A Robust Purity Method for Biotherapeutics Using New Peak Detection in an LC-MS-Based Multi-Attribute Method.

New peak detection (NPD), as part of the LC-MS-based multi-attribute method (MAM), allows for sensitive and unbiased detection of new or changing site-specific attributes between a sample and reference that is not possible with conventional UV or fluorescence detection-based methods. MAM with NPD can serve as a purity test that can establish whether a sample and the reference are similar. The broad implementation of NPD in the biopharmaceutical industry has been limited by the potential presence of false positives or artifacts, which increase the analysis time and can trigger unnecessary investigations of product quality. Our novel contributions to the success of NPD are the curation of false positives, use of the known peak list concept, pairwise analysis approach, and the development of a NPD system suitability control strategy. In this report, we also introduce a unique experimental design utilizing sequence variant co-mixes to measure NPD performance. We show that NPD has superior performance relative to conventional control system methods in the detection of an unexpected change as compared with the reference. NPD is a new frontier in purity testing that reduces subjectivity, need for analyst intervention, and potential for missing unexpected product quality changes.

Monitoring multiple quality attributes of a complex Fc-fusion protein during cell culture production processes by mD-LC-MS peptide mapping.

Fc-fusion proteins represent a successful class of biopharmaceutical products. They are considered highly heterogeneous products due to the common degradation of amino acids that occurs during their production in upstream and downstream processes (e.g., oxidation and deamidation) and, above all, their complex glycosylation profile. Multi-dimensional liquid chromatography-mass spectrometry (mD-LC-MS) has recently gained much interest for process analytical technology, enabling the integration of this analytical technology in production and purification environments. In this study, an online mD-LC-MS/MS peptide mapping method was developed for monitoring multiple quality attributes, including the N-glycosylation state of a complex Fc-fusion protein, which is made by combining two heavily glycosylated cytokines with an Fc domain. This fully automated workflow includes sample purification, reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis. Two immobilized enzyme cartridges based on trypsin and Lys-C protease were employed to generate a detailed glycosylation mapping, as trypsin allowed the identification of only one of four glycosylation sites, while Lys-C was more informative for two other sites. Site-specific glycosylation information such as antennarity, sialyation, and core fucosylation state was also determined. In addition to glycans, other post-translational modifications could be monitored simultaneously during the cell culture production processes by the mD-LC-MS/MS approach. In summary, the generated data demonstrate the applicability of mD-LC-MS for the monitoring and trending of multiple attributes for complex antibody formats over production processes in an automated and fast manner, compared to the complex and time-consuming traditional offline assays.

Real-time monitoring of antibody quality attributes for cell culture production processes in bioreactors via integration of an automated sampling technology with multi-dimensional liquid chromatography mass spectrometry.

Online monitoring of quality attributes (QAs) directly within the bioreactor can provide the basis for advanced modes of protein production including process intensification, smart manufacturing, and real-time release testing. The development of technologies to enable monitoring of QAs has been highly challenging due to the relative immaturity of commercial technologies for online analysis, generally low abundance of the attributes requiring highly specialized analytics not always amenable to automation, and the significant burden on development organizations to demonstrate the comparability and suitability of the online technologies resulting in low investment interest. In this study, we present for the first time a fully automated and highly flexible method for direct monitoring of QAs from the bioreactor. The method combines an automated sampling system and multi-dimensional (mD) LC-MS/MS technology to provide a means of quantifying post-translational modifications (PTMs) during the cell culture process and making real-time process decisions based on the resulting peptide mapping data. In doing so, a wide variety of PTMs can be identified and quantified including, but not limited to, oxidation, succinimidation, deamidation, isomerization, and glycosylation. The potential of this analytical workflow for the monitoring and trending of multiple attributes during cell culture production processes was first demonstrated with a standard IgG1 antibody over the production process. Then, the online workflow was applied to a complex format Fc-fusion protein to monitor sialylation. The ability to monitor sialylation offers a unique opportunity to develop process control schemes to ensure the final product meets quality specifications, showing the potential of this workflow in the context of online process analytical technology (PAT).

Novel markers to track oxidative polysorbate degradation in pharmaceutical formulations.

Polysorbates can undergo oxidative degradation in pharmaceutical formulations resulting in both soluble and insoluble degradation products. The insoluble degradants may precipitate to form subvisible and visible particulates, which are undesirable in liquid parenteral products. To date, no oxidation byproduct has been identified as an established marker to track Polysorbate 20 oxidation. Herein, we identified the aldehyde derivative of free fatty acid esters as a byproduct of polysorbate oxidation that can be derivatized using 2,4-dinitrophenylhydrazine and tracked analytically to monitor oxidative polysorbate degradation in pharmaceutical formulations.

Integrated experimental and theoretical approach for corrosion and wear evaluation of laser surface nitrided, Ti-6Al-4V biomaterial in physiological solution.

A laser based surface nitriding process was adopted to further enhance the osseo-integration, corrosion resistance, and tribological properties of the commonly used bioimplant alloy, Ti-6Al-4V. Earlier preliminary osteoblast, electrochemical, and corrosive wear studies of laser nitrided titanium in simulated body fluid clearly revealed improvement of cell adhesion as well as enhancement in corrosion and wear resistance but mostly lacked the in-depth fundamental understanding behind these improvements. Therefore, a novel integrated experimental and theoretical approach were implemented to understand the physical phenomena behind the improvements and establish the property-structure-processing correlation of nitrided surface. The first principle and thermodynamic calculations were employed to understand the thermodynamic, electronic, and elastic properties of TiN for enthalpy of formation, Gibbs free energy, density of states, and elastic properties of TiN were investigated. Additionally, open circuit potential and cyclic potentio-dynamic polarization tests were carried out in simulated body fluid to evaluate the corrosion resistance that in turn linked with the experimentally measured and computationally predicted surface energies of TiN. From these results, it is concluded that the enhancement in the corrosion resistance after laser nitriding is mainly attributed to the presence of covalent bonding via hybridization among Ti (p) and N (d) orbitals. Furthermore, mechanical properties, such as, Poisson׳s ratio, stiffness, Pugh׳s ductility criteria, and Vicker׳s hardness, predicted from first principle calculations were also correlated to the increase in wear resistance of TiN. All the above factors together seem to have contributed to significant improvement in both wear and corrosion performance of nitride surface compared to the bare Ti-6Al-4V in physiological environment indicating its suitability for bioimplant applications.

Laser induced nitrogen enhanced titanium surfaces for improved osseo-integration.

The osseo-integration, corrosion resistance, and tribological properties of the commonly used bioimplant alloy Ti-6Al-4V were enhanced using a laser-based surface nitridation process. The biomedical properties of the laser nitrided Ti-6Al-4V were investigated using experimental and computational methodologies. Electrochemical analysis of laser nitrided titanium in simulated body fluid (SBF) was performed to assess the biomedical characteristics in near-human body conditions. Additionally, the corrosive wear performance of these laser nitrided samples was evaluated using pin-on-disk geometry with a zirconia pin counter surface in SBF to mimic the biological scenario. Osteoblast studies were conducted to evaluate cell affinity towards titanium nitrided bioimplant material. Cells adhered to all substrates, with high viability. Initial cell adhesion was revealed by focal adhesion formation on all substrates. Cells can proliferate on samples treated with 1.89 and 2.12 × 10(6) J/m(2) laser conditions, while those treated with 1.70 × 10(6) J/m(2) inhibited proliferation. Thus, microstructural and phase observations, electrochemical analyses, corrosive wear evaluation, and cell behavior analysis of laser nitrided surface of bioimplant material (Ti-6Al-4V) indicated that laser nitriding greatly improves the performance of bioimplant material.