CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols.

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Generic Chemometric Models for Metabolite Concentration Prediction Based on Raman Spectra.

Chemometric models for on-line process monitoring have become well established in pharmaceutical bioprocesses. The main drawback is the required calibration effort and the inflexibility regarding system or process changes. So, a recalibration is necessary whenever the process or the setup changes even slightly. With a large and diverse Raman dataset, however, it was possible to generate generic partial least squares regression models to reliably predict the concentrations of important metabolic compounds, such as glucose-, lactate-, and glutamine-indifferent CHO cell cultivations. The data for calibration were collected from various cell cultures from different sites in different companies using different Raman spectrophotometers. In testing, the developed "generic" models were capable of predicting the concentrations of said compounds from a dilution series in FMX-8 mod medium, as well as from an independent CHO cell culture. These spectra were taken with a completely different setup and with different Raman spectrometers, demonstrating the model flexibility. The prediction errors for the tests were mostly in an acceptable range (<10% relative error). This demonstrates that, under the right circumstances and by choosing the calibration data carefully, it is possible to create generic and reliable chemometric models that are transferrable from one process to another without recalibration.

Non-invasive and time-resolved measurement of the respiration activity of Chinese hamster ovary cells enables prediction of key culture parameters in shake flasks.

BACKGROUND: Shake flasks are frequently used for mammalian cell suspension cultures. For process development and routine culture monitoring, information on culture behavior is needed early on. MAIN METHODS AND MAJOR RESULTS: Here, cell-specific oxygen uptake rates (qO2 ) of two CHO cell lines were determined from shake flask experiments by simultaneous measurement of oxygen transfer rates (OTR) and viable cell concentrations (VCC). For cell line one, qO2 decreased from 2.38·10-10  to 1.02·10-10  mmol cell-1  h-1 during batch growth. For cell line two, qO2 was constant (1.90·10-10  mmol h-1 ). Determined qO2 values were used to calculate the VCC from OTR data. Cumulated oxygen consumption and glucose consumption were correlated for both cell lines and enabled calculation of glucose concentrations from OTR data. IgG producing cell line one had an oxygen demand of ∼15 mmoloxygen gglucose -1 , cell line two consumed ∼5 mmoloxygen gglucose -1 . The established correlations for determination of VCC and glucose were successfully transferred to subsequent cultivations for both cell lines. Combined measurement of the OTR and the carbon dioxide transfer rate enabled quantitative determination of the lactate concentration (production and consumption) without sampling. CONCLUSIONS AND IMPLICATIONS: Taken together, non-invasive measurement of the respiration activity enabled time-resolved determination of key culture parameters for increased process understanding in shake flasks.

Time-Resolved Monitoring of the Oxygen Transfer Rate of Chinese Hamster Ovary Cells Provides Insights Into Culture Behavior in Shake Flasks.

Cultivations of mammalian cells are routinely conducted in shake flasks. In contrast to instrumented bioreactors, reliable options for non-invasive, time-resolved monitoring of the culture status in shake flasks are lacking. The Respiration Activity Monitoring Respiration Activity Monitoring System system was used to determine the oxygen transfer rate (OTR) in shake flasks. It was proven that the OTR could be regarded as equal to the oxygen uptake rate as the change of the dissolved oxygen concentration in the liquid phase over time was negligibly small. Thus, monitoring the oxygen transfer rate (OTR) was used to increase the information content from shake flask experiments. The OTR of a Chinese hamster ovary cell line was monitored by applying electrochemical sensors. Glass flasks stoppered with cotton plugs and polycarbonate flasks stoppered with vent-caps were compared in terms of mass transfer characteristics and culture behavior. Similar mass transfer resistances were determined for both sterile closures. The OTR was found to be well reproducible within one experiment (standard deviation <10%). It correlated with changes in cell viability and depletion of carbon sources, thus, giving more profound insights into the cultivation process. Culture behavior in glass and polycarbonate flasks was identical. Monitoring of the OTR was applied to a second culture medium. Media differed in the maximum OTR reached during cultivation and in the time when all carbon sources were depleted. By applying non-invasive, parallelized, time-resolved monitoring of the OTR, the information content and amount of data from shake flask experiments was significantly increased compared to manual sampling and offline analysis. The potential of the technology for early-stage process development was demonstrated.

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling.

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.

Aggregates in monoclonal antibody manufacturing processes.

Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients' health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product.

Effect of dialysis flux and membrane material on dyslipidaemia and inflammation in haemodialysis patients.

BACKGROUND: Dyslipidaemia, inflammation and oxidative stress are prominent risk factors that potentially cause vascular disease in haemodialysis patients. Dialysis modalities affect uraemic dyslipidaemia, possibly by modifying oxidative stress, but the effects of dialyser flux and membrane material on atherogenic remnant particles and oxidized low-density lipoproteins (LDL) are unknown. METHODS: We performed a randomized crossover study in 36 patients on haemodialysis to analyse the effect of dialyser flux and membrane material on plasma lipids, apolipoproteins and markers of inflammation and oxidative stress. Stable patients on low-flux dialysis with polysulphone for >/=6 weeks were assigned to high-flux polysulphone or high-flux modified cellulose with similar dialyser surface area and permeability characteristics and crossed over twice every 6 weeks. RESULTS: Thirty patients completed the study per protocol. Treatments with high-flux polysulphone and modified cellulose lowered serum triglyceride (by 20% and 10%, respectively; P<0.05) and remnant-like particle cholesterol by 32% (P<0.001) and 11% (NS) after the first 6 weeks of treatment. Oxidized LDL decreased significantly with high-flux polysulphone, but not with modified cellulose. Apolipoproteins CII and CIII were reduced, whereas the ratio CII/CIII was increased (all P<0.05). Acute-phase proteins and LDL and high-density lipoprotein cholesterol remained unaffected. CONCLUSIONS: This randomized crossover study demonstrates a potent effect of high-flux haemodialysis on uraemic dyslipidaemia. Polysulphone membrane material showed superiority on oxidatively modified LDL, an indicator of oxidative stress in haemodialysis patients.

A switch to high-flux helixone membranes reverses suppressed interferon-gamma production in patients on low-flux dialysis.

UNLABELLED: Long-term hemodialysis (HD) induces an inflammatory response and is associated with a suppressed cellular immune response manifested, in part, by impaired interferon (IFN-gamma) production. We investigated the effect of high-flux HD using the synthetic Helixone membrane and ultrafiltered dialysate on plasma levels of inflammatory mediators and on the whole blood production of IFN-gamma. METHODS: Twelve ESRD patients were dialyzed under low-flux HD (polysulfone F6) and again after 6 weeks of high-flux HD (Helixone FX100). Ultrafiltered bicarbonate dialysate without bacterial growth and no detectable endotoxin was used throughout the study. Plasma levels of urea, albumin, beta(2)-microglobulin (beta(2)-m), interleukin (IL)-6, C-reactive protein (CRP), IL-1 receptor antagonist (IL-1Ra), IL-18, and IL-18-binding protein (IL-18BP) were measured. In addition, the Staphylococcus epidermidis-induced production of IFN-gamma and IL-18 was assessed in whole blood cultures of HD patients as well as in 9 healthy subjects. RESULTS: Plasma levels of urea, albumin, IL-6, IL-1Ra and CRP were not significantly different between high-flux and low-flux HD. In contrast, beta(2)-m levels decreased significantly by 31% with high-flux Helixone (p < 0.002). Stimulated whole blood production of IFN-gamma was reduced in low-flux HD but increased to near normal levels after 6 weeks of high-flux HD. Plasma levels of free IL-18 and its specific inhibitor IL-18BP were not different between the two dialyzer membranes. CONCLUSION: Compared to low-flux polysulfone HD with ultrafiltered dialysate, high-flux HD with the synthetic Helixone membrane did not result in a significant change in plasma levels of proinflammatory (IL-6, CRP, IL-18) and anti-inflammatory (IL-1Ra, IL-18BP) cytokines. However, high-flux HD restores whole blood IFN-gamma production without significant changes in free IL-18. Therefore, the immune modulation in high-flux HD is likely due to removal of inhibitors of IFN-gamma production other than IL-18BP.