Online Coupling of Size Exclusion Chromatography to Capillary Enhanced Raman Spectroscopy for the Analysis of Proteins and Biopharmaceutical Drug Products.

The online coupling of size exclusion chromatography (SEC) to capillary enhanced Raman spectroscopy (CERS) based on a liquid core waveguide (LCW) flow cell was applied for the first time to assess the higher-order structure of different proteins. This setup allows recording of Raman spectra of the monomeric protein within complex mixtures, since SEC enables the separation of the monomeric protein from matrix components such as excipients of a biopharmaceutical product and higher molecular weight species (e.g., aggregates). The acquired Raman spectra were used for structural elucidation of well characterized proteins such as bovine serum albumin, hen egg white lysozyme, and ß-lactoglobulin and of the monoclonal antibody rituximab in a medicinal product. Additionally, the CERS detection of the disaccharide sucrose, which is used as a stabilizing excipient, was quantified to achieve a limit of detection (LOD) of 120 µg and a limit of quantification (LOQ) of 363 µg injected on the column.

In-use stability of ready-to-administer daratumumab subcutaneous injection solution in plastic syringes.

OBJECTIVE: In multiple myeloma patients, daratumumab is preferably injected subcutaneously. The summary of product characteristics of daratumumab subcutaneous injection solution specifies physicochemical stability for the prepared syringe for 24 hours at 2-8°C protected from light, and another 12 hours at room temperature (15-25°C) in ambient light conditions. The aim of this study was to determine the in-use stability of ready-to-administer daratumumab subcutaneous injection solution in different types of syringe and different conditions over a 28-day period. METHODS: Daratumumab subcutaneous (DARZALEX 1800 mg) injection solution was withdrawn into disposable three-piece Luer-Lock syringes (20 mL, 50 mL), capped, and stored light protected at 2-8°C or at room temperature (22±2°C) over a maximum period of 28 days. Samples were taken immediately after preparation (day 0) and after 2, 7, 14, 21, and 28 days. Physicochemical stability was determined by ion-exchange high-performance liquid chromatography (IE-HPLC) and size-exclusion high-performance liquid chromatography (SE-HPLC) with ultraviolet detection, pH measurement and visual inspection for particles or colour changes. RESULTS: In the IE-HPLC assay, peak areas and peak-to-peak area ratios remained unchanged over the whole study period, and showed no additional peaks of degraded daratumumab charge variants. In the SE-HPLC assay, neither a formation of aggregates nor of fragments was detected. Daratumumab monomer concentrations exceeded 95% of the initially measured concentrations over the entire test period. pH values remained constant. Test solutions remained clear, and no colour changes or visible particles were detected. All results were independent of storage conditions. CONCLUSION: Daratumumab subcutaneous injection solution proved to be physicochemically stable in capped three-piece plastic syringes for at least 28 days when stored light protected at 2-8°C or at room temperature (22±2°C). For microbiological reasons aseptic preparation and refrigerated storage are recommended. In-use stability of ready-to-administer daratumumab subcutaneous syringes prepared under appropriate aseptic conditions is given for 28 days.

Quality Control of Personalized Drug Products - Identity and Quantity of Monoclonal Antibodies as Active Pharmaceutical Ingredient.

Deliberate underdosing occurred in personalized preparations of drugs such as monoclonal antibodies as the active pharmaceutical ingredient in the past. To ensure the required quality standard and to prevent future fraud attempts at an early stage, a HPLC-DAD-HRMS method was established. Thereby, identity and quantity of the active ingredients bevacizumab, rituximab and trastuzumab were determined. The analysis of ten samples from seven pharmacies fulfilled the quality criteria and were therefore not objectionable.

Development and validation of a method for airborne monoclonal antibodies to quantify workplace exposure.

Modern therapy strategies are based on patient-specific treatment where the drug and dose are optimally adapted to the patient's needs. In recent drugs, monoclonal antibodies (mAbs) are increasingly used as active ingredients. Their patient-specific formulations are not part of the pharmaceutical industry's manufacturing process but are prepared from concentrates by pharmaceutical personnel. During the manufacturing process, however, active pharmaceutical ingredients are released in trace amounts or, in the case of accidents and spills, also in high concentrations. Regardless of the source of entry, mAbs can become airborne, be inhaled, and cause undesirable side-effects such as sensitization. To assess the risk for pharmaceutical personnel, a personal air sampling method was developed and validated for bevacizumab, cetuximab, daratumumab, omalizumab, rituximab and trastuzumab. The method is based on the combination of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The analytical method achieves a limit of detection of 0.30-8.8 ng mL-1, recoveries of 83-96 % (intra-day assay) and 75-89 % (inter-day assay), with no detectable carry-over. A polycarbonate filter proved suitable for sampling airborne monoclonal antibodies, as it achieved 80-104 % recovery across all mAbs. It also showed concentration-independent desorption efficiency. The sampling duration can be up to 480 min without negatively affecting the recovery. MAbs are stable on the polycarbonate filter at 5 °C for 3 days (recovery: 94 % ± 5 %) and at - 20 °C for 14 days (recovery: 97 % ± 4 %). Our method demonstrated that there is a potential for release when handling monoclonal antibodies. However, this can be reduced below the limit of detection by using pressure equalization systems (spikes).

Comparison of originator and biosimilar monoclonal antibodies using HRMS, Fc affinity chromatography, and 2D-HPLC.

Due to the complex manufacturing process of therapeutic monoclonal antibodies, it is hardly possible to produce an identical copy of the original product (originator). Consequently, follow-on products (biosimilars) must demonstrate their efficacy being similar to the originator in terms of structure and function. During this process, a variety of analytical methods are required for this purpose. This study focuses on three particularly relevant analytical techniques: high-resolution mass spectrometry, fragment crystallisable (Fc) affinity chromatography, and two-dimensional peptide mapping. Each analytical method proved able to identify specific differences between originator and biosimilar. High-resolution mass spectrometry was used to characterize the glycan pattern. It was shown that a trastuzumab biosimilar did not have the G0:G0F sugar modification identified in the originator. The application of affinity chromatography to rituximab showed that originator and biosimilar interacted differently with the immobilized Fc receptor. Furthermore, 2D-HPLC peptide mapping demonstrated the influence of orthogonality of separation dimensions, leading to differentiation of a rituximab originator and biosimilar.

Development of a multidimensional online method for the characterization and quantification of monoclonal antibodies using immobilized flow-through enzyme reactors.

Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20 min. Nevertheless, a previous denaturation of the protein is required, which also needs 20 min. Recoveries were determined at various concentrations and were 100% ± 1% at 100 ng on column, 96% ± 7% at 250 ng on column and 98% ± 2% at 450 ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50 ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3 ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60 °C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS).

Quality control of cytostatic drug preparations-comparison of workflow and performance of Raman/UV and high-performance liquid chromatography coupled with diode array detection (HPLC-DAD).

The drugs used for treatment during chemotherapy are manufactured individually for each patient in specialised pharmacies. Thorough quality control to confirm the identity of the delivered active pharmaceutical ingredient and the final concentration of the prepared application solution is not standardized yet except for optical or gravimetric testing. However, solution stability problems, counterfeit drugs, and erroneous or deliberate underdosage may occur and negatively influence the quality of the product and could cause severe health risks for the patient. To take a step towards analytical quality control, an on-site analytical instrument using Raman and UV absorption spectroscopy was employed and the results were compared to high-performance liquid chromatography coupled to diode array detection. Within the scope of the technology evaluation, the uncertainty of measurement was determined for the analysis of the five frequently used cytostatic drugs 5-fluorouracil, cyclophosphamide, gemcitabine, irinotecan and paclitaxel. The Raman/UV technique (2.0-3.2% uncertainty of measurement; level of confidence: 95%) achieves a combined uncertainty of measurement comparable to HPLC-DAD (1.7-3.2% uncertainty of measurement; level of confidence: 95%) for the substances 5-fluorouracil, cyclophosphamide and gemcitabine. However, the uncertainty of measurement for the substances irinotecan and paclitaxel is three times higher when the Raman/UV technique is used. This is due to the fact that the Raman/UV technique analyses the undiluted sample; therefore, the sample has a higher viscosity and tendency to foam. Out of 136 patient-specific preparations analysed within this study, 96% had a deviation of less than 10% from the target content.

Development of an analytical method to assess the occupational health risk of therapeutic monoclonal antibodies using LC-HRMS.

Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1 µg per sample for daratumumab and 25 µg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.