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The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C-terminal lysine-containing peptides from Lys-C digest by on-column derivatization of lysines with 2-methoxy-4,5-dihydro-1H-imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post-source decay carried out on derivatized peptides gave rise almost exclusively to y-series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine-containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on-column derivatization protocol is designed to be carried out on C18 Spin Tubes or Cleanup C18 Pipette Tips. We observed that use of buffered D2O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between epsilon-amino or cysteine thiol groups and 2-methoxy-4,5-dihydro-1H-imidazole. Complete tagging of the peptides by on-column reaction could be obtained when using D2O, as compared to water-based reaction. Methylation of cysteine residues was enhanced when beta-mercaptoethanol was added in the reactant solution.

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Current methods for determination of impurities with different charge-to-volume ratio are limited especially in terms of sensitivity and precision. The main goal of this research was to establish a quantitative method for determination of impurities with charges differing from that of recombinant human granulocyte colony-stimulating factor (rhG-CSF, filgrastim) with superior precision and sensitivity compared to existing methods. A CZE method has been developed, optimized, and validated for a purity assessment of filgrastim in liquid pharmaceutical formulations. Optimal separation of filgrastim from the related impurities with different charges was achieved on a 50 μm id fused-silica capillary of a total length of 80.5 cm. A BGE that contains 100 mM phosphoric acid adjusted to pH 7.0 with triethanolamine was used. The applied voltage was 20 kV while the temperature was maintained at 25°C. UV detection was set to 200 nm. Method was validated in terms of selectivity/specificity, linearity, precision, LOD, LOQ, stability, and robustness. Linearity was observed in the concentration range of 6-600 μg/mL and the LOQ was determined to be 0.3% relative to the concentration of filgrastim of 0.6 mg/mL. Other validation parameters were also found to be acceptable; thus the method was successfully applied for a quantitative purity assessment of filgrastim in a finished drug product.

Background Biosimilars are follow-on versions of peptide biological drugs, and differences in manufacturing and formulation can result in variations in physicochemical and clinical profiles. The European Medicines Agency (EMA) has set stringent standards (Ph Eur) that must be met for the approval of a biosimilar. PurposeStandards of manufacture may differ between biosimilars approved via EMA pathways, and copy biologicals that lack approval pathways. Therefore, we undertook comparative characterisation tests of a range of biosimilar products from different global regions to determine if variations exist. This study is the first of its kind. Materials and Methods Samples of Nivestim (Ni), Neupogen (Ne), Tevagrastim (T), Ratiograstim and Zarzio (Z) were obtained from the EU region; and Leucostim (L), GeSysin (G), Filgen (F) and Neukine (Nk) were obtained from the Middle East and Africa (MENA) region. All samples were within the expiry date. Samples were analysed for impurities using iso-electric focussing (IEF) to identify differences in charge, size-exclusion high-performance liquid chromatography (SEC-HPLC) to identify differences in higher molecular weight impurities, reverse phase HPLC (RP-HPLC) to identify differences in total and individual related impurities, and ion chromatography (IC) to detect differences in f-met filgrastim and related, more acidic, impurities. Results All biosimilars met EMA standards for IEF and SEC-HPLC analysis. Total impurities (RP-HLPC) for the EU products were in the range 1.8–2.6% and within EMA requirements (≤3.5%); however, the MENA samples contained impurities in the range 5.9% (G) – 8.2% (L), which is beyond the Ph Eur range. IC analysis revealed f-met and acidic impurities to be <0.20% for most EU products (threshold 1.0%) and 0.4% for Ne. However, for MENA compounds, these impurities comprised 0.4% (Nk) – 1.7% (G) of the samples. Conclusions Copy biologicals from MENA have higher levels of impurities than biosimilars from the EU and do not meet EMA standards for approval. No conflict of interest.