Rapid preparation of 1-vinylimidazole based non-affinity polymers for the highly-selective purification of antibodies from multiple biological sources.

There is an urgent need for developing advanced purification techniques with the merits of low cost and satisfactory capacity in order to meet the challenges in the current downstream purification of monoclonal antibodies (mAbs). Herein, a simple and inexpensive nitrogen heterocycle molecule, 1-vinylimidazole (VIM), was proposed as the capture ligand of antibodies for the first time. The corresponding VIM-based non-affinity polymeric material (polyVIM) was then fabricated via a one-step polymerization for use in the highly selective purification of antibodies. Compared to the previously reported materials, this novel material exhibited many advantages without clearly sacrificing selectivity, such as a simpler and faster fabrication (within 1.5 h), comparable or even higher binding capacity (saturated static adsorption capacity > 190 mg/g polymer, dynamic binding capacity about 31.62 mg/g polymer), lower non-specific protein adsorption, and much lower cost. Notably, the polyVIM can effectively purify the antibodies from multiple biological sources with high purity (95.4% for mAbs in the cell culture medium, 93.3% for hIgG in the human serum), with an acceptable recovery (91.6% for mAbs, 77.0% for hIgG), and good reusability (> 10 times). Moreover, the target ELISA binding assay and NFAT-luc reporter gene assay demonstrated that the enriched antibodies can well maintain their binding activity and bioactivity during the whole purification process. The excellent performance of the polyVIM material may be attributed to the high recognition ability of VIM for antibodies, as well as the biocompatible and antifouling properties of the porous polymer. This study provides a promising alternative material for the purification of mAbs in downstream processes and the enrichment of hIgG in human serum.

Antibody drug separation using thermoresponsive anionic polymer brush modified beads with optimised electrostatic and hydrophobic interactions.

Antibody drugs play an important role in biopharmaceuticals, because of the specificity for target biomolecules and reduction of side effects. Thus, separation and analysis techniques for these antibody drugs have increased in importance. In the present study, we develop functional chromatography matrices for antibody drug separation and analysis. Three types of polymers, poly(N-isopropylacrylamide (NIPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-N-phenyl acrylamide (PhAAm)), P(NIPAAm-co-AMPS-co-n-butyl methacrylate (BMA)), and P(NIPAAm-co-AMPS-co-tert-butylacrylamide (tBAAm)), were modified on silica beads through atom transfer radical polymerisation. Rituximab elution profiles were observed using the prepared beads-packed column. Rituximab adsorption at high temperature and elution at low temperature from the column were observed, as a result of the temperature-modulated electrostatic and hydrophobic interactions. Using the column, rituximab purification from contaminants was performed simply by changing the temperature. Additionally, three types of antibody drugs were separated using the column through temperature-modulated hydrophobic and electrostatic interactions. These results demonstrate that the temperature-responsive column can be applied for the separation and analysis of biopharmaceuticals through a simple control of the column temperature.

Evaluation of additives on reversed-phase chromatography of monoclonal antibodies using a 1000 Šstationary phase.

A wide pore (1000 Å) diphenyl stationary phase was evaluated for the analysis of monoclonal antibodies (mAbs), comparing a conventional mobile phase of acetonitrile-water containing overall 0.1% trifluoracetic acid (TFA) with a similar mobile phase incorporating in addition 5% butanol. Alternatively, TFA was replaced by ammonium formate (AF) buffer (pH 3.0) and by methane sulfonic acid. Addition of 5% butanol to the mobile phase reduces the minimum temperature at which suitable UV analysis of the mAbs can be obtained from about 70 °C with TFA alone to about 60 °C thus potentially improving column lifetime and reducing the possibility of sample degradation. AF buffers produce satisfactory UV sensitivity at 70 °C and have the advantage of reducing signal suppression in mass spectrometry (MS). Some peak tailing was noted in comparison with TFA separations. Methane sulfonic acid at the same molar concentration as TFA produced the best chromatographic peaks, maintaining reasonable UV sensitivity down to 50 °C, also giving acceptable results even at only 3 mM concentration of the additive. The good results with this additive were attributed to its stronger acidity and consequent suppression of the ionisation of column silanols. Surprisingly, peak response (as measured by the size of the peaks) was rather poorly correlated with the peak capacity of the gradient analysis. A possible explanation is self-deactivation of active column sites by a portion of the sample.

Development of an LC-MS/MS method with protein G purification strategy for quantifying bevacizumab in human plasma.

Biopharmaceutical products such as protein drugs and monoclonal antibodies (mAb) are currently of great interest with monoclonal antibody drugs being one of the fastest growing categories of biopharmaceutical products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gained high interest for measuring mAb drugs in biological samples in recent years due to its high selectivity. Bevacizumab is a humanized immunoglobulin G (IgG) mAb drug against human vascular endothelial cell growth factor A (VEGF-A). It is used for treating many types of cancers. Recent studies have indicated that clinical outcomes vary among patients treated with bevacizumab and produce various side effects, such as vascular disorders. In this study, we developed an LC-MS/MS method to quantify bevacizumab concentration. We selected readily available and economic materials for sample preparation to facilitate its wider use in clinical fields.-Protein G was used to trap bevacizumab from human plasma. In place of an extended stable isotope-labeled internal standard (SIL-IS), the IgG-based drug-IS tocilizumab was used because of its better calibration performance. The method was validated in terms of its precision, accuracy, linearity, and sensitivity. The accuracies which were expressed as percentage recoveries for three concentration levels were within 92.8 ± 3.2 to 112.7 ± 4.5%. Repeatability and intermediate precision in terms of peak area ratios were lower than 5.2 and 12.9% RSD, respectively. The application to patients' sample measurements revealed a wide individual variability of drug concentrations, and the proposed simple and general method may facilitate personalized medicine for improving therapeutic efficacy and safety. Graphical abstract ᅟ.

Quality Control of Therapeutic Monoclonal Antibodies at the Hospital After Their Compounding and Before Their Administration to Patients.

Monoclonal antibodies (mAbs) are widely used in cancer therapy and recently many new mAbs have gained EMA and FDA approvals for oncology indications. Here we describe a highly reproducible CZE method, relying on a cationic coating allowing separation and identification of a complex mixture of four compounded mAbs widely used in cancer therapy (cetuximab, rituximab, bevacizumab, and trastuzumab).