The spectra were recorded with a 0

The spectra were recorded with a 0.1 second integration time per 1 nm increment. could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical eCF506 methods presented in this study were able to detect and characterize trastuzumab aggregates. Key words: immunoglobulin, aggregation, stability, protein, trastuzumab, herceptin?, methodology Introduction During storage and before eCF506 administration, therapeutic proteins are subject to degradation by many factors such as deamidation, racemization, isomerization, oxidation and aggregation.1,2 Stabilization of protein pharmaceuticals for long-term storage is often achieved by lyophilization.3 Special precautions should be taken to make sure the integrity of the product during reconstitution and until administration. The presence of seeds in the reconstituted answer can lead to the formation of visible particles.4 To avoid aggregate formation, the nature of the solutions used for reconstitution and for dilution before administration must be carefully chosen. Both the excipients used during the lyophilization process and the excipients used during the reconstitution process should make sure the stability of the Rabbit polyclonal to ACMSD protein. The reconstitution process in itself can also influence aggregation. Reconstitution instructions for therapeutic immunoglobulins often state that shaking should be avoided and in several cases (e.g., trastuzumab, infliximab, palivizumab and efalizumab) state that the solvent should be added slowly. A slow dissolution process has been shown to decrease the aggregate content of a protein after reconstitution.5 Shaking causes foaming if detergents are present in the formulation, hampering a precise dosage of the drug. Furthermore, shaking can induce aggregation by increasing the water-air interface and by inducing mechanical stress.6,7 As underlined in the Package eCF506 Insert,8 trastuzumab (Herceptin?) may be sensitive to mechanical stress induced by agitation or rapid expulsion from a syringe. Inappropriate protein handling can thus occur at several stages preceding administration, which can result in aggregation, further leading to reduced efficacy and potential eCF506 side effects.9C11 Analytical techniques should be able to detect the changes in the aggregation state of therapeutic proteins resulting from mishandling. In the present paper, a combination of analytical techniques was evaluated on trastuzumab, a therapeutic immunoglobulin whose sales value is ranked among the top biotech drugs.12,13 The FDA-approved Package Insert8 says that after reconstitution, trastuzumab must be diluted in an infusion bag containing 0.9% sodium chloride. The use of 5% dextrose is usually prohibited without providing further details. The explanation is however available in the Summary of Product Characteristics approved by the EMEA,14 which says that dextrose solutions cause aggregation of the protein. Asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) measurements were performed on trastuzumab diluted in 0.9% sodium chloride or in 5% dextrose, a solution causing aggregation. Our data show that if the analytical conditions are carefully chosen, the methods presented in this paper allow the detection and characterization of trastuzumab aggregates in 5% dextrose. Results Investigation of trastuzumab aggregation by FFF. Trastuzumab was diluted in 0.9% sodium chloride or 5% dextrose (final concentration 1.28 mg/ml) and different FFF experimental conditions were tested to investigate protein aggregation. Characterization was performed on both solutions within their published lifetime of 24 hours after dilution. When trastuzumab samples were injected in the FFF channel using a standard separation method (Table 1, Method 1) and with 0.9% sodium chloride as running buffer (carrier liquid), no difference could be seen between trastuzumab diluted in sodium chloride and diluted in dextrose (Fig. 1). The main peak had a molecular weight of 160 kDa. A limited number of small aggregates, mostly dimers, could be detected after 13 minutes of elution: 0.69% for trastuzumab diluted in 0.9% sodium chloride (NaCl-trastuzumab) and 0.64% for trastuzumab diluted in 5% dextrose (dextrose-trastuzumab). Based on the data presented in Physique 1, one could conclude that no significant aggregates were present in dextrose-trastuzumab solutions. Open in a separate window Physique 1 Trastuzumab diluted in 0.9% NaCl (A) and in 5% dextrose (B) analysed by FFF. Trastuzumab concentration was 1.28 mg/ml and the analysis was performed with 0.9% NaCl as the running buffer (carrier liquid). Separation started at 5 minutes elution time and stopped at 14 minutes elution time. Molecular weights (thick lines, left scale) are superimposed around the UV signals (thin lines, right scale). The monomer peak at 11.5 minutes shows a eCF506 molecular weight of 160 kDa. The.