Multi-attribute method performance profile for quality control of monoclonal antibody therapeutics.

Multi-attribute method (MAM) using peptide map analysis with high resolution mass spectrometry is increasingly common in product characterization and the identification of critical quality attributes (CQAs) of biotherapeutic proteins. Capable of providing structural information specific to amino acid residues, quantifying relative abundance of product variants or degradants, and detecting profile changes between product lots, a robust MAM can replace multiple traditional methods that generate profile-based information for product release and stability testing. In an effort to provide informative and efficient analytical monitoring for monoclonal antibody (mAb) products, from early development to manufacturing quality control, we describe the desired MAM performance profile and address the major scientific challenges in MAM method validation. Furthermore, to support fast speed investigational product development, we describe a platform method validation strategy and results of an optimized MAM workflow. This strategy is applied to support the use of MAM for multiple mAb products with similar structures and physicochemical properties, requiring minimal product-specific method validation activities. Three mAb products were used to demonstrate MAM performance for common and representative product quality attributes. Method validation design and acceptance criteria were guided by the Analytical Target Profile concept, as well as relevant regulatory guidelines to ensure the method is fit-for-purpose. A comprehensive system suitability control strategy was developed, and reported here, to ensure adequate performance of the method including sample preparation, instrument operation, and data analysis. Our results demonstrated sufficient method performance for the characteristics required for quantitative measurement of product variants and degradants.

Ruthenium coordination preferences in imidazole-containing systems revealed by electrospray ionization mass spectrometry and molecular modeling: Possible cues for the surprising stability of the Ru (III)/tris (hydroxymethyl)-aminomethane/imidazole complexes.

Ruthenium is a platinoid that exhibits a range of unique chemical properties in solution, which are exploited in a variety of applications, including luminescent probes, anticancer therapies, and artificial photosynthesis. This paper focuses on a recently demonstrated ability of this metal in its +3 oxidation state to form highly stable complexes with tris (hydroxymethyl)aminomethane (H2 NC(CH2 OH)3 , Tris-base or T) and imidazole (Im) ligands, where a single RuIII cation is coordinated by two molecules of each T and Im. High-resolution electrospray ionization mass spectrometry (ESI MS) is used to characterize RuIII complexes formed by placing a RuII complex [(NH3 )5 RuII Cl]Cl in a Tris buffer under aerobic conditions. The most abundant ionic species in ESI MS represent mononuclear complexes containing an oxidized form of the metal, ie, [Xn RuIII T2 - 2H]+ , where X could be an additional T (n = 1) or NH3 (n = 0-2). Di- and tri-metal complexes also give rise to a series of abundant ions, with the highest mass ion representing a metal complex with an empirical formula Ru3 C24 O21 N6 H66 (interpreted as cyclo(T2 RuO)3 , a cyclic oxo-bridged structure, where the coordination sphere of each metal is completed by two T ligands). The empirical formulae of the binuclear species are consistent with the structures representing acyclic fragments of cyclo(T2 RuO)3 with addition of various combinations of ammonia and dioxygen as ligands. Addition of histidine in large molar excess to this solution results in complete disassembly of poly-nuclear complexes and gives rise to a variety of ionic species in the ESI mass spectrum with a general formula [RuIII Hisk Tm (NH3 )n - 2H]+ , where k = 0 to 2, m = 0 to 3, and n = 0 to 4. Ammonia adducts are present for all observed combinations of k and m, except k = m = 2, suggesting that [His2 RuIII T2 - 2H]+ represents a complex with a fully completed coordination sphere. The observed cornucopia of RuIII complexes formed in the presence of histidine is in stark contrast to the previously reported selective reactivity of imidazole, which interacts with the metal by preserving the RuT2 core and giving rise to a single abundant ruthenium complex (represented by [Im2 RuIII T2 - 2H]+ in ESI mass spectra). Surprisingly, the behavior of a hexa-histidine peptide (HHHHHH) is similar to that of a single imidazole, rather than a single histidine amino acid: The RuT2 core is preserved, with the following ionic species observed in ESI mass spectra: [HHHHHH·(RuIII T2 )m - (3m-1)H]+ (m = 1-3). The remarkable selectivity of the imidazole interaction with the RuIII T2 core is rationalized using energetic considerations at the quantum mechanical level of theory.

Exploiting His-Tags for Absolute Quantitation of Exogenous Recombinant Proteins in Biological Matrices: Ruthenium as a Protein Tracer.

Metal labeling and ICP MS detection offer an alternative to commonly accepted techniques that are currently used to quantitate exogenous proteins in vivo, but modifying the protein surface with metal-containing groups inevitably changes its biophysical properties and is likely to affect trafficking and biodistribution. The approach explored in this work takes advantage of the presence of hexa-histidine tags in many recombinant proteins, which have high affinity toward a range of metals. While many divalent metals bind to poly histidine sequences reversibly, oxidation of imidazole-bound CoII or RuII is known to result in a dramatic increase of the binding strength. In order to evaluate the feasibility of using imidazole-bound metal oxidation as a means of attaching permanent tags to polyhistidine segments, a synthetic peptide YPDFEDYWMKHHHHHH was used as a model. RuII can be oxidized under ambient (aerobic) conditions, allowing any oxidation damage to the peptide beyond the metal-binding site to be avoided. The resulting peptide-RuIII complex is very stable, with the single hexa-histidine segment capable of accommodating up to three metal ions. Localization of RuIII within the hexa-histidine segment of the peptide was confirmed by tandem mass spectrometry. The RuIII/peptide binding appears to be irreversible, with both low- and high-molecular weight biologically relevant scavengers failing to strip the metal from the peptide. Application of this protocol to labeling a recombinant form of an 80 kDa protein transferrin allowed RuIII to be selectively placed within the His-tag segment. The metal label remained stable in the presence of ubiquitous scavengers and did not interfere with the receptor binding, while allowing the protein to be readily detected in serum at sub-nM concentrations. The results of this work suggest that ruthenium lends itself as an ideal metal tag for selective labeling of His-tag containing recombinant proteins to enable their sensitive detection and quantitation with ICP MS.

Quantitative Determination of Protein-Ligand Affinity by Size Exclusion Chromatography Directly Coupled to High-Resolution Native Mass Spectrometry.

High throughput protein-ligand interaction screening assays employing mass spectrometric detection are widely used in early stage drug discovery. Mass spectrometry-based screening approaches employ a target protein added to a pool of small-molecule compounds, and binding is assessed by measuring ligands denatured from the complexes. Direct analysis of protein-ligand interactions using native mass spectrometry has been demonstrated but is not widely used due to the detection limit on protein size, the requirement of volatile buffers, and the necessity for specialized instrumentation to preserve weak interactions under native conditions. Here we present a robust, quantitative, and automated online size-exclusion chromatography-native mass spectrometry (SEC-nMS) platform for measuring affinities of noncovalent protein-small-molecule interactions on an Orbitrap mass spectrometer. Indoleamine 2,3-dioxygenase 1, a catabolic enzyme, and inhibitory ligands were employed as a demonstration of the method. Efficient separation and elution enabled preservation of protein-ligand complexes and increased throughput. The high sensitivity and intra charge state resolution at high m/ z offered by the Exactive Plus EMR Orbitrap allowed for protein ligand affinity quantitation and resolved individual compounds close in mass. Vc50 values determined via collision-induced dissociation experiments enabled the evaluation of complex stability in the gas phase and were found to be independent of the extent of complex formation. For the first time, Vc50 determinations were achieved on an inline SEC-nMS platform. Systematic comparison of our method with optimized chip-based nanoelectrospray infusion served as a reference for ligand screening and affinity quantitation and further revealed the advantages of SEC-MS.

Synthesis of oxazoles from enamides via phenyliodine diacetate-mediated intramolecular oxidative cyclization.

A group of functionalized oxazoles were synthesized in moderate to good yields from enamides via phenyliodine diacetate (PIDA)-mediated intramolecular cyclization. The main advantageous features of the present method include its broad substrate scope and the heavy-metal-free characteristic of the oxidative carbon-oxygen bond formation process.

Identification of ligand binding site on RXRγ using molecular docking and dynamics methods.

Retinoid X receptors (RXRα, ß and γ) are recently known to be cancer chemotherapies targets. The ligand binding domains of RXRs have been crystallized, but the information of RXRγ ligand binding site is not yet available due to the lack of liganded complex. A thorough understanding of the ligand binding sites is essential to study RXRs and may result in cancer therapeutic breakthrough. Thus we aimed to study the RXRγ ligand binding site and find out the differences between the three subtypes. Alignment and molecular simulation were carried out for identifying the RXRγ ligand binding site, characterizing the RXRγ ligand binding mode and comparing the three RXRs. The result has indicated that the RXRγ ligand binding site is defined by helices H5, H10, ß-sheet s1 and the end loop. Besides hydrophobic interactions, the ligand 9-cis retinoic acid interacts with RXRγ through a hydrogen bond with Ala106, a salt bridge with Arg95 and the π-π interactions with Phe217 and Phe218. The binding modes exhibit some similarities among RXRs, such as the interactions with Arg95 and Ala106. Nonetheless, owing to the absence of Ile47, Cys48, Ala50, Ala51 and residues 225∼237 in the active site, the binding pocket in RXRγ is two times larger than those of RXRα and RXRß. Meanwhile, spatial effects of Trp84, Arg95, Ala106, Phe217 and Phe218 help to create a differently shaped binding pocket as compared to those of RXRα and RXRß. Consequently, the ligand in RXRγ undergoes a "standing" posing which is distinct from the other two RXRs.