic proteins formulated under acidic conditions.

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D(H)) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s(w) and D(H), reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D(H) and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M(z)) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I(-) > Br(-) > Cl(-) > F(-)) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxxCTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.

Intraneuronal deposition of alpha-synuclein as fibrils and oxidative stress are both implicated in the pathogenesis of Parkinson's disease. We found that the critical rate-limiting step in nucleation of alpha-synuclein fibrils under physiological conditions is the oxidative formation and accumulation of a dimeric, dityrosine cross-linked prenucleus. Dimer formation is accelerated for the pathogenic A30P and A53T mutant alpha-synucleins, because of their greater propensity to self-interact, which is reflected in the smaller values of the osmotic second virial coefficient compared to that of wild-type synuclein. Our finding that oxidation is an essential step in alpha-synuclein aggregation supports a mechanism of Parkinson's disease pathogenesis in which the separately studied pathogenic factors of oxidative stress and alpha-synuclein aggregation converge at the critical step of alpha-synuclein dimer formation.

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of proteins. It exists as a covalent dimer in solution, with the 15 kDa monomers linked by an interchain disulfide bond through the Cys101 residues. Sedimentation equilibrium and velocity experiments demonstrated that, after removal of the interchain disulfide bond, GDNF remains as a non-covalent dimer and is stable at pH 7.0. To investigate the effect of the intermolecular disulfide on the structure and stability of GDNF, we compared the solution structures of the wild-type protein and a cysteine-101 to alanine (C101A) mutant using Fourier transform infrared (FTIR), FT-Raman and circular dichroism (CD) spectroscopy and sedimentation analysis. The elimination of the intermolecular disulfide bond causes only minor changes (approximately 4%) in the secondary structures of GDNF. The far- and near-UV CD spectra demonstrated that the secondary and tertiary structures were similar for both wild-type and C101A GDNF. Heparin binding and sedimentation velocity experiments also indicated that the folded structure of the wild-type and C101A GDNF are indistinguishable. The thermal stability of GDNF does not appear to be affected by the absence of the interchain disulfide bond and the biological activity of the C101A mutant is identical with that of the wild-type protein. However, small but significant changes in side chain conformations of tyrosine and aliphatic residues were observed by FT-Raman spectroscopy upon removal of the intermolecular disulfide bond, which may reflect structural changes in the area of dimeric contact. By comparing the Raman spectrum of wild-type GDNF with that of the C101A analog, we identified the conformation of the intermolecular disulfide as trans-gauche-trans geometry. These results indicate that GDNF is an active, properly folded molecule in the absence of the interchain disulfide bond.

Purpose: BDNF, a noncovalent homodimer, was modified by covalently attaching polyethylene glycol (PEG) with an average molecular weight of 20kDa to the N-terminal methionine. Stability of modified BDNF (PEG-BDNF) in aqueous solution was compared to BDNF after storage at elevated temperature in the presence and absence of NaCl., Methods: SDS-PAGE. Light Scattering and Size Exclusion Chromatography were used to assess conformational stability and chemical degradation. In addition, CD spectroscopy was used to follow changes in secondary and tertiary structures upon thermal stress of the protein., Results: NaCl containing formulations are more stable than NaCl-free formulations. In NaCl-free formulations, the main degradation product of BDNF and PEG-BDNF had a molecular weight of monomer that was more chemically degraded than the dimer. Additionally, the degradation of PEG-BDNF occurred at an accelerated rate compared to BDNF in NaCl-free environments., Conclusions: The addition of NaCl to formulations enhances the shelf-life and conformational stability of both BDNF and PEG-BDNF.

Erythropoietin (EPO) derived from Escherichia coli is unstable to elevated temperature and tends to aggregate with time, making it unsuitable for high-resolution structure analysis. The mammalian EPO contains about 40% carbohydrate, which makes this protein more stable and less prone to aggregate than non-glycosylated E.coli-derived EPO, but makes it unsuitable for high-resolution analysis owing to its size and flexibility. In an attempt to decrease the aggregation of E.coli-derived EPO, the three asparagine residues at positions 24, 38 and 83 were mutated to lysine residues. In the native protein, these residues are the sites of N-linked glycosylation, which suggests that they should be located on the surface of the protein and should not be involved in interactions in the hydrophobic protein core. Therefore, the substitution of basic amino acids for these neutral asparagine residues is not expected to affect the protein structure, but should increase the isoelectric point of the protein and its net positive charge, decreasing its tendency to aggregate at or below neutral pH due to electrostatic interactions. No apparent alterations in receptor binding, as determined by both cell-surface receptor competition assay and in vitro receptor dimerization experiments, were observed when these mutations were introduced into the EPO sequence. However, this mutant protein displayed a significant increase in stability to heat treatment and to storage, relative to the wild-type molecule. This resulted in a greater number of observable cross peaks in the mutant EPO in 2D NOESY experiments. However, the mutant was similar to the wild-type in stability when urea was used as a denaturant. This indicates that the introduced mutations resulted in a decrease in aggregation with heating or with prolonged incubation at ambient temperature, without changing the conformational stability or the receptor binding affinity of the mutant protein. This approach of placing charged residues at sites where N-glycosylation occurs in vivo could be applied to other systems as well.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.

Interferon regulatory factor 4 (IRF-4), an intracellular, multidomain protein, is a member of the interferon regulatory factor family and a lymphoid-specific transcription factor that can form a ternary complex with DNA and the transcription factor PU.1. Recombinant human IRF-4 was expressed in Escherichia coli and purified from the soluble cell extract and the insoluble inclusion bodies. The inclusion bodies were solubilized with guanidinium-hydrochloride and sequentially buffer exchanged into urea- and then NaCl-containing solutions. This two-step process for the removal of the denaturants was the critical step to allow for the correct folding of IRF-4. Following purification through immobilized metal affinity, hydrophobic interaction, and gel permeation chromatographies, the renatured protein was shown to be structurally and physically equivalent to a sample of IRF-4 produced in the soluble fraction of E. coli cells. This was confirmed by near and far UV circular dichroism analysis, including thermal stability analysis. The purified IRF-4 was also shown to be capable of binding DNA in a PU.1-dependent manner by electrophoretic mobility shift analysis. The protein folding and purification methods are suitable for producing large quantities of full-length IRF-4., (Copyright 1999 Academic Press.)

Purpose: The present study was performed to examine the effect of solution conditions on the reversibility of the thermal denaturation of megakaryocyte growth and development factor (rHuMGDF)., Methods: Changes in the far UV CD spectra of rHuMGDF with temperature were used to monitor the thermal denaturation of the protein, and the recovery of folded protein following a return to room temperature. The effect of protein concentration, scan rate, and buffer composition on thermal denaturation and on the reversibility were determined. Surface tension measurements were used to determine the effect of this unfolding reaction on the surface adsorption of the protein. Sedimentation velocity was used to assess recovery of native monomer and the size of soluble aggregates. In addition, monomeric protein remaining in solution after incubation at 37 degrees C for 2 weeks in either 10 mM imidazole of 10 mM phosphate was determined., Results: In phosphate buffer the rHuMGDF irreversibly precipitates upon unfolding under all the conditions examined. In imidazole the unfolding is at least partially reversible, with no visible precipitate seen; the degree of reversibility increased by lowering both protein and salt concentrations, and the amount of time spent at elevated temperature. In order to compare thermal unfolding occuring with different degrees of reversibility, the melting temperature was defined as the temperature at which melting begins. The melting temperature itself is relatively independent of the buffer composition, or experimental conditions. At low protein concentrations the protein stabilizer sucrose had a marginal effect on the thermal transition of rHuMGDF, while at protein concentrations of about 2 mg/ml the inclusion of sucrose increased the apparent melting temperature by about 4 degrees C, to that seen at low protein concentrations, but had little effect on the reversibility of denaturation. Inclusion of 1 or 2 M urea did not affect the reaction. Surface tension measurements of rHuMGDF solutions showed little difference before and after melting, and in the presence or absence of sucrose. When unfolding is irreversible, the MGDF appears to form soluble aggregates of tetramers to 14-mers, while under reversible conditions native monomer is recovered. More monomeric MGDF remained in solution following storage for 2 weeks at 37 degrees C in imidazole than in phosphate, in both the presence and absence of sucrose., Conclusions: These results can be explained by assuming that thermal denaturation proceeds as a two-step reaction, the first step being the equilibrium between folded and unfolded states, while the second step is a slow irreversible aggregation. The different buffer systems affect the rate of the aggregation step, but not the intrinsic thermal stability nor the rate of the unfolding step.

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.

Chemical modification and mutagenesis of methionines in recombinant human granulocyte colony-stimulating factor (G-CSF) were investigated. Selective oxidation of G-CSF by H2O2 and t-butyl hydroperoxide leads to generation of different oxidized forms. Four modified forms were isolated and shown to contain 1 to 4 oxidized methionyl residues. All methionines in G-CSF are reactive, with reaction kinetics following the order of Met1>Met138>Met127>>>Met122. H2O2 oxidation of Met122 is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met127. All oxidized forms retain gross G-CSF conformation similar to that of the native molecule and are able to bind the soluble G-CSF receptor. However, G-CSF form oxidized at both Met127 and Met122 is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met1-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met138 is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met127 is less accessible to solvent and Met122 is near the hydrophobic core. Oxidation at both Met127 and Met122 results in alterations of G-CSF structure that affect the apparent molecular size, polarity, and stability and lead to the loss of G-CSF biological function. G-CSF variants with Leu replacement at Met127 or at Met138 are not completely resistant to oxidation-induced inactivation, while the variant with Leu replacement at both sites is more stable and can retain in vitro biological activity following oxidation., (Copyright 1999 Academic Press.)

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties., (Copyright 1999 Academic Press.)

The agouti-related protein gene (Agrp) is a novel gene implicated in the control of feeding behavior. The hypothalamic expression of Agrp is regulated by leptin, and overexpression of Agrp in transgenic animals results in obesity and diabetes. By analogy with the known actions of agouti, these data suggest a role for the Agrp gene product in the regulation of melanocortin receptors expressed in the central nervous system. The availability of recombinant, highly purified protein is required to fully address this potential interaction. A nearly full-length form of AGRP (MKd5-AGRP) was expressed in the cytosolic or soluble fraction of Escherichia coli and appeared as large intermolecular disulfide-bonded aggregates. Following oxidation, refolding, and purification, this protein was soluble, and eluted as a single symmetric peak on RP-HPLC. Circular dichroism studies indicated that the purified protein contains primarily random coil and beta-sheet secondary structure. Sedimentation velocity studies at neutral pH demonstrated that MKd5-AGRP is monomeric at low micromolar concentrations. Mobility shifts observed using SDS-PAGE under reducing and nonreducing conditions for bacterially expressed and mammalian expressed AGRP were identical, an indication of a similar disulfide structure. The purification to homogeneity of a second, truncated form of AGRP (Md65-AGRP) which was expressed in the insoluble or inclusion body fraction is also described. Both forms act as competitive antagonists of alpha-melanocyte stimulating hormone (alpha-MSH) at melanocortin-3 (MC-3) and melanocortin-4 receptors (MC-4). The demonstration that AGRP is an endogenous antagonist with respect to these receptors is a unique mechanism within the central nervous system, and has important implications in the control of feeding.

The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.

The effect of pH and urea on the conformation of recombinant human megakaryocyte growth and development factor (rHuMGDF) was determined by circular dichroism, intrinsic fluorescence spectroscopy, and equilibrium ultracentrifugation. The conformation of rHuMGDF was dependent on pH and urea concentration. Multiple folding forms were evidenced by multiple pH-induced transitions and urea-induced equilibrium transitions that deviated from a simple two-state process. In neutral to alkaline pH, rHuMGDF exists as a monomer, but an acid-induced conformational state self-associates to form a soluble aggregate. A folding intermediate(s) was observed with a more stable secondary structure than tertiary structure and was dependent on the pH of the urea-induced denaturation. The differences in the stabilities of the folding states were most distinct in the pH range of 4.5 to 6.5. The presence of intermediates in the folding pathway of rHuMGDF are similar to findings of previous studies of related growth factors that share a common three-dimensional structure.

A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.

Stem cell factor (SCF) is thought to be a member of the four-helical bundle cytokine superfamily, and exists in solution as a noncovalent homodimer. It is the ligand for Kit, a tyrosine kinase type III receptor. The interaction of SCF and Kit affects early hematopoietic progenitors, as well as gametocytes, melanocytes, and mast cells. Upon binding of SCF the Kit undergoes dimerization and transphosphorylation. Circular dichroism (CD), intrinsic fluorescence, and Fourier transform infrared (FTIR) spectroscopy were used for conformational analyses of free SCF, soluble Kit (sKit), and the complex. The sKit consisted of the extracellular domain of Kit, contained five Ig-like domains, and was prepared from the conditioned media of transfected Chinese hamster ovary cells. With these techniques, a reproducible conformational change was seen upon ligand/receptor binding. The far-UV CD and FTIR spectroscopy indicated a slight increase in the alpha-helical content. The near-UV CD and fluorescence spectra showed changes in the environments of the aromatic amino acids. The thermal denaturation of SCF was not affected by complex formation, while the melting temperature of sKit increased only a few degrees when binding SCF. This indicates that binding is temperature dependent, consistent with titration calorimetry results published previously which demonstrated that there is a large enthalpy of binding. The conformational changes which accompany SCF/sKit binding could play a role in the receptor dimerization and signal transduction which follow.

Purpose: To investigate the role and importance of the four methionines in recombinant human leptin, and the effect of methionine oxidation in leptin structural stability and biological activity., Methods: Oxidized leptin derivatives were prepared in the presence of H2O2 and t-butylhydroperoxide, separated by RP-HPLC, and characterized by peptide mapping and LC/MS. Their biophysical and biological properties were studied., Results: Six major species of oxidized leptins were detected: two mono-oxidized, one di-oxidized, two tri-oxidized, and one tetra-oxidized. Further oxidation at cystine disulfide was also detected. Kinetic analysis indicated that oxidation at Met1 and Met69 proceeded first and independently. In 48 mM t-butylhydroperoxide, the pseudo first-order rate constants, k1 and k69, were 1.5 x 10(-3) and 2.3 x 10(-4) min-1. No change in the secondary or tertiary structure was detected for Met1 mono-oxidized and Met1, Met69 di-oxidized leptins. The Met1 mono-oxidized leptin retained full potency as compared to native leptin. A slight decrease of thermostability and a significant loss of the in vitro bioactivity were observed for Met1, Met69 di-oxidized leptin. Both Met55 and Met137 were not oxidized in t-butylhydroperoxide but only in H2O2. They appeared to be much less accessible to oxidation and might interact with the hydrophobic core structure of the leptin molecule., Conclusions: The oxidation of leptin occurred in the order of Met1 > Met69 >> Met55 approximately Met137, and the importance for maintaining leptin structural integrity was Met55 approximately Met137 >> Met69 approximately Met1. Met69, but not Met1, plays a critical role in the protein stability and activity.