David A. Keire

Characterization of currently marketed heparin products: key tests for quality assurance

AbstractDuring the 2007–2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500xa0MHz 1H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007–2008 crisis.n FigureA plot the 1.90 to 2.30 ppm region of an overlay of the 500 MHz 1H-NMR spectra of 20 mg samples of a heparin sodium API alone or spiked with 1.0%, 5.0% or 10.0% weight percent of OSCS and the same API alone or containing 1.0%, 5.0% or 10.0% of DS. Signals associated with the presence of the spiked OSCS or DS in heparin are denoted.

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.

Physicochemical characterization of complex drug substances: evaluation of structural similarities and differences of protamine sulfate from various sources.

The purpose of this study was to characterize and evaluate differences of protamine sulfate, a highly basic peptide drug, obtained from five different sources, using orthogonal thermal and spectroscopic analytical methods. Thermogravimetric analysis and modulated differential scanning calorimetry showed that all five protamine sulfate samples had different moisture contents and glass transition and melting temperatures when temperature was modulated from 25 to 270°C. Protamine sulfate from source III had the highest residual moisture content (4.7u2009±u20090.2%) at 105°C, resulting in the lowest glass transition (109.7°C) and melting (184.2°C) temperatures compared with the other four sources. By Fourier-transform infrared (FTIR) spectroscopy, the five sources of protamine sulfate had indistinguishable spectra, and the spectra were consistent with a predominantly random coil conformation in solution and a minor population in a β-sheet conformation (~12%). Circular dichroism spectropolarimetry confirmed the FTIR results with prominent minima at 206xa0nm observed for all five sources. Finally, proton (1H) nuclear magnetic resonance spectroscopy showed that all five protamine sulfate sources had identical spectra with backbone amide chemical shifts between 8.20 and 8.80xa0ppm, consistent with proteins with predominantly random coil conformation. In conclusion, thermal analyses showed differences in the thermal behavior of the five sources of protamine sulfate, while spectroscopic analyses showed the samples had a predominantly random coil conformation with a small amount of β-sheet present.

Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure

Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures (e.g., oligomerized mAb). Here, several commonly available analytical methods, i.e., size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR.

NMR profiling of biomolecules at natural abundance using 2D 1H–15N and 1H–13C multiplicity-separated (MS) HSQC spectra

2D NMR (1)H-X (X=(15)N or (13)C) HSQC spectra contain cross-peaks for all XHn moieties. Multiplicity-edited(1)H-(13)C HSQC pulse sequences generate opposite signs between peaks of CH(2) and CH/CH(3) at a cost of lower signal-to-noise due to the (13)C T(2) relaxation during an additional 1/(1)JCH period. Such CHn-editing experiments are useful in assignment of chemical shifts and have been successfully applied to small molecules and small proteins (e.g. ubiquitin) dissolved in deuterated solvents where, generally, peak overlap is minimal. By contrast, for larger biomolecules, peak overlap in 2D HSQC spectra is unavoidable and peaks with opposite phases cancel each other out in the edited spectra. However, there is an increasing need for using NMR to profile biomolecules at natural abundance dissolved in water (e.g., protein therapeutics) where NMR experiments beyond 2D are impractical. Therefore, the existing 2D multiplicity-edited HSQC methods must be improved to acquire data on nuclei other than (13)C (i.e.(15)N), to resolve more peaks, to reduce T(2) losses and to accommodate water suppression approaches. To meet these needs, a multiplicity-separated(1)H-X HSQC (MS-HSQC) experiment was developed and tested on 500 and 700 MHz NMR spectrometers equipped with room temperature probes using RNase A (14 kDa) and retroviral capsid (26 kDa) proteins dissolved in 95% H(2)O/5% D(2)O. In this pulse sequence, the 1/(1)JXH editing-period is incorporated in to the semi-constant time (semi-CT) X resonance chemical shift evolution period, which increases sensitivity, and importantly, the sum and the difference of the interleaved (1)J(XH)-active and the (1)J(XH)-inactive HSQC experiments yield two separate spectra for XH(2) and XH/XH(3). Furthermore we demonstrate improved water suppression using triple xyz-gradients instead of the more widely used z-gradient only water-suppression approach.

Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins

&NA; An NMR HSQC method has recently been proposed for the quantitative determination of the mono‐ and disaccharide subunits of heparin and low molecular weight heparins (LMWH). The focus of the current study was the validation of this procedure to make the 2D‐NMR method suitable for pharmaceutical quality control applications. Pre‐validation work investigated the effects of several experimental parameters to assess robustness and to optimize critical factors. Important experimental parameters were pulse sequence selection, equilibration interval between pulse trains and temperature. These observations were needed so that the NMR method was sufficiently understood to enable continuous improvement. A standard validation study on heparin then examined linearity, repeatability, intermediate precision and limits of detection and quantitation; selected validation parameters were also determined for LMWH. HighlightsRobust heparin mono‐ and di‐saccharide composition measurements are a key tests for ensuring drug quality.NMR HSQC data enables quick and detailed heparin compositional analysis with minimal sample treatment.A robustness investigation and method validation showed the NMR technique was suitable for pharmaceutical quality assessment purposes.Signal to noise measurements provided a metric for inclusion of peaks in composition analysis and for the proposal of a System Suitability Test.Important NMR factors for optimal performance were pulse sequence, relaxation delay and temperature control.

Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim

&NA; The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter‐laboratory study performed using filgrastim drug products demonstrated that two‐dimensional (2D)‐NMR 1HN‐15NH heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR ‘cold probes.’ Here, the applicability of the 2D‐NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter‐laboratory precision of the 2D‐NMR method when implemented on room temperature probes. HighlightsThe 2D‐NMR method has been extended to NMRs equipped with room temperature probes.High statistical precision was achieved comparable to experiments with cold probes.Rapid acquisition methods can reduce experimental time from 7 days to 3 days.

One- and Two-Dimensional NMR Techniques for Biopharmaceuticals

Nuclear magnetic resonance (NMR) spectroscopy is routinely used by chemists to assist in the identification of the atomic structure of their molecule of interest. However, although NMR spectroscopy is a widely used analytical tool, the application of NMR techniques in the biopharmaceutical industry has been limited by the perception that the approach is too complex or insensitive. In order to illustrate the utility of the technique, we provide a description of the physical basis of NMR, the instrument design and the factors that impact the data. These concepts are then demonstrated using practical examples of the application of NMR to biotherapeutics. This chapter is intended for biologists and biochemists that have little or no formal training or knowledge in NMR spectroscopy. An overview of the important principles (without the use of mathematics) is laid out to provide the reader with the concepts needed to understand the types of information obtainable and the limitations and the advantages of NMR spectroscopy of biologics. The authors hope that a better understanding of NMR methods for biotherapeutic products will dissipate arguments against its adoption while helping to identify how NMR can contribute to a drug development program and the manufacturing process.

Rational Selection, Criticality Assessment, and Tiering of Quality Attributes and Test Methods for Analytical Similarity Evaluation of Biosimilars

Leading regulatory agencies recommend biosimilar assessment to proceed in a stepwise fashion, starting with a detailed analytical comparison of the structural and functional properties of the proposed biosimilar and reference product. The degree of analytical similarity determines the degree of residual uncertainty that must be addressed through downstream in vivo studies. Substantive evidence of similarity from comprehensive analytical testing may justify a targeted clinical development plan, and thus enable a shorter path to licensing. The importance of a careful design of the analytical similarity study program therefore should not be underestimated. Designing a state-of-the-art analytical similarity study meeting current regulatory requirements in regions such as the USA and EU requires a methodical approach, consisting of specific steps that far precede the work on the actual analytical study protocol. This white paper discusses scientific and methodological considerations on the process of attribute and test method selection, criticality assessment, and subsequent assignment of analytical measures to US FDA’s three tiers of analytical similarity assessment. Case examples of selection of critical quality attributes and analytical methods for similarity exercises are provided to illustrate the practical implementation of the principles discussed.

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 (DM) between the two brands (inter-brand) and distance ratio (DR) among the different lots (intra-brand). The PCA on 1D inter-brand spectral comparison yielded a DM value of 213. In comparing 2D spectra, the Tucker3 analysis yielded the highest differentiability value (DMxa0=xa0305) in the comparisons made followed by PCA (DMxa0=xa0255) then the GI method (DMxa0=xa040). 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.