Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals.

Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.

Characterization of pharmaceutical polymorphs by isothermal calorimetry.

A great number of pharmaceutical substances exist in crystalline solid-state. Because of the complexity of their chemical structure many different polymorphs of a given substance can exist. Polymorphic forms of solid pharmaceuticals influence not only their dissolution behavior, i.e. bioavailability but also their solid-state stability. It is well known that only one polymorphic form is thermodynamically stable and all other metastable forms will convert, eventually, to the more stable form. Hence it is essential to choose the most suitable polymorphic form in the early stage of pharmaceutical development. The following article reviews the recent applications of solution calorimetry that allows characterization of pharmaceutical polymorphs through accurate determination of enthalpy of solution. Each crystalline form possesses a defined enthalpy of solution, therefore solution calorimetry is used for the quantitative analysis of the desired polymorphic form and determination of enthalpy of transition corresponding to the difference in enthalpies of solution for a polymorphic pair. More recently this technique has been applied to the estimation of thermodynamic transition temperature, which is useful for the evaluation of thermodynamic stability relationships between polymorphs. This article will also describe the kinetics and thermodynamics of polymorphic transitions, from a metastable form to the thermodynamically stable form, through studies using ampoule-based isothermal microcalorimetry. Such studies are particularly useful when metastable forms are to be selected in order to enhance bioavailability. If the metastable form, or the pharmaceutical product containing it, can be shown to be sufficiently stable, it could then be used in a formulation where its therapeutic effects could be exploited.

[Simple method for quantitative determination of active polymorphous and amorphous drugs in drug products by fourier transform-Raman spectroscopy].

The feasibility of a simple Fourier transform (FT)-Raman spectroscopic method for the quantitative determination of unexpected active drug polymorphs or amorphous in drug products was explored. In this study, calibration samples were prepared by physically mixing drug substances with their polymorphs or amorphous, without using excipients. A partial least-squares (PLS) method was applied to the quantitative analysis of the FT-Raman spectra obtained. As model drug products, compound A drug substances (form alpha) containing several ratios of polymorph, (form beta), were physically mixed with excipients to prepare powder samples corresponding to compound A tablets (30 and 120 mg). The mixture of compound B, form I and amorphous, were also mixed with excipients to mimic powdered compound B tablets (32 mg). Satisfactory relationships between the theoretical contents of polymorph or amorphous and determined contents were obtained; quantitation limits were 5-10%. In the case of powder samples corresponding to compound A tablets (30 mg) and compound B tablets (32 mg), differentiating the FT-Raman spectra prior to PLS analysis was required to improve the precision of the determinations.

Matrix media selection for the determination of residual solvents in pharmaceuticals by static headspace gas chromatography.

The influence of the matrix medium used for the determination of residual solvents in pharmaceuticals by static headspace gas chromatography was investigated. The purpose of this paper is to propose a guide for the choice of a matrix medium suitable for the determination of residual solvents of interest. Dimethylsulfoxide (DMSO), N,N-dimethyformamide (DMF), N,N-dimethylacetamide (DMA), benzyl alcohol (BA), 1,3-dimethyl-2-imidazolidinone (DMI) and water were studied as matrix media, and seventeen solvents used for the synthesis and purification of drug substances were used as target analytes. The peak shape of each analyte was not affected by the matrix medium, whereas the peak intensities for all solvents were strongly affected by the matrix medium; for example, the peak intensity of methanol in a BA matrix was more than four times that in a DMSO matrix. With a few exceptions, the peak intensities are approximately doubled for every 20 degrees C rise in equilibrium temperature between 80 and 140 degrees C, and there is no difference in this behavior among the matrix media. In addition, the formation of artifacts from the matrix media, upon heating in a headspace sampling apparatus, was investigated. Artifacts were also formed following ultrasonication of sample solutions used to increase dissolution of the sample into the matrix medium selected. These artifacts included benzene and toluene which were restricted as Class 1 and 2 toxic solvents in the ICH guideline.

A kinetic and thermodynamic study of seratrodast polymorphic transition by isothermal microcalorimetry.

The development of isothermal microcalorimetry to a study of the kinetic and thermodynamics of polymorphic transitions in seratrodast ((+/-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoic acid) Form II is reported. Sieved samples of Form II were allowed to convert to Form I, in a reaction vessel of an isothermal microcalorimeter, under 13, 31, 63 and 93% relative humidity (RH) between 48 and 65 degrees C. The power (Phi, in Watts) versus time curves from the microcalorimeter were integrated into the heat output (q, in Joules) versus time curves to yield fractional extent of Form I converted versus time curves. The change in enthalpy (-5.70 kJmol(-1)) agreed very closely with that obtained by differential scanning calorimetry and solution calorimetry, which indicated that the power measured by the microcalorimeter was due only to the Form II-to-Form I transition. Application of the theoretical kinetic method [J. Am. Ceram. Soc. 55 (1972) 74] revealed that the transition took place via a two-dimensional growth of nuclei mechanism at all the studied relative humidities and temperatures. The rate constant increased with increasing RH and temperature, and with decreasing the particle size of sample. The activation energies obtained from Arrhenius plots were 292, 290, 280 and 284 kJmol(-1), and the extrapolated rate constants at 25 degrees C were also 3.01 x 10(-10), 3.11 x 10(-10), 9.65 x 10(-10) and 3.84 x 10(-9)s(-1) for 13, 31, 63 and 93% RH, respectively.

Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin.

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.06% and 0.95%, respectively, when using IgG(4) as a model. Furthermore, RSDs of charge variant compositions were less than 5.0%. These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution, and it is applicable to quality testing and detailed characterization in the pharmaceutical industry. In addition, it should be noticed that the method provided non-linear pH gradient within the tested ranges, from pI 9.50 to 3.78, and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF. This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values.

A novel method for estimation of transition temperature for polymorphic pairs in pharmaceuticals using heat of solution and solubility data.

A novel method for thermodynamic stability studies of polymorphic drug substances has been developed. In order to estimate the transition temperature for an enantiotropic polymorphic pair, a formula for calculating the temperature at which the solubilities of each polymorph become equal has been derived with heat of solution and solubility as the variables. This formula is based on the assumption that van't Hoff plots (logarithmic solubility versus reciprocal of absolute temperature plots) of each polymorph show a straight line (heat of solution is independent of temperature) whose slope can be expressed as a function of heat of solution. The transition temperatures for seratrodast, acetazolamide and carbamazepine polymorphic pairs calculated by the formula were in good agreement with the results of previous studies. Furthermore, the calculated transition temperature for the indomethacin polymorphic pair was above the melting point, an unrealistic temperature range, suggesting that these polymorphs are monotropically related. Since this formula requires solubility data at only one arbitrary temperature other than heat of solution data for both polymorphs in a polymorphic pair, the proposed method is much faster than the conventional method requiring solubility data at five or more different temperatures for the preparation of van't Hoff plots.