Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays.

Propranolol is a widely used ß-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.

Regulatory Experiences with Root Causes and Risk Factors for Nitrosamine Impurities in Pharmaceuticals.

N-Nitrosamines (also referred to as nitrosamines) are a class of substances, many of which are highly potent mutagenic agents which have been classified as probable human carcinogens. Nitrosamine impurities have been a concern within the pharmaceutical industry and by regulatory authorities worldwide since June 2018, when regulators were informed of the presence of N-nitrosodimethylamine (NDMA) in the angiotensin-II receptor blocker (ARB) medicine, valsartan.  Since that time, regulatory authorities have collaborated to share information and knowledge on issues related to nitrosamines with a goal of promoting convergence on technical issues and reducing and mitigating patient exposure to harmful nitrosamine impurities in human drug products. This paper shares current scientific information from a quality perspective on risk factors and potential root causes for nitrosamine impurities, as well as recommendations for risk mitigation and control strategies.

Comprehensive N-Glycan Mapping using Parallel Reaction Monitoring LC-MS/MS.

PURPOSE: Glycan composition can impact a biotherapeutic's safety and efficacy. For example, changes in the relative abundance of different glycan attributes like afucosylation, galactosylation or high-mannose content can change the properties or functions of a monoclonal antibody (mAb). While established methods can effectively characterize major glycan species in biotherapeutic drug products, there is still a need for more sensitive and specific methods that can effectively monitor low abundance species which may impact mAb function. METHODS: Glycans released from two mAbs, adalimumab and trastuzumab, were derivatized with Rapifluor-MS™. Glycans were separated using HILIC and detected using either fluorescence (FLD) or mass spectrometry (MS). A parallel reaction monitoring (PRM) workflow was used for the MS analysis. RESULTS AND CONCLUSION: FLD analysis identified 18 and 19 glycan peaks in adalimumab and trastuzumab, respectively. Glycan identities were determined using MS-analysis and a high number of FLD peaks containing co-eluting glycan species were observed. PRM analysis quantified 38 and 39 glycan species in adalimumab and trastuzumab, respectively, and the increase in glycans that could be identified was due to superior sensitivity and selectivity compared to FLD. Notably, many low abundance glycans identified by PRM included species that were not reported in other studies. PRM also offered several additional advantages; unique structural features could be identified using the collected MS/MS spectra and de-coupling MS acquisition and data processing simplified the transfer of methods between instruments. The results established PRM as a precise, informative tool for glycan analysis and quantitation.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems. In this study, we employed human lymphoblastoid TK6 cells transduced with human cytochrome P450 (CYP) 2A6 to evaluate the genotoxicity of six nitrosamines that have been identified as impurities in drug products: N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA). Using flow cytometry-based assays, we found that 24-h treatment with NDEA, NEIPA, NMBA, and NMPA caused concentration-dependent increases in the phosphorylation of histone H2A.X (γH2A.X) in CYP2A6-expressing TK6 cells. Metabolism of these four nitrosamines by CYP2A6 also caused significant increases in micronucleus frequency as well as G2/M phase cell-cycle arrest. In addition, nuclear P53 activation was found in CYP2A6-expressing TK6 cells exposed to NDEA, NEIPA, and NMPA. Overall, the genotoxic potency of the six nitrosamine impurities in our test system was NMPA > NDEA ≈ NEIPA > NMBA > NDBA ≈ NDIPA. This study provides new information on the genotoxic potential of nitrosamines in human cells, complementing test results generated from traditional assays and partially addressing the issue of the relevance of nitrosamine genotoxicity for humans. The metabolically competent human cell system reported here may be a useful model for risk assessment of nitrosamine impurities found in drugs.

Multiphase Drug Distribution and Exchange in Oil-in-Water Nanoemulsion Revealed by High-Resolution 19F qNMR.

An oil-in-water (o/w) nanoemulsion (NE), composed of oil globules, stabilized by a surfactant, and dispersed in an aqueous phase, is increasingly developed in complex drug formulation. Kinetically stable NEs are used to formulate hydrophobic drugs and typically provide higher dosage strengths and better content uniformity. However, little is known accurately about drug distribution in its multiphase solution, especially for the possible drug presence in the surfactant (s) phase, the interface layer between the dispersed oil (o) and the continuous water (w) phases. Here, high-resolution 19F quantitative NMR spectroscopy was applied directly and noninvasively on an o/w NE drug product containing difluprednate (DFPN). The well-resolved 19F peaks of DFPN depended on the shielding molecules in each phase, which revealed mass-balanced DFPN distribution in multiple phases of (w), (s), and (o) of NE globules at a quantity of 1.8 ± 0.1, 35 ± 2, and 59 ± 3% per labeled content, respectively. Furthermore, the dilution-dependent 19F peak line broadening and shift suggested a millisecond dynamic exchange between the NE and the less-noticed smaller but thermodynamically stable microemulsion (ME) globules in NE solution. The high-resolution NMR result revealed that the drug availability could be quickly achieved using an o/w NE formulation because of the drug multiphase distribution and the ME-assisted fast drug exchange among globules.

International Regulatory Collaboration on the Analysis of Nitrosamines in Metformin-Containing Medicines.

Recalls of some batches of metformin have occurred due to the detection of N-nitrosodimethylamine (NDMA) in amounts above the acceptable intake (AI) of 96 ng per day. Prior to the recalls, an international regulatory laboratory network had been monitoring drugs for nitrosamine impurities with each laboratory independently developing and validating multiple analytical procedures to detect and measure nitrosamines in metformin drugs used in their jurisdictions. Here, we provide an overview of the analysis of metformin active pharmaceutical ingredients (APIs) and drug products with 1090 samples (875 finished dosage forms (FDFs) and 215 API samples) tested beginning in November of 2019 through July of 2020. Samples were obtained internationally by a variety of approaches, including purchased, received from firms via information requests or selected by regional regulatory authorities (either at wholesalers or during GMP inspections). Only one nitrosamine (NDMA) was detected and was only present in some batches of metformin products. For API samples, 213 out of 215 lots tested had no measurable level of NDMA. For FDF samples tested, the number of batches with NDMA above the AI amount for patient safety was 17.8% (156/875). Based on these data, although the presence of NDMA was of concern, 82.2% of the samples of metformin drug products tested met quality and safety standards for patients. Regulatory agencies continue to collaborate extensively and work with marketing authorization holders to understand root causes of nitrosamine formation and agree on corrective actions to mitigate the presence of NDMA in future metformin batches.

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

In Vitro Analysis of N-Nitrosodimethylamine (NDMA) Formation From Ranitidine Under Simulated Gastrointestinal Conditions.

Importance: A publication reported that N-nitrosodimethylamine (NDMA), a probable human carcinogen, was formed when ranitidine and nitrite were added to simulated gastric fluid. However, the nitrite concentrations used were greater than the range detected in acidic gastric fluid in prior clinical studies. Objective: To characterize NDMA formation following the addition of ranitidine to simulated gastric fluid using combinations of fluid volume, pH levels, and nitrite concentrations, including physiologic levels. Design, Setting, and Participants: One 150-mg ranitidine tablet was added to 50 or 250 mL of simulated gastric fluid with a range of nitrite concentrations from the upper range of physiologic (100 µmol/L) to higher concentrations (10 000 µmol/L) with a range of pH levels. NDMA amounts were assessed with a liquid chromatography-mass spectrometry method. Main Outcomes and Measures: NDMA detected in simulated gastric fluid 2 hours after adding ranitidine. Results: At a supraphysiologic nitrite concentration (ie, 10 000 µmol/L), the mean (SD) amount of NDMA detected in 50 mL simulated gastric fluid 2 hours after adding ranitidine increased from 222 (12) ng at pH 5 to 11 822 (434) ng at pH 1.2. Subsequent experiments with 50 mL of simulated gastric fluid at pH 1.2 with no added nitrite detected a mean (SD) of 22 (2) ng of NDMA, which is the background amount present in the ranitidine tablets. Similarly, at the upper range of physiologic nitrite (ie, 100 µmol/L) or at nitrite concentrations as much as 50-fold greater (1000 or 5000 µmol/L) only background mean (SD) amounts of NDMA were observed (21 [3] ng, 24 [2] ng, or 24 [3] ng, respectively). With 250 mL of simulated gastric fluid, no NDMA was detected at the upper physiologic range (100 µmol/L) or 10-fold physiologic (1000 µmol/L) nitrite concentrations, while NDMA was detected (mean [SD] level, 7353 [183] ng) at a 50-fold physiologic nitrite concentration (5000 µmol/L). Conclusions and Relevance: In this in vitro study of ranitidine tablets added to simulated gastric fluid with different nitrite concentrations, ranitidine conversion to NDMA was not detected until nitrite was 5000 µmol/L, which is 50-fold greater than the upper range of physiologic gastric nitrite concentrations at acidic pH. These findings suggest that ranitidine is not converted to NDMA in gastric fluid at physiologic conditions.

A Real-Time NMR Method for Measurement of In Vitro Aggregation Kinetics Of Degarelix Drug Products.

Degarelix is a gonadotropin-releasing hormone (GnRH) receptor antagonist. Upon contact with physiological fluid, degarelix undergoes quick gelation and forms a depot at the site of injection providing sustained release. The molecular gelling kinetics is a critical physiochemical quality attribute of degarelix products that may impact drug delivery. However, high-resolution and drug substance (DS)-specific analytical methods for characterizing gelling kinetics of degarelix are still lacking. Accordingly, the current study focused on developing NMR-based methods to characterize in vitro initial aggregation of degarelix in Firmagon® drug product (DP). The high-precision real-time NMR method was demonstrated to quickly differentiate lot to lot differences in degarelix aggregation kinetics, and to reveal the effects of degarelix concentration, pH, salt, and temperature on the kinetics. The results could be useful for quality assurance of degarelix products and facilitate complex generic drug development. The real-time NMR method developed here could also be adopted to other complex DPs that have varied aggregation and release properties.

Minor N-Glycan Mapping of Monoclonal Antibody Therapeutics Using Middle-Down NMR Spectroscopy.

The N-glycosylation pattern of Asn-297 may have impacts on monoclonal antibody (mAb) drug plasma clearance, antibody-dependent cell mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Notably, the changes in the relative abundance of certain minor glycans, like the afucosylation, high-mannose, or galactosylation are known to change mAb properties and functions. Here, a middle-down NMR spectroscopy based analytical procedure was applied to assess the composition and structure of glycans on adalimumab and trastuzumab without glycan cleavage from the mAbs. The anomeric 2D 1H-13C spectra showed distinct patterns that could be used to profile and differentiate mAb glycan compositions. Specifically, the anomeric C1/H1 resonances from N-acetylglucosamine (GlcNAc2 and -5) and mannose (Man4) were identified as characteristic peaks for key glycan anomeric linkages and branching states. They were also utilized for measuring the relative abundance of minor glycans of total afucosylation (aFuc%), high mannose (HM%), and branch specific galactosylation (Gal1-3% and Gal1-6%). The obtained total aFuc% value of 11-12% was similar between the two mAbs; however, trastuzumab had significantly lower level of high mannose and a higher level of galactosylation than adalimumab. Overall, the 2D-NMR measurements provided functionally relevant mAb glycan composition and structure information. The method was deemed fit-for-purpose for assessment of these mAb quality attributes and involved fewer chemical preparation steps than the classical approaches that cleave glycans prior to making measurements.

Sedimentation Velocity Analytical Ultracentrifugation Analysis of Marketed Rituximab Drug Product Size Distribution.

PURPOSE: Analytical methods suitable for intact drug products are often necessary to evaluate the equivalence in physicochemical properties between two drug products (DP) containing the same drug substance (DS), e.g., an innovator biologic drug and its proposed biosimilar. Analytical Ultracentrifugation (AUC) is a biophysics technique applied to the analysis of size and shape of biomolecules. However, the application of AUC to formulated monoclonal antibody (mAb) DP at high concentration has not been reported. METHODS: A sedimentation velocity (SV) AUC procedure with a short-pathlength centerpiece was applied to two marketed rituximab DPs, Rituxan® (US) and Reditux® (India), without any buffer exchange or dilution. Detailed precision analysis was performed. RESULTS: Highly reproducible sedimentation coefficient values (S) and peak areas were obtained for the dominant (> 84%) monomeric rituximab peak. The minor mAb fragment peaks had large variation in both S values and peak areas (3-12%). The identification of oligomer peaks was only reproducible once the abundance was higher than 2%. CONCLUSIONS: SV-AUC provides an orthogonal characterization tool for protein size distribution, composition and assay, which could be informative for biosimilar drug developers who mostly only have access to formulated mAb. However, AUC needs thorough validation on its accuracy, precision and sensitivity.

A Cautionary Tale: Quantitative LC-HRMS Analytical Procedures for the Analysis of N-Nitrosodimethylamine in Metformin.

A private testing laboratory reported in a Citizen Petition (CP) to FDA that 16 of 38 metformin drug products they tested had N-nitrosodimethyl amine (NDMA) amounts above the allowable intake (AI) of 96 ng/day. Because the FDA had been monitoring drugs for nitrosamines, orthogonal analytical procedures had been developed, validated and applied to detect the following nitrosamines in metformin drug products (if present): (i) NDMA (with a dedicated method) or (ii) NDMA (with a second confirmatory method), N-nitroso-diethylamine (NDEA), N-ethyl-N-nitroso-2-propanamine (NEIPA), N-nitroso-diisopropylamine (NDIPA), N-nitroso-di-n-propylamine (NDPA), N-nitroso-methylphenylamine (NMPA), N-nitroso-di-n-butylamine (NDBA) and N-nitroso-N-methyl-4-aminobutyric acid (NMBA). In contrast to the private laboratory results, FDA testing on the same set of 38 samples with orthogonal procedures observed amounts over the AI in only 8 of the 38 products and generally observed lower values than reported by the private testing laboratory. As described here, the investigation into the cause of the discrepancy revealed that N,N-dimethylformamide (DMF) can interfere with NDMA measurements. The data showed that the use of sufficient mass accuracy in the data acquisition and appropriate mass tolerance setting in the data processing to assure the selectivity of mass spectrometry measurements of NDMA in the presence of co-eluting DMF was necessary to prevent overestimation of the level of NDMA in metformin drug products. Overall, care should be taken to assure the necessary specificity in analytical procedures for adequate assessment of the nitrosamine level in drug products that also contain DMF or other potential interfering substances.

Multiplexed Comparative Analysis of Intact Glycopeptides Using Electron-Transfer Dissociation and Synchronous Precursor Selection Based Triple-Stage Mass Spectrometry.

A recently developed synchronous precursor selection (SPS) mass spectrometry to the third (MS3) protocol enables more accurate multiplexed quantification of proteins/peptides using tandem mass tags (TMT) through comparison of reporter ion intensities at the MS3 level. However, challenges still exist for TMT-based simultaneous quantification and identification of intact glycopeptides due to inefficient peptide backbone fragmentation when using collision-induced dissociation (CID). To overcome this limitation, here we report an improved SPS/ETD workflow for TMT-based intact glycopeptide quantification and identification. The SPS/ETD approach was implemented on an Orbitrap Tribrid mass spectrometer and begins with selection of a parent ion in the MS scan, followed by tandem mass spectrometry (MS2) fragmentation by CID in the ion trap. Following MS2 fragmentation, SPS enables simultaneous isolation of the top 10 MS2 fragment ions for further higher energy collisional dissociation (HCD) fragmentation with the resulting MS3 fragments detected in an Orbitrap analyzer. Here, in addition to the standard SPS workflow, an electron-transfer dissociation (ETD) MS2 was performed and analyzed in the ion trap. The resultant ETD and CID spectra were used for the identification of the intact glycopeptides, while the quantitative comparison of site-specific glycans was achieved utilizing TMT reporter ions from HCD MS3 spectra. For intact glycopeptides, through systematic optimization and evaluation using a glycoprotein interference model, the SPS/ETD approach was demonstrated to offer improved accuracy, precision, and sensitivity compared to traditional data-dependent MS2 quantification, while maintaining the glycopeptide identification capability. Finally, this workflow was applied for the site-specific quantitative comparison of the glycoforms for two therapeutic enzymes (Cerezyme and VPRIV) and their different lots. The results demonstrate that this workflow is suitable for TMT-based intact glycopeptide characterization of glycoproteins.

An NMR Protocol for In Vitro Paclitaxel Release from an Albumin-Bound Nanoparticle Formulation.

The paclitaxel protein-bound particles for injectable suspension (marketed under the brand name Abraxane®) contains nanosized complexes of paclitaxel and albumin. The molecular interaction between paclitaxel and albumin within the higher-order nanostructure is analytically challenging to assess, as is any correlation of differences to differences in therapeutic effect. However, because the higher-order nanostructures may affect the paclitaxel release, a suitable in vitro assay to detect potential differences in paclitaxel release between comparator lots and products is desirable. Herein, solution NMR spectroscopy with a T2-filtering technique was developed to detect paclitaxel signal while suppressing albumin signals to follow the released paclitaxel in the NMR tube upon dilution. The non-invasive nature of NMR allows for precise measurement of a full range of dilution-induced drug release percentage from 14 to 92% without any sample extraction. The critical concentration of the drug product (DP) at 50% of release was 0.63 ± 0.04 mg/mL in PBS buffer. In addition, 2D diffusion ordered NMR spectroscopy (DOSY) results revealed that the released paclitaxel experiencing slightly slowed diffusion rates than free paclitaxel, which was attributed to paclitaxel in equilibrium with albumin-bound states. Collectively, the dilution-based NMR method offered an analytical approach to investigate physicochemical attributes of complex injectable products with minimal needed sample preparation and perturbation to nanoparticle formulation.

An NMR-Based Similarity Metric for Higher Order Structure Quality Assessment Among U.S. Marketed Insulin Therapeutics.

Protein or peptide higher order structure (HOS) is a quality attribute that could affect therapeutic efficacy and safety. Where appropriate, the HOS similarity between a proposed follow-on product and the reference listed drug should be demonstrated during regulatory assessment. Establishing quantitative HOS similarity for 2 drug substances, manufactured by different processes, has been challenging. Herein, HOS differences among U.S. marketed insulin drug products (DPs) were quantified using nuclear magnetic resonance spectra and principal component analysis (PCA). Then, the unitless Mahalanobis distance (DM) in PCA space was calculated between insulin analog reference listed drugs and their recently approved follow-on products, and all DM values were 3.29 or less. By contrast, a larger DM value of 20.5 was obtained between the 2 insulin human DPs independently approved. However, upon mass-balanced and reversible dialysis of the 2 insulin human DPs against the same buffers, the DM value was reduced to 1.19 or less. Thus, the observed range of nuclear magnetic resonance-PCA-derived DM values can be used as a robust and sensitive measure of HOS similarity. Overall, the DM values of 3.3 for DP and 1.2 for drug substances using insulin therapeutics represented realistic and achievable similarity metrics for developing generic or biosimilar drugs, quality assurance, or control.

Raman mapping of fentanyl transdermal delivery systems with off-label modifications.

Raman mapping is a powerful and emerging tool in characterization of pharmaceuticals and provides non-destructive chemical and structural identification with minimal sample preparation. One pharmaceutical form that is suitable but has not been studied in-depth with Raman mapping is transdermal delivery systems (TDS). TDS are dosage forms designed to deliver a therapeutically effective amount of active pharmaceutical ingredient (API) across a patient's skin. To enhance drug delivery through the skin, the API in the formulation is often close to a saturated or supersaturated state. Thus, improper use or off-label modifications can lead to occurrence of unwanted API changes, specifically, crystallization over time. Here, off-label modifications were mimicked on a set of fentanyl drug-in-adhesive TDS sold on the U.S. market by four different manufacturers via die cutting, and then the die cut TDS were investigated through confocal Raman mapping for structural and chemical changes. Using Multivariate Curve Resolution (MCR), not only was morphological and chemical characterization of transdermal systems provided, but also fentanyl crystals in certain products due to off-label modifications were identified. The chemometric model used in analysis of Raman maps allowed precise identification of fentanyl as the crystalline material as confirmed by the hit-quality-index correlation of component spectra from the chemometric model with library spectra of a fentanyl reference standard. The results show that confocal Raman mapping with MCR can be utilized in assessing pharmaceutical quality of TDS. This method has the potential to be widely used in characterization of such systems as an alternative to existing techniques.

1D and 2D-HSQC NMR: Two Methods to Distinguish and Characterize Heparin From Different Animal and Tissue Sources.

The US Food and Drug Administration has encouraged the reintroduction of bovine heparin drug product to the US market to mitigate the risks of heparin shortages and potential adulteration or contamination of the primary source which is porcine heparin. Here, a 1D-NMR method was applied to compare heparin sodium of bovine intestinal origin with that of bovine lung, porcine, or ovine intestinal origin. The results showed that a simple 1D test using NMR signal intensity ratios among diagnostic signals of the proton spectra uniquely identified the origin of heparin and concomitantly could be used to assure the correct sample labeling. However, a limitation of the use of only mono-dimensional spectra is that these spectra may not provide sufficiently detailed information on the composition of heparin batches to adequately determine the quality of this complex product. As an alternative, a higher resolution quantitative 2D-HSQC method was used to calculate the percentage of mono- and disaccharides, distinguish the origin of heparin and, simultaneously, assess the heparin composition. The 2D-HSQC method is proposed to provide sufficient information to evaluate the quality of industrial production process used to make the drug substance. Together, the 1D and 2D data produced by these measurements can be used to assure the identity and purity of this widely used drug.

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry.

Protein therapeutic higher order structure (HOS) is a quality attribute that can be assessed to help predict shelf life. To model product shelf-life values, possible sample-dependent pathways of degradation that may affect drug efficacy or safety need to be evaluated. As changes in drug thermal stability over time can be correlated with an increased risk of HOS perturbations, the effect of long-term storage on the product should be measured as a function of temperature. Here, complementary high-resolution mass spectrometry methods for HOS analysis were used to identify storage-dependent changes of biotherapeutics (bevacizumab (Avastin), trastuzumab (Herceptin), rituximab (Rituxan), and the NIST reference material 8671 (NISTmAb)) under accelerated or manufacturer-recommended storage conditions. Collision-induced unfolding ion mobility-mass spectrometry data showed changes in monoclonal antibody folded stability profiles that were consistent with the appearance of a characteristic unfolded population. Orthogonal hydrogen-deuterium exchange-mass spectrometry data revealed that the observed changes in unfolding occurred in parallel to changes in HOS localized to the periphery of the hinge region. Using intact reverse-phase liquid chromatography-mass spectrometry, we identified several mass species indicative of peptide backbone hydrolysis, located between the variable and constant domains of the heavy chain of bevacizumab. Taken together, our data highlighted the capability of these approaches to identify age- or temperature-dependent changes in biotherapeutic HOS.