Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation.

Open image in new window

Graphical Abstract ?

     

Wavelet Galerkin method for eigenvalue problem of a compact integral operator

We consider the approximation of eigenfunctions of a compact integral operator with a smooth kernel by the Galerkin method using wavelet bases. By truncating the Galerkin operator, we obtain a sparse representation of a matrix eigenvalue problem. We prove that the error bounds for the eigenvalues and for the distance between the spectral subspaces are of the orders O(nμ^-2nr) and O(μ^-nr), respectively, where μ⁻ⁿ denotes the norm of the partition and r denotes the order of the wavelet basis functions. By iterating the eigenvectors, we show that the error bounds for the eigenvectors are of the order O(nμ^-2nr). We illustrate our results with numerical results.     

Free vibration of nonhomogeneous Timoshenko nanobeams

Free vibration of nonhomogeneous nanobeams based on nonlocal Timoshenko beam theory has been studied using boundary characteristic orthogonal polynomial functions in the Rayleigh–Ritz method. Orthogonal polynomial functions satisfying essential boundary conditions have been generated with the help of Gram–Schmidt Process. Nonhomogeneity of nanobeams is assumed to arise due to linear and quadratic variations in Young’s modulus and density of the nanobeams with space coordinate. The lowest three frequency parameters of nanobeams subjected to different boundary conditions have been computed for various values of nonhomogeneous parameters to demonstrate the effect of each parameters on the frequency parameters. A detailed investigation has been reported for all the possible cases of variations in Young’s modulus and density to analyze the numerical results for different scaling effect parameters and four types of boundary conditions. Present results are compared with the results in special cases and are found to be in good agreement.     

Racemic 1,2‐diphenylbut‐3‐yn‐2‐ol

Molecules of the title compound, C₁₆H₁₄O, are chiral and crystallize in space group P with Z′ = 2, and with one R and one S mol­ecule in the asymmetric unit. The conformations of the phenyl rings in the two independent mol­ecules differ slightly. Supramolecular organization in the crystal is via tetrameric O—H?H(O) hydrogen‐bonded synthons formed separately by each conformer. These tetrameric synthons stack along the c axis via C[triple‐bond]C—H?O(H) hydrogen bonds. The only link between the conformer stacks is provided by weaker C_methylene—H and C_phenyl—H interactions with π_arene density.     

Asymmetric dearomatization of the furan ring promoted by conjugate organolithium addition to (menthyloxy)(3-furyl)carbene complexes of chromium

The sequential low-temperature addition reaction of an organolithium compound and methyl triflate to (menthyloxy)(3-furyl)carbene complexes of chromium and tungsten proceeded with excellent regioselectivity (1,4-addition) and diastereoselectivity (2,3-trans disposition of the nucleophile and electrophile groups) to afford new 2,3-disubstituted (2,3-dihydro-3-furyl)carbene complexes. In addition, a high degree of diastereofacial selectivity was achieved by employing alkenyllithium compounds. After detachment of both the metal fragment and the chiral auxiliary group, trisubstituted 2,3-dihydrofuran derivatives containing a quaternary stereogenic center at the C3 position were obtained. The characterization, including X-ray crystallography, of a novel type of stable four-membered chelate (eta(2)-alkene)tetracarbonylcarbene complex of chromium is also reported.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of zafirlukast, a selective leukotriene antagonist in human plasma: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of zafirlukast (ZFK) with 500 microL human plasma using valdecoxib as an internal standard (IS). The API-4,000 LC-MS/MS was operated under multiple reaction-monitoring mode using the electrospray ionization technique. The assay procedure involved extraction of ZFK and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with 10 mm ammonium acetate (pH 6.4):acetonitrile (20:80, v/v) on a Hypersil BDS C(18) column. The total chromatographic run time was 2.0 min and the elution of ZFK and IS occurred at approximately 1.11 and 1.58 min, respectively. The MS/MS ion transitions monitored were 574.2 --> 462.1 for ZFK and 313.3 --> 118.1 for IS. The method was proved to be accurate and precise at a linearity range of 0.15-600 ng/mL with a correlation coefficient (r) of >or=0.999. The method was rugged with 0.15 ng/mL as lower limit of quantitation. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 20 mg ZFK tablet.     

Analysis of the MAP/G a ,b /1/N Queue

The Markovian arrival process (MAP) is used to represent the bursty and correlated traffic arising in modern telecommunication network. In this paper, we consider a single server finite capacity queue with general bulk service rule in which arrivals are governed by MAP and service times are arbitrarily distributed. The distributions of the number of customers in the queue at arbitrary, post-departure and pre-arrival epochs have been obtained using the supplementary variable and the embedded Markov chain techniques. Computational procedure has been given when the service time distribution is of phase type.     

Embeddings of canonical modules and resolutions of connected sums

For an ideal $I_{m,n}$ generated by all square-free monomials of degree m in a polynomial ring R with n variables, we obtain a specific embedding of a canonical module of $R/I_{m,n}$ to $R/I_{m,n}$ itself. The construction of this explicit embedding depends on a minimal free R-resolution of an ideal generated by $I_{m,n}$. Using this embedding, we give a resolution of connected sums of several copies of certain Artin $\mathsf{k}$-algebras where $\mathsf{k}$ is a field.     

Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by ‘bottom‐up’ mass spectrometry with electron transfer dissociation

Aspartic acid formed by nonenzymatic deamidation of asparagine often isomerizes to isoaspartic acid through a succinimide intermediate. Accumulation of isoaspartic acid initiates aggregation and degradation in proteins. Deamidation at the antigen‐binding region reduces the efficacy and also upregulates immunogenicity of monoclonal antibodies. We report an improved ‘bottom‐up’ tandem mass spectrometric method to detect and decipher the position of isoaspartate formation in therapeutic immunoglobulin gamma in a single chromatographic run. Differentiation between aspartate and isoaspartate residues through collision‐induced tandem mass spectrometry is formidable due to their identical mass. Signature backbone cleavage ions, c_n + 57 and z_l–n ? 57, produced upon radical‐mediated fragmentation, were used to delineate the site of isomerization. It is more conclusive than monitoring the relative peak intensity and the decrease in hydrophobicity of the isoaspartate‐containing peptide in a chromatographic elution. Collectively, this methodology provides a useful tool to monitor deamidation and isomerization in biopharmaceuticals during their production, downstream processing and storage. Copyright © 2010 John Wiley & Sons, Ltd.