Olivier Mozziconacci

Do Not Drop: Mechanical Shock in Vials Causes Cavitation, Protein Aggregation, and Particle Formation

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

UV photodegradation of murine growth hormone: Chemical analysis and immunogenicity consequences

During manufacturing, therapeutic proteins may be exposed to ultraviolet (UV) radiation. Such exposure is of concern because UV radiation may cause photooxidative damage to proteins, which in turn could lead to physical changes such as aggregation and enhanced immunogenicity. We exposed murine growth hormone (mGH) to controlled doses of UV radiation, and examined the resulting chemical, physical and immunogenic changes in the protein. mGH chemical structure was analyzed by mass spectrometry after UV irradiation. Photooxidation products detected by mass spectrometry included methionine sulfoxide formed at Met[127] and Met[149] residues, and, tentatively assigned by MS/MS analysis, ether cross-links between original Ser[78] and Cys[188], and Cys[206] and Ser[213], and a thioether cross-link between Cys[17] and Cys[78] residues, transformation of Cys[189] into Ala, and various hydrolytic fragments. Physical damage to UV-irradiated mGH was monitored by infrared spectrometry, chromatographic analyses, and particle counting by micro-flow imaging. UV radiation caused mGH to aggregate, forming insoluble microparticles containing mGH with non-native secondary structure. When administered subcutaneously to Balb/c or Nude Balb/c mice, UV-irradiated mGH provoked antibodies that cross-reacted with unmodified mGH in a fashion consistent with a T-cell dependent immune response. In wildtype Balb/c mice, titers for anti-mGH IgG1 antibodies increased with increasing UV radiation doses.

Comparative Evaluation of the Chemical Stability of 4 Well-Defined Immunoglobulin G1-Fc Glycoforms

As part of a series of articles in this special issue evaluating model IgG1-Fc glycoforms for biosimilarity analysis, 3 well-defined IgG1-Fc glycoforms (high mannose-Fc, Man5-Fc, and N-acetylglucosamine-Fc) and a nonglycosylated Fc protein (N297Q-Fc) were examined in this work to elucidate chemical degradation pathways. The 4 proteins underwent a combination of accelerated thermal stability studies and 4 independent forced degradation studies (UV light, metal-catalyzed oxidation, peroxyl radicals, and hydrogen peroxide) at pH 6.0. Our results highlight chemical degradations at Asn315, Met428, Trp277, and Trp313. A cross-comparison of the different Fc glycoforms, stress conditions, and the observed chemical reactions revealed that both the deamidation of Asn315 and the transformation of Trp277 into glycine hydroperoxide were glycan dependent during incubation for 3 months at 40 °C. Our data will show that different glycans not only affect chemical degradation differently but also do lead to different impurity profiles, which can affect chemical degradation.

Degradation Mechanisms of Polysorbate 20 Differentiated by (18)O-labeling and Mass Spectrometry.

ABSTRACTPurposeTo investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways.MethodsUltra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20.ResultsThe analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via “acyl-cleavage” of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation.ConclusionsThe 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

Fluorogenic Tagging Methodology Applied to Characterize Oxidized Tyrosine and Phenylalanine in an Immunoglobulin Monoclonal Antibody

PurposeMetal-catalyzed oxidation (MCO) of proteins is of primary concern in the development of biotherapeutics as it represents a prominent degradation pathway with potential undesired biological and biotherapeutic consequences.MethodsWe developed a fluorogenic derivatization methodology to study the MCO of IgG1 using a model oxidation system, CuCl2/L-ascorbic acid.ResultsBesides the oxidation of Met, Trp and His residues, we detected significant oxidation of Phe and Tyr in IgG1.ConclusionThe fluorogenic derivatization method provides an alternative approach for the rapid detection of oxidized Tyr and Phe as their benzoxazole derivatives by fluorescence spectrometry and size exclusion chromatography coupled to fluorescence detection.

Photolysis of Recombinant Human Insulin in the Solid State: Formation of a Dithiohemiacetal Product at the C-Terminal Disulfide Bond

ABSTRACTPurposeExposure of protein pharmaceuticals to light can result in chemical and physical modifications, potentially leading to loss of potency, aggregation, and/or immunogenicity. To correlate these potential consequences with molecular changes, the nature of photoproducts and their mechanisms of formation must be characterized. The present study focuses on the photochemical degradation of insulin in the solid state.MethodsSolid insulin was characterized by solid-state NMR, polarized optical microscopy and scanning electron microscopy; various insulin preparations were exposed to UV light prior to product analysis by mass spectrometry.ResultsUV-exposure of solid human insulin results in photodissociation of the C-terminal intrachain disulfide bond, leading to formation of a CysS• thiyl radical pair which ultimately disproportionates into thiol and thioaldehyde species. The high reactivity of the thioaldehyde and proximity to the thiol allow the formation of a dithiohemiacetal structure. Dithiohemiacetal is formed during the UV-exposure of both crystalline and amorphous insulin.ConclusionsDithiohemiacetals represent novel structures generated through the photochemical modification of disulfide bonds. This is the first time that such structure is identified during the photolysis of a protein in the solid state.

Chemical degradation of proteins in the solid state with a focus on photochemical reactions.

Protein pharmaceuticals comprise an increasing fraction of marketed products but the limited solution stability of proteins requires considerable research effort to prepare stable formulations. An alternative is solid formulation, as proteins in the solid state are thermodynamically less susceptible to degradation. Nevertheless, within the time of storage a large panel of kinetically controlled degradation reactions can occur such as, e.g., hydrolysis reactions, the formation of diketopiperazine, condensation and aggregation reactions. These mechanisms of degradation in protein solids are relatively well covered by the literature. Considerably less is known about oxidative and photochemical reactions of solid proteins. This review will provide an overview over photolytic and non-photolytic degradation reactions, and specially emphasize mechanistic details on how solid structure may affect the interaction of protein solids with light.

Photodegradation of Oxytocin and Thermal Stability of Photoproducts

Photodegradation of oxytocin at λ = 253.7 nm and λ ≥ 290 nm results in the transformation of the intrachain disulfide bond into predominantly dithiohemiacetal and thioether. Especially the dithiohemiacetal is sensitive to further degradation by light and/or elevated temperature, implying that the combination of an initial photostress and a subsequent heat stress can yield products significantly different compared with those observed under heat stress only.

The Botanical Drug Substance Crofelemer as a Model System for Comparative Characterization of Complex Mixture Drugs

Crofelemer is a botanical polymeric proanthocyanidin that inhibits chloride channel activity and is used clinically for treating HIV-associated secretory diarrhea. Crofelemer lots may exhibit significant physicochemical variation due to the natural source of the raw material. A variety of physical, chemical, and biological assays were used to identify potential critical quality attributes (CQAs) of crofelemer, which may be useful in characterizing differently sourced and processed drug products. Crofelemer drug substance was extracted from tablets of one commercial drug product lot, fractionated, and subjected to accelerated thermal degradation studies to produce derivative lots with variations in chemical and physical composition potentially representative of manufacturing and raw material variation. Liquid chromatography, UV absorbance spectroscopy, mass spectrometry, and nuclear magnetic resonance analysis revealed substantial changes in the composition of derivative lots. A chloride channel inhibition cell-based bioassay suggested that substantial changes in crofelemer composition did not necessarily result in major changes to bioactivity. In 2 companion papers, machine learning and data mining approaches were applied to the analytical and biological data sets presented herein, along with chemical stability data sets derived from forced degradation studies, to develop an integrated mathematical model that can identify CQAs which are most relevant in distinguishing between different populations of crofelemer.

Site-Specific Hydrolysis Reaction C-Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG-1

The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).