Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed. Mass spectrometry methods provide rationale for targeted removal strategies through the provision of both qualitative and quantitative HCP information. A high pH, low pH, reversed-phase data independent 2D-LC-MS(E) proteomic platform was applied to determine HCP repertoires in the Protein A purified monoclonal antibody (mAb) samples as a function of culture harvest time, elution buffer used for DSP and also following inclusion of additional DSP steps. Critical quality attributes (CQAs) were examined for mAbs purified with different Protein A elution buffers to ensure that the selected buffers not only minimized the HCP profile but also exhibited no adverse effect on product quality. Results indicated that an arginine based Protein A elution buffer minimized the levels of HCPs identified and quantified in a purified mAb sample and also demonstrated no impact on product CQAs. It was also observed that mAbs harvested at later stages of cell culture contained higher concentrations of HCPs but that these were successfully removed by the addition of DSP steps complementary to Protein A purification. Taken together, our results showed how mass spectrometry based methods for HCP determination in conjunction with careful consideration of processing parameters such as harvest time, Protein A elution buffers, and subsequent DSP steps can reduce the HCP repertoire of therapeutic mAbs.

Anti-idiotypic monoclonal antibodies (mAb) have been evaluated for actively induced immunotherapy with encouraging results. However, rational combination of cancer vaccines with chemotherapy may improve the therapeutic efficacy of these two approaches used separately. The main objective of this study was to evaluate the antitumor effect of the co-administration of 1E10 (Racotumomab), a monoclonal anti-idiotype tumor vaccine against an IgM mAb, named P3 that reacts specifically with NeuGc-containing gangliosides and low-dose Cyclophosphamide in a mammary carcinoma model. F3II tumor-bearing mice were immunized subcutaneously with 100 microg of 1E10 mAb in Alum or with 150 mg/m(2) of Cyclophosphamide intravenously 7 days after the tumor inoculation. While a limited antitumor effect was induced by a single 1E10 mAb immunization; its co-administration with low-dose Cyclophosphamide reduced significantly the F3II mammary carcinoma growth. That response was comparable with the co-administration of the standard high-dose chemotherapy for breast cancer based on 60 mg/m(2) of Doxorubicin and 600 mg/m(2) of Cyclophosphamide, without toxicity signs. Combinatorial chemo-immunotherapy promoted the CD8(+) lymphocytes tumor infiltration and enhanced tumor apoptosis. Furthermore, 1E10 mAb immunization potentiated the antiangiogenic effect of low-dose Cyclophosphamide. Additionally, splenic myeloid cells Gr1(+)/CD11b(+) associated with a suppressor phenotype were significantly reduced in F3II tumor-bearing mice immunized with 1E10 mAb alone or in combination with low-dose Cyclophosphamide. This data may provide a rational for chemo-immunotherapy combinations with potential medical implications in breast cancer.

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A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.