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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Chemical Structure and Composition of Major Glycans Covalently Linked to Therapeutic Monoclonal Antibodies by Middle-Down Nuclear Magnetic Resonance.

Glycosylation of monoclonal antibodies (mAbs) is a critical quality attribute that can impact mAb drug efficacy and safety. The mAb glycans are inherently heterogeneous in chemical structure and composition of monosaccharides. The established fluorescence or mass-spectrometry (MS) detection methods for glycosylation evaluation may require multiple steps of glycan cleavage or extensive digestion of the mAb, chemical labeling of the glycans, column separation and report the chemical identity of glycans indirectly through retention time and molecular weight values. In demonstrating chemical structure similarity and comparability among mAb drugs, orthogonal analytical methods for measuring glycan chemistry are needed to ensure the quality of drug products. Here, a "middle-down" NMR method is developed as a proof-of-concept approach to measure the domain-specific glycosylation of marketed mAb drugs without cleavage of the glycan moieties. Complete glycan 1H/13C chemical shift assignments were obtained at 13C natural abundance from commercial standard glycans that allowed unambiguous determination of the chemical structure, glycosidic linkage position, and anomeric configuration of each monosaccharide in the major N-glycan scaffolds found in mAb molecules. The analysis of glycan anomeric peaks in two-dimensional (2D) 1H-13C NMR spectra yielded metrics for clinically important mAb quality attributes (i.e., galactosylation (Gal%) and fucosylation (Fuc%)), consistent with literature results using a standard glycan-mapping method. Therefore, the middle-down NMR method provided a facile orthogonal measurement for mAb glycosylation characterization with improved chemical information content on glycan structure determination and quantification, compared to standard approaches.

Application of Ultra-Centrifugation and Bench-Top 19F NMR for Measuring Drug Phase Partitioning for the Ophthalmic Oil-in-Water Emulsion Products.

Generic drug products are expected to have the same active pharmaceutical ingredient (API) (Q1) with the same content (Q2) and microstructure arrangement (Q3) as the innovator product. In complex oil-in-water emulsion drugs, the hydrophobic API is mainly formulated in oil droplets stabilized by surfactant and micelles composed of extra surfactant molecules. The API phase partition in oil and water (mainly micelle) is a critical quality attribute (CQA) of emulsion product in demonstrating physicochemical equivalence using difluprednate (DFPN) emulsion product Durezol® as a model, we developed a novel low-field benchtop NMR method to demonstrate its applicability in measuring DFPN phase partition for ophthalmic oil-in-water emulsion products. Low-field 19F spectra were collected for DFPN in formulation, in water phase and oil phase after separation from ultra-centrifugation. The NMR data showed the mass balance of DFPN before and after phase separation. The average water phase content of different Durezol® lots was 32 ± 3% with 1% variation from method reproducibility test. The partition results were 52 ± 2% for the in-house control products prepared in Q1/Q2 equivalence to Durezol® but by a different process. The significant difference in DFPN-phase partition between Durezol® and the in-house formulation demonstrated manufacture difference readily changed the API partition. The newly developed ultra-centrifugation and 19F NMR by benchtop instrument is a simple, robust, and sensitive analytical method for ophthalmic emulsion drug product development and control.

Chemometric Methods to Quantify 1D and 2D NMR Spectral Differences Among Similar Protein Therapeutics.

NMR spectroscopy is an emerging analytical tool for measuring complex drug product qualities, e.g., protein higher order structure (HOS) or heparin chemical composition. Most drug NMR spectra have been visually analyzed; however, NMR spectra are inherently quantitative and multivariate and thus suitable for chemometric analysis. Therefore, quantitative measurements derived from chemometric comparisons between spectra could be a key step in establishing acceptance criteria for a new generic drug or a new batch after manufacture change. To measure the capability of chemometric methods to differentiate comparator NMR spectra, we calculated inter-spectra difference metrics on 1D/2D spectra of two insulin drugs, Humulin R® and Novolin R®, from different manufacturers. Both insulin drugs have an identical drug substance but differ in formulation. Chemometric methods (i.e., principal component analysis (PCA), 3-way Tucker3 or graph invariant (GI)) were performed to calculate Mahalanobis distance (D M) between the two brands (inter-brand) and distance ratio (D R) among the different lots (intra-brand). The PCA on 1D inter-brand spectral comparison yielded a D M value of 213. In comparing 2D spectra, the Tucker3 analysis yielded the highest differentiability value (D M = 305) in the comparisons made followed by PCA (D M = 255) then the GI method (D M = 40). In conclusion, drug quality comparisons among different lots might benefit from PCA on 1D spectra for rapidly comparing many samples, while higher resolution but more time-consuming 2D-NMR-data-based comparisons using Tucker3 analysis or PCA provide a greater level of assurance for drug structural similarity evaluation between drug brands.

Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin.

Because of the complexity and global nature of the heparin supply chain, the control of heparin quality during manufacturing steps is essential to ensure the safety of the final active pharmaceutical ingredient (API). For this reason, there is a need to develop consistent analytical methods able to assess the quality of heparin early in production (i.e., as the crude heparin before it is purified to API under cGMP conditions). Although a number of analytical techniques have been applied to characterize heparin APIs, few of them have been applied for crude heparin structure and composition analyses. Here, to address this issue, NMR spectroscopy and chemometrics were applied to characterize 88 crude heparin samples. The samples were also analyzed by strong anion exchange HPLC (SAX-HPLC) as an orthogonal check of the purity levels of the crudes analyzed by NMR. The HPLC data showed that the chemometric analysis of the NMR data differentiated the samples based on their purity. These orthogonal approaches differentiated samples according their glycosaminoglycan (GAG) composition and their mono and disaccharide composition and structure for each GAG family (e.g., heparin/heparan, dermatan sulfate, and chondroitin sulfate A). Moreover, quantitative HSQC and multivariate analysis (PCA) were used to distinguish between crude heparin of different animal and tissue sources.

Synthesis and detection of N-sulfonated oversulfated chondroitin sulfate in marketplace heparin.

N-sulfonated oversulfated chondroitin sulfate (NS-OSCS), recently reported as a potential threat to the heparin supply, was prepared along with its intermediate derivatives. All compounds were spiked into marketplace heparin and subjected to United States Pharmacopeia (USP) identification assays for heparin (proton nuclear magnetic resonance [(1)H NMR], chromatographic identity, % galactosamine [%GalN], anti-factor IIa potency, and anti-factor Xa/IIa ratio). The U.S. Food and Drug Administration (FDA) strong-anionic exchange high-performance liquid chromatography (SAX-HPLC) method resolved NS-OSCS from heparin and OSCS and had a limit of detection of 0.26% (w/w) NS-OSCS. The %GalN test was sensitive to the presence of NS-OSCS in heparin. Therefore, current USP heparin monograph tests (i.e., SAX-HPLC and %GalN) detect the presence of NS-OSCS in heparin.

Modern analytics for naturally derived complex drug substances: NMR and MS tests for protamine sulfate from chum salmon.

This work describes orthogonal NMR and MS tests for the structure and composition of the drug protamine sulfate derived from chum salmon. The spectral response pattern obtained by 1D-(1)H-NMR and MS methods from salmon protamine, a mixture of four predominant peptide chains, is dependent on the amino acid sequence and abundance of each peptide. Thus, an assay was developed based on the ratios of alanine, glycine and arginine amino acid residue NMR peaks (relative to the arginine CδH proton signal) in this mixture that are unique to the salmon source. In addition, MS analysis provided sensitive sequence determination and impurity analysis based on shifts from exact masses. Spectra from protamine sulfate active pharmaceutical ingredient (API) suppliers and from a formulated drug product purchased from the US market were examined. Based on these marketplace survey data, NMR acceptance criteria for chum salmon derived protamine sulfate could be based on the absence of aromatic amino acid signals and on ratios of Ala ßH/Arg δH, Gly αH/Arg δH and Arg αH/Arg δH integrated areas of 2.4 ± 1%, 9.4 ± 3% and 50 ± 5%, respectively. For MS, acceptance criteria based on the presence of specific mass to charge (m/z) ratio peaks (m/z = +8 of 530.455, 540.841, 532.208 and 508.950) could be used for the four major peptides present in the mixture with relative abundances of 17 ± 1%, 31 ± 2%, 27 ± 1% and 25 ± 3%, respectively. The specificity of the combined NMR and MS assay was tested by comparison to data obtained from herring protamine which contains a different mixture of peptides with related amino acid sequences. Both assays were able to clearly distinguish protamine derived from these different natural sources.

Modern analytics for synthetically derived complex drug substances: NMR, AFFF-MALS, and MS tests for glatiramer acetate.

Glatiramer acetate (GA) is a mixture of synthetic copolymers consisting of four amino acids (glutamic acid, lysine, alanine, and tyrosine) with a labeled molecular weight range of 5000 to 9000 Da. GA is marketed as Copaxone™ by Teva for the treatment of multiple sclerosis. Here, the agency has evaluated the structure and composition of GA and a commercially available comparator, Copolymer-1. Modern analytical technologies which can characterize these complex mixtures are desirable for analysis of their comparability and structural "sameness." In the studies herein, a molecular fingerprinting approach is taken using mass-accurate mass spectrometry (MS) analysis, nuclear magnetic resonance (NMR) (1D-(1)H-NMR, 1D-(13)C-NMR, and 2D NMR), and asymmetric field flow fractionation (AFFF) coupled with multi-angle light scattering (MALS) for an in-depth characterization of three lots of the marketplace drug and a formulated sample of the comparator. Statistical analyses were applied to the MS and AFFF-MALS data to assess these methods' ability to detect analytical differences in the mixtures. The combination of multiple orthogonal measurements by liquid chromatography coupled with MS (LC-MS), AFFF-MALS, and NMR on the same sample set was found to be fit for the intended purpose of distinguishing analytical differences between these complex mixtures of peptide chains.

Chemoenzymatic synthesis and structural characterization of 2-O-sulfated glucuronic acid-containing heparan sulfate hexasaccharides.

Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. Nuclear magnetic resonance analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin (AT) binding affinities of a GlcA2S- and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to AT, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate.

Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers.

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733-737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310-312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D (1)H,(15)N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.