Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry.

Monitoring of host cell proteins (HCPs) during the manufacturing of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug products. Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. Here, we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process. While we also explored spectral library-free DIA interpretation, the spectral library-based approach still showed highest accuracy and reproducibility (coefficients of variation < 10%) with a sensitivity down to the sub-ng/mg mAb level. Thus, this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry / 药物分析学报

Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosor-bent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of pro-teins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that de-livers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more con-ventional standard protein spikes and the DIA approach was benchmarked against a classical data-dependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation＜10％)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

Analytical Quality by Design, Life Cycle Management, and Method Control.

Analytical methods are utilized throughout the biopharmaceutical and vaccines industries to conduct research and development, and to help control manufacturing inputs and outputs. These analytical methods should continuously provide quality data to support decisions while managing the remaining of risk and uncertainty. Analytical quality by design (AQbD) can provide a systematic framework to achieve a continuously validated, robust assay as well as life cycle management. AQbD is rooted in ICH guidelines Q8 and Q9 that were translated to the analytical space through several white papers as well as upcoming USP 1220 and ICH Q14. In this white paper, we expand on the previously published concepts of AQbD by providing additional context for implementation in relation to ICH Q14. Using illustrative examples, we describe the AQbD workflow, its relation to traditional approaches, and potential pathways for ongoing, real-time verification. We will also discuss challenges with respect to implementation and regulatory strategies.

In vitro permeation of chromium species through porcine and human skin as determined by capillary electrophoresis-inductively coupled plasma-sector field mass spectrometry.

Since the species that trigger chromium allergy are not yet known, it is important to gain more of an insight into the mechanism of chromium transport through the skin and into the relationship between chromium allergy and chromium species. In vitro permeation studies with porcine and human skin were performed using a Franz static diffusion cell. Investigations attempted to elucidate (i) which Cr compounds are able to permeate through skin, (ii) the influence the Cr concentration in the donor solution has on the Cr permeation, and (iii) the effect that the time of exposure to the donor solution has on Cr permeation. Capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry (CE-ICP-SFMS) was used to separate and quantify the Cr species in the receptor fluid. 50 mmol L(-1) phosphate buffer (pH 2.5) was used for CE separation, and two different electrophoretic runs were carried out (in the positive and negative modes). Pneumatic nebulization (PN)-ICP-SFMS was used in order to quantify the total amount of Cr absorbed by the skin after microwave-assisted acid digestion of the tissue. Cr(VI) was found to pass most easily through the skin. Nevertheless, Cr(VI) was also shown to be absorbed more efficiently by the skin than Cr(III), an observation attributed to a more pronounced rejection of the positively charged Cr(III) ions by the skin barrier. These results were in good agreement with in vitro permeation studies previously reported in the literature in which other analytical techniques were used. Differences observed in the permeation of Cr following the application of aqueous Cr donor solutions and Cr-containing simulated sweat donor solutions are also described.

Kangaroo vs. porcine aortic valves: calcification potential after glutaraldehyde fixation.

The aim of this study was to evaluate and compare the calcification potential of kangaroo and porcine aortic valves after glutaraldehyde fixation at both low (0.6%) and high (2.0%) concentrations of glutaraldehyde in the rat subcutaneous model. To our knowledge this is the first report comparing the time-related, progressive calcification of these two species in the rat subcutaneous model. Twenty-two Sprague-Dawley rats were each implanted with two aortic valve leaflets (porcine and kangaroo) after fixation in 0.6% glutaraldehyde and two aortic valve leaflets (porcine and kangaroo) after fixation in 2% glutaraldehyde respectively. Animals were sacrificed after 24 h and thereafter weekly for up to 10 weeks after implantation. Calcium content was determined using inductively coupled plasma-mass spectrometry and confirmed histologically. Mean calcium content per milligram of tissue (dry weight) treated with 0.6 and 2% glutaraldehyde was 116.2 and 110.4 microg/mg tissue for kangaroo and 95.0 and 106.8 microg/mg tissue for porcine valves. Calcium content increased significantly over time (8.8 microg/mg tissue per week) and was not significantly different between groups. Regression analysis of calcification over time showed no significant difference in calcification of valves treated with 0.6 or 2% glutaraldehyde within and between the two species. Using the subcutaneous model, we did not detect a difference in calcification potential between kangaroo and porcine aortic valves treated with either high or low concentrations of glutaraldehyde.

Identification of the major selenium compound, Se-Methionine, in three yeast (Saccharomyces cerevisiae) dietary supplements by on-line narrowbore liquid chromatography-electrospray tandem mass spectrometry.

On-line monitoring of six Se-compounds was accomplished by using an XTerra MS C18 column coupled to electrospray tandem mass spectrometry (ES-MS-MS). In view of the nature of the compounds, the positively charged ion pairing agent tetraethylammoniumchloride (TEACl) was added to the mobile phase. The HPLC-ES-MS-MS method was optimized with six commercially available Se-compounds. Substitution of the analytical column by the narrowbore type significantly enhanced the sensitivity of the method. We were able to detect the m/z of these six molecules on-line. Furthermore, all product ions could be monitored. The method was applied to three different yeast-based supplements. They were submitted to proteolytic digestion and screened for their Se-content by HPLC-HG-AFS (hydride generation-atomic fluorescence spectrometry). By application of on-line narrowbore HPLC-electrospray tandem mass spectrometry, the main compound present in these three supplements, Se-Methionine, could be measured on its m/z and its product ions. The method can be further extended for on-line measurement of different Se-species in complex matrices

Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the detection of chromium species after incubation of chromium in simulated sweat.

The presence of chromium in chromium-tanned leather represents a considerable health problem since it can lead to chronic allergic contact dermatitis. Apart from trivalent chromium (Cr(III)), which is used for tanning, leather often contains hexavalent chromium (Cr(VI)), resulting from the oxidation of Cr(III) during the tanning process. This study deals with the chromium compounds in simulated sweat when brought into contact with Cr(III) or Cr(VI) and with chromium-tanned leathers. A capillary electrophoresis (CE) method was developed, with inductively coupled plasma-sector field-mass spectrometry (ICP-SF-MS) for element-specific detection. Two different electrophoretic runs, applying once the positive and once the negative polarity mode, were necessary for the detection of positively and negatively charged chromium species. Although sometimes described in the literature, a pre-run derivatization of the chromium-species was not performed here to prevent species transformation. 50 mmol.L(-1) sodium phosphate at a pH of 2.5 was used as CE separation buffer and as make-up liquid for the CE-ICP-SF-MS interface. When applied to simulated sweat samples incubated with Cr(VI), this method showed that methionine is responsible for the reduction of Cr(VI) into Cr(III), which, at its turn, forms a complex with lactic acid. In the case of sweat plus Cr(III), the latter step was also seen. Applied to simulated sweat in contact with leather samples, the method developed showed the presence of the former species among a much more complex pattern.

Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation.

Vanadium speciation in serum by means of blue native gel electrophoresis.

Slab-gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium-protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the (48)V radiotracer and the phosphor-screen technology. The method was first developed using transferrin, incubated with (48)V, as a model. After it was proved that the vanadium-transferrin complex was stable during separation, the method was validated by separating serum incubated with (48)V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations.

Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anion-exchange chromatography.

Male Wistar rats were intraperitoneally injected with [(48)V]vanadium tracer to (1) investigate the distribution of vanadium over different tissues and (2) study the distribution of vanadium over the proteins and peptides in serum, packed cells and homogenates of tissues by means of liquid chromatography experiments (size exclusion, ion exchange). Target organs were primarily kidney, bone, spleen and liver. In serum we found that vanadium was mainly bound to transferrin; however, a small amount was also bound to albumin. Besides these two complexes, a significant part of vanadium occurred as readily exchangeable ("free") vanadium. In packed cells, vanadium is mainly bound to hemoglobin and to two abundant low molecular mass complexes. The chromatograms of tissues (kidney, liver, testes, spleen and lung) show similar high molecular mass complexes (vanadium co-elutes with ferritin, transferrin and hemoglobin). Between the low molecular mass complexes there are similar peaks for spleen, testes and kidneys on the one hand, and liver and lung on the other hand, albeit the differences are small. In the case of lung, there is an additional low molecular mass peak.