Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Fast pyrolysis of 13C-labeled cellobioses: gaining insights into the mechanisms of fast pyrolysis of carbohydrates.

A fast-pyrolysis probe/tandem mass spectrometer combination was utilized to determine the initial fast-pyrolysis products for four different selectively (13)C-labeled cellobiose molecules. Several products are shown to result entirely from fragmentation of the reducing end of cellobiose, leaving the nonreducing end intact in these products. These findings are in disagreement with mechanisms proposed previously. Quantum chemical calculations were used to identify feasible low-energy pathways for several products. These results provide insights into the mechanisms of fast pyrolysis of cellulose.

Development of a high-throughput laser-induced acoustic desorption probe and raster sampling for laser-induced acoustic desorption/atmospheric pressure chemical ionization.

Laser-induced acoustic desorption (LIAD) was recently coupled to atmospheric pressure chemical ionization (APCI) and shown to be of great utility for the analysis of a variety of thermally labile nonpolar analytes that are not amenable to ionization via electrospray ionization, such as nonvolatile hydrocarbons. Despite these advancements, LIAD still suffered from several limitations, including only being able to sample a small fraction of the analyte molecules deposited on a Ti foil for desorption, poor reproducibility, as well as limited laser power throughput to the backside of the foil. These limitations severely hinder the analysis of especially challenging analytes, such as asphaltenes. To address these issues, a novel high-throughput LIAD probe and an assembly for raster sampling of a LIAD foil were designed, constructed, and tested. The new probe design allows 98% of the initial laser power to be realized at the backside of the foil over the 25% achieved previously, thus improving reproducibility and allowing for the analysis of large nonvolatile analytes, including asphaltenes. The raster assembly provided a 5.7 fold increase in the surface area of a LIAD foil that could be sampled and improved reproducibility and sensitivity for LIAD experiments. The raster assembly can also improve throughput as foils containing multiple analytes can be prepared and analyzed.

Laser-induced acoustic desorption (LIAD) mass spectrometry.

Large thermally labile molecules were not amenable to mass spectrometric analysis until the development of atmospheric pressure evaporation/ionization methods, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), since attempts to evaporate these molecules by heating induces degradation of the sample. While ESI and MALDI are relatively soft desorption/ionization techniques, they are both limited to preferential ionization of acidic and basic analytes. This limitation has been the driving force for the development of other soft desorption/ionization techniques. One such method employs laser-induced acoustic desorption (LIAD) to evaporate neutral sample molecules into mass spectrometers. LIAD utilizes acoustic waves generated by a laser pulse in a thin metal foil. The acoustic waves travel through the foil and cause desorption of neutral molecules that have been deposited on the opposite side of the foil. One of the advantages of LIAD is that it desorbs low-energy molecules that can be ionized by a variety of methods, thus allowing the analysis of large molecules that are not amenable to ESI and MALDI. This review covers the generation of acoustic waves in foils via a laser pulse, the parameters affecting the generation of acoustic waves, possible mechanisms for desorption of neutral molecules, as well as the various uses of LIAD by mass spectrometrists. The conditions used to generate acoustic or stress waves in solid materials consist of three regimes: thermal, ablative, and constrained. Each regime is discussed, in addition to the mechanisms that lead to the ablation of the metal from the foil and generation of acoustic waves for two of the regimes. Previously proposed desorption mechanisms for LIAD are presented along with the flaws associated with some of them. Various experimental parameters, such as the exact characteristics of the laser pulse and foil used, are discussed. The internal and kinetic energy of the neutral desorbed molecules are also considered. Our research group has been instrumental in the development and use of LIAD. For example, we have systematically examined the influence of many parameters, such as the type of the foil and its thickness, as well as the analyte layer's thickness, on the efficiency of desorption of neutral molecules. The coupling of LIAD with different instruments and ionization techniques allows for broad use of LIAD in our research laboratories. The most important applications involve analytes that cannot be analyzed by using other mass spectrometric methods, such as large saturated hydrocarbons and heavy hydrocarbon fractions of petroleum. We also use LIAD to characterize lipids, peptides, and oligonucleotides. Fundamental research on the reactions of charged mono-, bi-, and polyradicals with biopolymers, especially oligonucleotides, also requires the use of LIAD, as well as thermochemical measurements for neutral biopolymers. These are but a few of the uses of LIAD in our research group.