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MacH H.,Merck And Co. | Arvinte T.,University of Geneva | Arvinte T.,Therapeomic Inc.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2011

As the share of therapeutic proteins in the arsenal of modern medicine continue increasing, relatively little progress has been made in the development of analytical methods that would address specific needs encountered during the development of these new drugs. Consequently, the researchers resort to adaptation of existing instrumentation to meet the demands of rigorous bioprocess and formulation development. In this report, we present a number of such adaptations as well as new instruments that allow efficient and precise measurement of critical parameters throughout the development stage. The techniques include use of atomic force microscopy to visualize proteinacious sub-visible particles, use of extrinsic fluorescent dyes to visualize protein aggregates, particle tracking analysis, determination of the concentration of monoclonal antibodies by the analysis of second-derivative UV spectra, flow cytometry for the determination of subvisible particle counts, high-throughput fluorescence spectroscopy to study phase separation phenomena, an adaptation of a high-pressure liquid chromatography (HPLC) system for the measurement of solution viscosity and a variable-speed streamlined analytical ultracentrifugation method. An ex vivo model for understanding the factors that affect bioavailability after subcutaneous injections is also described. Most of these approaches allow not only a more precise insight into the nature of the formulated proteins, but also offer increased throughput while minimizing sample requirements. © 2011 Elsevier B.V. All rights reserved.

Arvinte T.,Therapeomic Inc. | Arvinte T.,University of Geneva | Palais C.,Therapeomic Inc. | Green-Trexler E.,Merck And Co. | And 4 more authors.
mAbs | Year: 2013

Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin® (trastuzumab) or Avastin® (bevacizumab) but not Remicade® (infliximab). The aggregates in the plasma-Herceptin®-5% dextrose solution were globular, size range 0.5-9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin®-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin ®, Avastin® and Remicade® were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin® or Avastin® with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux® (cetuximab), whereas no binding was measured for Humira® (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum. © 2013 Landes Bioscience.

Peters B.J.M.,St. Antonius Hospital | Capelle M.A.H.,Therapeomic Inc. | Arvinte T.,Therapeomic Inc. | Arvinte T.,University of Geneva | Van De Garde E.M.W.,St. Antonius Hospital
mAbs | Year: 2013

Automation robots have recently come to the market as an alternative for manual compounding of drugs for intravenous administration. our aim was to assess whether robotic compounding can be performed with monoclonal antibodies (mAbs) without influencing the aggregation state of the proteins. Three frequently used mAbs were studied: infliximab (Remicade®, Janssen Biotech) and trastuzumab (Herceptin®, Roche) in lyophilized form, and bevacizumab (Avastin®, Roche) as a liquid formulation stored at 2°C to 8°C. The effects of different procedures to prepare the patient doses on antibody aggregation were evaluated. Remicade® and Herceptin® were reconstituted both manually and by a robotic arm (i.v. STATION®, Health Robotics). Additionally, the influence of vigorous shaking during reconstitution was investigated. The effects of rapid aspiration and dispensing on antibody aggregation were investigated for all three mAbs. Aggregation state was assessed by UV-Vis absorbance, 90° light scatter, fluorescence spectroscopy, Nile red fluorescence microscopy, and field flow fractionation without cross and focus flow. Robotic reconstituted samples showed similar findings compared with manual reconstitution if performed exactly according to the summary of product characteristics (SPC). Vials that were vigorously shaken showed a significant increase in aggregates. Similarly, rapid aspiration/dispense cycles resulted in a strong increase in the number and sizes of aggregates for all three mAbs; this result was observed after just one rapid aspiration/dispense cycle. Our study showed that robotic compounding of mAbs is feasible if the robot is exactly programmed according to the SPC, indicating that robotic compounding can be used to achieve reproducible high-quality compounding for delicate formulations. © 2013 Landes Bioscience.

Mueller C.,University of Geneva | Mueller C.,Center Pharmapeptides | Capelle M.A.H.,Therapeomic Inc. | Arvinte T.,University of Geneva | And 4 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2011

Protein aggregation, which is triggered by various factors, is still one of the most prevalent problems encountered during all stages of protein formulation development. In this publication, we present novel excipients, tryptophan-mPEGs (Trp-mPEGs) of 2 and 5 kDa molecular weight and suggest their use in protein formulation. The synthesis and physico-chemical characterization of the excipients are described. Possible cytotoxic and hemolytic activities of the Trp-mPEGs were examined. Turbidity, 90° static light scatter, intrinsic fluorescence, fluorescence after staining the samples with Nile Red and fluorescence microscopy were used to study the inhibitory effect of the Trp-mPEGs on the aggregation of salmon calcitonin (sCT) in different buffer systems and at various molar ratios. Aggregation of sCT was reduced significantly with increasing concentrations of Trp-mPEG 2 kDa. A 10-fold molar excess of Trp-mPEG 2 kDa suppressed almost completely the aggregation of sCT in 10 mM sodium citrate buffer (pH 6) for up to 70 h. Trp-mPEG 5 kDa also reduced the aggregation of sCT, though less pronounced than Trp-mPEG 2 kDa. Low aggregation of sCT was measured after approximately 10 days in 10 mM sodium citrate buffer, pH 5, with a 10-fold molar excess of Trp-mPEG 2 kDa. This paper shows that Trp-mPEGs are potent excipients in reducing the aggregation of sCT. Trp-mPEGs are superior to dansyl-PEGs concerning the stabilization of sCT in a harsh environment, wherein sCT is prone to aggregation. Trp-mPEGs might therefore also be used for stabilization of other biopharmaceuticals prone to aggregation. © 2011 Elsevier B.V. All rights reserved.

Patois E.,University of Geneva | Patois E.,University of Lausanne | Capelle M.A.H.,Therapeomic Inc. | Gurny R.,University of Geneva | And 4 more authors.
Vaccine | Year: 2011

The stability of different seasonal influenza vaccines was investigated by spectroscopy and microscopy methods before and after the following stress-conditions: (i) 2 and 4 weeks storage at 25°C, (ii) 1 day storage at 37°C and (iii) one freeze-thaw cycle. The subunit vaccine Influvac ® (Solvay Pharma) and the split vaccine Mutagrip ® (Sanofi Pasteur) were affected by all stresses. The split vaccine Fluarix ® (GlaxoSmithKline) was affected only by storage at 25°C. The virosomal vaccine Inflexal ® V (Berna Biotech) was stable after the temperature stresses but aggregated after one freeze-thaw cycle. This study provides new insights into commercial vaccines of low antigen concentration and highlights the importance of using multiple techniques to assess vaccine stability. © 2011 Elsevier Ltd.

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