Assessment of change in the basic variants composition of trastuzumab during dilution in saline for administration.

Postproduction handling of drug products during preparation or clinical use may affect the structure and efficacy of the drug and perhaps remain unnoticed. Since chemical modifications can impact the product's structure, stability, and biological activity, this study investigates the impact of elevated temperature and subtle shift in pH on the drug product post-dilution in saline. The mAb sample diluted in saline for administration was stressed at elevated temperature and slightly acidic pH condition. Extended stability studies were performed and monitored for size and charge heterogeneity. Size heterogeneity shows no significant changes, whereas charge heterogeneity shows an increase in basic variants and a reduction in main species. Further, basic variants were isolated and characterized to identify the type and site of chemical modification. Intact mass analysis and peptide mapping identify that the basic variants were attributed mainly to the isomerization of HC Asp102 into iso-Asp or its succinimide intermediate. Four basic variants were found to exhibit similar structural properties as the main and control samples. However, basic variants showed reduced binding affinity to HER2 receptor, while there was no significant difference in FcRn binding. The results indicate that modification in the HC Asp102, which is present in the CDR, affects antigen binding and thus can influence the potency of the drug product. Hence, with the conventional stability studies required to license the drug product, including in-use or extended stability studies to mimic the postproduction handling would be desirable.

Near UV Photodegradation Mechanisms of Amino Acid Excipients: Formation of the Carbon Dioxide Radical Anion from Aspartate and Fe(III).

Carbon dioxide radical anion (•CO2-) is a powerful reducing agent that can reduce protein disulfide bonds and convert molecular oxygen to superoxide. Therefore, the generation of •CO2- can be detrimental to pharmaceutical formulations. Iron is among the most prevalent impurities in formulations, where Fe(III) chelates of histidine (His) can produce •CO2- upon exposure to near-UV light (Zhang and Schöneich, Eur. J. Pharm. Biopharm. 2023, 190, 231-241). Here, we monitor by spin-trapping in combination with electron paramagnetic resonance spectroscopy and/or high-performance liquid chromatography-mass spectrometry analysis the photochemical formation of •CO2- for a series of common amino acid excipients, including arginine (Arg), methionine (Met), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), and lysine (Lys). Our results indicate that in the presence of Fe(III), Asp, and Glu produce significant yields of •CO2- under photoirradiation with near-UV light. Notably, Asp demonstrates the highest efficiency of •CO2- generation compared with that of the other amino acid excipients. Stable isotope labeling indicates that •CO2- exclusively originates from the α-carboxyl group of Asp. Mechanistic studies reveal two possible pathways for •CO2- formation, which involve either a ß-carboxyl radical or an amino radical cation intermediate.

Molecular Dynamics Simulation of an Iron(III) Binding Site on the Fc Domain of IgG1 Relevant for Visible Light-Induced Protein Fragmentation.

Molecular dynamics simulations were employed to investigate the interaction between Fe(III) and an iron-binding site composed of THR259, ASP252, and GLU261 on the Fc domain of an IgG1. The goal was to provide microscopic mechanistic information for the photochemical, iron-dependent site-specific oxidative fragmentation of IgG1 at THR259 reported in our previous paper. The distance between Fe(III) and residues of interest as well as the binding pocket size was examined for both protonated and deprotonated THR259. The Fe(III) binding free energy (ΔG) was estimated by using an umbrella sampling approach. The pKa shift of the THR259 hydroxyl group caused by the presence of nearby Fe(III) was estimated based on a thermodynamic cycle. The simulation results show that Fe(III) resides inside the proposed binding pocket and profoundly changes the pocket configuration. The ΔG values indicate that the pocket possesses a strong binding affinity for Fe(III). Furthermore, Fe(III) profoundly lowers the pKa value of the THR259 hydroxyl group by 5.4 pKa units.

Protection against Aß-induced neuronal damage by KU-32: PDHK1 inhibition as important target.

A feature of most neurodegenerative diseases is the presence of "mis-folded proteins" that form aggregates, suggesting suboptimal activity of neuronal molecular chaperones. Heat shock protein 90 (Hsp90) is the master regulator of cell responses to "proteotoxic" stresses. Some Hsp90 modulators activate cascades leading to upregulation of additional chaperones. Novobiocin is a modulator at the C-terminal ATP-binding site of Hsp90. Of several novobiocin analogs synthesized and tested for protection against amyloid beta (Aß)-induced neuronal death, "KU-32" was the most potent in protecting primary neurons, but did not increase expression of other chaperones believed to help clear misfolded proteins. However, KU-32 reversed Aß-induced superoxide formation, activated Complex I of the electron transfer chain in mitochondria, and blocked the Aß-induced inhibition of Complex I in neuroblastoma cells. A mechanism for these effects of KU-32 on mitochondrial metabolism appeared to be the inhibition of pyruvate dehydrogenase kinase (PDHK), both in isolated brain mitochondria and in SH-SY5Y cells. PDHK inhibition by the classic enzyme inhibitor, dichloroacetate, led to neuroprotection from Aß25-35-induced cell injury similarly to KU-32. Inhibition of PDHK in neurons would lead to activation of the PDH complex, increased acetyl-CoA generation, stimulation of the tricarboxylic acid cycle and Complex I in the electron transfer chain, and enhanced oxidative phosphorylation. A focus of future studies may be on the potential value of PDHK as a target in AD therapy.

Near UV light photo-degradation of histidine buffer: Mechanisms and role of Fe(III).

Pharmaceutical formulations are sensitive to light-induced degradation. Recent studies have attributed some of the light sensitivity to the presence of Fe(III), the most prevalent metal leachable from pharmaceutical containers. Histidine (His) can promote Fe(III) leaching from stainless steel, especially at elevated storage temperatures. Since there is the chance that combinations of His and Fe(III) are present in pharmaceutical formulations, we investigated the photo-degradation mechanisms of Fe(III)-containing His buffer during expsoure to near UV light. Our results indicate the formation of carbon dioxide radical anion (•CO2-), a powerful reductant, and other photoproducts such as aldehydes and His-derived radicals. The generation of •CO2- can be promoted by increasing concentrations of Fe(III) and inhibited by the addition of the Fe(III) chelator EDTA. Mechanistically, product formation can be rationalized by photo-induced ligand-to-metal-charge-transfer (LMCT), followed by a series of radical transformations of reaction intermediates.

Photo-induced site-specific oxidative fragmentation of IgG1 mediated by iron(III)-containing histidine buffer: Mechanistic studies and excipient effects.

Fragmentation may compromise the clinical efficacy and safety profile of monoclonal antibodies (mAbs). We recently reported that Fe(III)-containing histidine (His) buffer mediates site-specific mAb fragmentation within the Fc domain when exposed to visible light (Y. Zhang and C. Schöneich, Mol. Pharm. 2023, 20, 650-662). Here, we show that this fragmentation proceeds even more efficiently under near-UV light. Several formulation strategies were applied in an attempt to reduce the photo-induced fragmentation. In solution formulations, the fragmentation can be mitigated by reducing the concentration of His buffer, adding Fe(III)-chelating agents, and replacing His with other amino acids. Fragmentation can be almost completely inhibited by formulating the protein in the lyophilized state. Mechanistically, His plays a critical role in the fragmentation process, likely due to its affinity for Fe(II), driving a photo-redox reaction towards product formation.

Primary Processes of Free Radical Formation in Pharmaceutical Formulations of Therapeutic Proteins.

Oxidation represents a major pathway for the chemical degradation of pharmaceutical formulations. Few specific details are available on the mechanisms that trigger oxidation reactions in these formulations, specifically with respect to the formation of free radicals. Hence, these mechanisms must be formulated based on information on impurities and stress factors resulting from manufacturing, transportation and storage. In more detail, this article focusses on autoxidation, metal-catalyzed oxidation, photo-degradation and radicals generated from cavitation as a result of mechanical stress. Emphasis is placed on probable rather than theoretically possible pathways.

Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review.

Over the past few decades, there has been a tremendous increase in the utilization of therapeutic peptides. Therapeutic peptides are usually administered via the parenteral route, requiring an aqueous formulation. Unfortunately, peptides are often unstable in aqueous solutions, affecting stability and bioactivity. Although a stable and dry formulation for reconstitution might be designed, from a pharmaco-economic and practical convenience point of view, a peptide formulation in an aqueous liquid form is preferred. Designing formulation strategies that optimize peptide stability may improve bioavailability and increase therapeutic efficacy. This literature review provides an overview of various degradation pathways and formulation strategies to stabilize therapeutic peptides in aqueous solutions. First, we introduce the major peptide stability issues in liquid formulations and the degradation mechanisms. Then, we present a variety of known strategies to inhibit or slow down peptide degradation. Overall, the most practical approaches to peptide stabilization are pH optimization and selecting the appropriate type of buffer. Other practical strategies to reduce peptide degradation rates in solution are the application of co-solvency, air exclusion, viscosity enhancement, PEGylation, and using polyol excipients.

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody.

Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metal-catalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

Visible Light Induces Site-Specific Oxidative Heavy Chain Fragmentation of a Monoclonal Antibody (IgG1) Mediated by an Iron(III)-Containing Histidine Buffer.

Fragmentation of therapeutic monoclonal antibodies represents a critical quality attribute. Here, we report a novel visible light-induced heavy chain fragmentation of IgG1 mediated by an Fe(III)-containing histidine (His) buffer. Based on non-reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis and mass spectrometry analysis, IgG1 fragments with apparent molecular weights of ∼130, ∼110, and ∼22 kDa were detected in photo-irradiated samples and were mechanistically rationalized with an oxidative cleavage at Thr259. Specifically, the reactions are proposed to involve the generation of an intermediary alkoxyl radical, which undergoes ß-cleavage to yield a glycyl radical. The latter either converts into Gly or adds oxygen and follows a peroxyl radical chemistry. The cleavage process requires the presence of His, while only negligible yields of cleavage products are formed when His is replaced by acetate, succinate, or phosphate buffer. Importantly, the fragmentation can be prevented by ethylenediaminetetraacetic acid (EDTA) only when the EDTA concentrations are in significant excess over the concentrations of Fe(III) and proteins, suggesting a strong binding between Fe(III) and IgG1.

The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case.

The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles.

Near-UV and Visible Light Degradation of Iron (III)-Containing Citrate Buffer: Formation of Carbon Dioxide Radical Anion via Fragmentation of a Sterically Hindered Alkoxyl Radical.

Citrate is a commonly used buffer in pharmaceutical formulations which forms complexes with adventitious metals such as Fe3+. Fe3+-citrate complexes can act as potent photosensitizers under near-UV and visible light exposure, and recent studies reported evidence for the photo-production of a powerful reductant, carbon dioxide radical anion (•CO2-), from Fe3+-citrate complexes (Subelzu, N.; Schöneich, N., Mol. Pharm. 2020, 17, 4163-4179). The mechanisms of •CO2- formation are currently unknown but must be established to devise strategies against •CO2- formation in pharmaceutical formulations which rely on the use of citrate buffer. In this study, we first established complementary evidence for the photolytic generation of •CO2- from Fe3+-citrate through spin trapping and electron paramagnetic resonance (EPR) spectroscopy, and subsequently used spin trapping in conjunction with tandem mass spectrometry (MS/MS) for mechanistic studies on the pathways of •CO2- formation. Experiments with stable isotope-labeled citrate suggest that the central carboxylate group of citrate is the major source of •CO2-. Competition studies with various inhibitors (alcohols and dimethyl sulfoxide) reveal two mechanisms of •CO2- formation, where one pathway involves ß-cleavage of a sterically hindered alkoxyl radical generated from the hydroxyl group of citrate.

Impact of various factors on the kinetics of non-enzymatic fragmentation of a monoclonal antibody.

Non-enzymatic hinge fragmentation of monoclonal antibodies (mAb) is considered a critical quality attribute since it changes the primary sequence of the proteins, thereby leading to structural changes which can affect stability, function, and efficacy. While peptide bonds are exceptionally stable under physiological conditions, reactive side chains of a few residues, the flexibility of the backbone, and physicochemical parameters such as pH, temperature, and the reaction of radicals and metal ions can promote the cleavage of peptide bonds. In this study, the relative extent and rate of fragmentation are compared with respect to the presence of several different factors (including hydrogen peroxide, metal ion, and temperature) as measured by size exclusion chromatography. A kinetic model of monomer degradation as a function of time (mAb only) is created. In the presence of either H2O2 or Cu2+, or both, the reaction kinetics follow different orders depending on the reaction conditions. The half-life for peptide bond cleavage of the mAb hinge region was 385 days at 40 °C and decreases to 250, 48, and 45 days in the presence of H2O2, Cu2+, and a combination of H2O2 and Cu2+, respectively. A temperature dependence of peptide bond cleavage at 35 °C, 40 °C, 45 °C, and 50 °C showed Arrhenius behavior with an apparent activation energy of 76.9 ± 16.4 kJ/mol. The reaction rates obtained from the Arrhenius equation were then extrapolated to predict fragmentation rates under real storage conditions (e.g., at 2-8 °C). We demonstrate that trace levels of impurities including peroxide left after surface sterilization or degradation of non-ionic surfactants or metal ions from the buffer components can significantly affect the stability of a mAb.

Advanced Oxidation Processes in Pharmaceutical Formulations: Photo-Fenton Degradation of Peptides and Proteins.

Formulations of therapeutic proteins are sensitive to photo-degradation by near UV and visible light. Mechanistically, especially the processes leading to protein modification under visible light exposure are not understood. Potentially, these processes may be triggered by a ligand to metal charge transfer in excipient-metal complexes. This article summarizes recent analytical and mechanistic work on such reactions under experimental conditions relevant to pharmaceutical formulations.

Intra-Micellar and Extra-Micellar Oxidation in Phosphate and Histidine Buffers Containing Polysorbate 80.

Polysorbate is a key excipient included in formulations of therapeutic proteins to help prevent aggregation and surface adsorption. The stability of both polysorbate and therapeutic proteins can be compromised by oxidative degradation. In general, polysorbate is added to formulations at concentrations above the critical micelle concentration (cmc). To date, however, few experiments have quantitatively addressed the extent of extra- and intra-micellar oxidation of polysorbate in pharmaceutically relevant buffers. This study utilizes 2,2'-azobis(2-methylpropionamidine)dihydrochloride (AAPH), a peroxyl radical-generating initiator, C11-BODIPY(581/591), a lipid peroxidation probe, and fluorescence spectroscopy to reveal that both intra- and extra-micellar oxidation proceed in pharmaceutically relevant phosphate and histidine buffers. It is further demonstrated that the relative extent of oxidation observed in the intra- and extra-micellar compartments is similar irrespective of the buffer system.

Near UV and Visible Light Photo-Degradation Mechanisms in Citrate Buffer: One-Electron Reduction of Peptide and Protein Disulfides promotes Oxidation and Cis/Trans Isomerization of Unsaturated Fatty Acids of Polysorbate 80.

Citrate is a common buffer for slightly acidic pH ranges of protein formulations. In the presence of iron, citrate buffer undergoes photo-degradation induced by near UV and visible light. Recent studies (Subelzu and Schöneich, Mol. Pharm. 2020, 17, 4163-4179) have documented that such photo-degradation results in the formation of carbon dioxide radical anion (•CO2-), a strong reductant which reduces Fe3+, O2, and disulfide bonds. In the present study we show that near UV and visible light photo-degradation of citrate in the presence of iron can induce reductive peptide and protein disulfide cleavage as well as free radical damage of a surfactant, polysorbate 80 (PS80). Reductive disulfide cleavage is most likely caused by efficient electron transfer from carbon dioxide radical anions to disulfides, resulting in the generation of thiol/thiolate and thiyl radicals. The latter can react with mono- and polyunsaturated fatty acids of PS80 to cause cis/trans isomerization and/or oxidation. Representative products generated by cis/trans isomerization and oxidation of oleic acid esters have been detected by HPLC-MS analysis. Further evidence for reductive disulfide cleavage was obtained through the analysis of free thiols. The oxidation of PS80 can also be the consequence of reactive oxygen species (ROS) generation through the reduction of O2 by carbon dioxide radical anions and/or intermediary Fe2+.

Oxidation and Deamidation of Monoclonal Antibody Products: Potential Impact on Stability, Biological Activity, and Efficacy.

The role in human health of therapeutic proteins in general, and monoclonal antibodies (mAbs) in particular, has been significant and is continuously evolving. A considerable amount of time and resources are invested first in mAb product development and then in clinical examination of the product. Physical and chemical degradation can occur during manufacturing, processing, storage, handling, and administration. Therapeutic proteins may undergo various chemical degradation processes, including oxidation, deamidation, isomerization, hydrolysis, deglycosylation, racemization, disulfide bond breakage and formation, Maillard reaction, and ß-elimination. Oxidation and deamidation are the most common chemical degradation processes of mAbs, which may result in changes in physical properties, such as hydrophobicity, charge, secondary or/and tertiary structure, and may lower the thermodynamic or kinetic barrier to unfold. This may predispose the product to aggregation and other chemical modifications, which can alter the binding affinity, half-life, and efficacy of the product. This review summarizes major findings from the past decade on the impact of oxidation and deamidation on the stability, biological activity, and efficacy of mAb products. Mechanisms of action, influencing factors, characterization tools, clinical impact, and risk mitigation strategies have been addressed.

Analysis of N15-rat growth hormone after incubation with rat subcutaneous tissue and immune cells using ultra-pressure chromatography-mass spectrometry.

Therapeutic proteins (TPs) are exposed to various immune cells like macrophages and neutrophils, especially after subcutaneous (SC) administration. It is well known that the immune cells can generate reactive oxygen species (ROS) and this may lead to oxidation of TPs. The oxidation can occur in the SC tissue after SC administration, during distribution to the immune organs like lymph nodes and spleen, and even in the blood circulation. The oxidation can lead to alteration of their pharmacokinetics and efficacy. Therefore, it is important to study the oxidation of TPs in the biological matrices using ultra-pressure chromatography-mass spectrometry. Rat growth hormone (rGH) was selected as a test protein due to its similarity with human growth hormone (hGH), which is widely used for treatment of growth hormone deficiency. In this manuscript, we have summarized sample processing strategy and ultra-pressure chromatography-mass spectrometry methodology to identify rGH and its degradation products after ex-vivo incubation with rat SC tissue, and in vitro incubation with rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as a model foreign host species. We did not observe oxidation of rGH in these biological matrices. This could be due to very minor yields of oxidation products, lack of sensitivity of the mass spectrometry method, loss of protein during sample processing, rapid turnover of oxidized protein or a combination of all factors.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Pharmaceutical Excipients Enhance Iron-Dependent Photo-Degradation in Pharmaceutical Buffers by near UV and Visible Light: Tyrosine Modification by Reactions of the Antioxidant Methionine in Citrate Buffer.

PURPOSE: To evaluate the effect of excipients, including sugars and amino acids, on photo-degradation reactions in pharmaceutical buffers induced by near UV and visible light. METHODS: Solutions of citrate or acetate buffers, containing 1 or 50 µM Fe3+, the model peptides methionine enkephalin (MEn), leucine enkephalin (LEn) or proctolin peptide (ProP), in the presence of commonly used amino acids or sugars, were photo-irradiated with near UV or visible light. The oxidation products were analyzed by reverse-phase HPLC and HPLC-MS/MS. RESULTS: The sugars mannitol, sucrose and trehalose, and the amino acids Arg, Lys, and His significantly promote the oxidation of peptide Met to peptide Met sulfoxide. These excipients do not increase the yields of hydrogen peroxide, suggesting that other oxidants such as peroxyl radicals are responsible for the oxidation of peptide Met. The addition of free Met reduces the oxidation of peptide Met, but, in citrate buffer, causes the addition of Met oxidation products to Tyr residues of the target peptides. CONCLUSIONS: Commonly used excipients enhance the light-induced oxidation of amino acids in model peptides.