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 subcloning of a clonal cell line demonstrates cellular heterogeneity that does not impact process consistency or robustness.

During development of a cell line intended to support production of an IgG2 monoclonal antibody, a sequence variant caused by a genetic mutation was identified in the bulk drug substance. Gene copy number analysis together with the level of the observed variant in genomic DNA indicated that the master cell bank was a mixed population of cells; some harboring the variant copy and some mutation free. Since the cell bank had been single-cell cloned, this variant could be used as a biomarker to demonstrate either that the bank was not derived from a single cell, or that the variant was a result of a post-cloning genetic event, leading to a mixed population of cells. The sequence variant was only present in a small percentage of subclones, confirming the hypothesis that the cell bank was indeed a mixed population. Interrogation of subclones via Southern blot analysis revealed that almost all subclones had very similar transgene integrant structures, suggesting that the cell bank was likely derived from a single cell, and the cellular event that yielded the sequence variant was a post-cloning event. Further, there were likely several other post-cloning events that impacted transgene loci, leading to a population of related, yet genetically distinct cells comprising the cell bank. Despite this, the heterogeneous bank performed consistently in a bioprocess across generational age with comparable product quality. These results experimentally demonstrate the heterogeneity of a cell bank derived from a single cell, and its relationship to process consistency. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:602-612, 2018.

Characterization and Higher-Order Structure Assessment of an Interchain Cysteine-Based ADC: Impact of Drug Loading and Distribution on the Mechanism of Aggregation.

The impact of drug loading and distribution on higher order structure and physical stability of an interchain cysteine-based antibody drug conjugate (ADC) has been studied. An IgG1 mAb was conjugated with a cytotoxic auristatin payload following the reduction of interchain disulfides. The 2-D LC-MS analysis shows that there is a preference for certain isomers within the various drug to antibody ratios (DARs). The physical stability of the unconjugated monoclonal antibody, the ADC, and isolated conjugated species with specific DAR, were compared using calorimetric, thermal, chemical denaturation and molecular modeling techniques, as well as techniques to assess hydrophobicity. The DAR was determined to have a significant impact on the biophysical properties and stability of the ADC. The CH2 domain was significantly perturbed in the DAR6 species, which was attributable to quaternary structural changes as assessed by molecular modeling. At accelerated storage temperatures, the DAR6 rapidly forms higher molecular mass species, whereas the DAR2 and the unconjugated mAb were largely stable. Chemical denaturation study indicates that DAR6 may form multimers while DAR2 and DAR4 primarily exist in monomeric forms in solution at ambient conditions. The physical state differences were correlated with a dramatic increase in the hydrophobicity and a reduction in the surface tension of the DAR6 compared to lower DAR species. Molecular modeling of the various DAR species and their conformers demonstrates that the auristatin-based linker payload directly contributes to the hydrophobicity of the ADC molecule. Higher order structural characterization provides insight into the impact of conjugation on the conformational and colloidal factors that determine the physical stability of cysteine-based ADCs, with implications for process and formulation development.

Characterization of Intact Antibody Drug Conjugate Variants Using Microfluidic Capillary Electrophoresis-Mass Spectrometry.

In this work, we utilize capillary electrophoresis-mass spectrometry (CE-MS) in an integrated microfluidic platform to analyze an intact, lysine-linked antibody drug conjugate (ADC) in order to assess post translational modifications and drug load variants. The initial charge heterogeneity of the unconjugated IgG-2 monoclonal antibody (mAb) was assessed by separating intact charge variants. Three main charge variants were resolved in the CE dimension. These variants were attributed to pyroglutamic acid formation and decarboxylation on the primary structure of the mAb through characteristic mass shifts and changes in electrophoretic mobility. Additionally, glycoforms of the antibody charge variants were identified in the deconvoluted mass spectra. The observed glycoforms and their distribution compared favorably to a released N-glycan analysis performed on the mAb. After conjugation, the ADC was analyzed using the same microchip CE-MS method. The addition of a drug load resulted in a decrease in mobility and an increase in mass of 3145 Da. Five main species that differed in their respective drug-to-antibody ratios (DAR) were fully resolved in the CE separation, with each DAR displaying the same variant population observed on the unconjugated mAb. A DAR range of 0-4 was observed with an average of 1.7 drug loads. The DAR distribution generated from the microfluidic CE-MS data compared favorably to results from infusion-ESI-MS and imaging CE (iCE) analysis of the ADC, techniques commonly used for intact mAb and ADC characterization.

Hybrid capillary/microfluidic system for comprehensive online liquid chromatography-capillary electrophoresis-electrospray ionization-mass spectrometry.

A hybrid multidimensional separation system was made by coupling capillary liquid chromatography (LC) to a microfluidic device. The microfluidic device integrated flow splitting, capillary electrophoresis (CE), electroosmotic pumping, and electrospray ionization (ESI) emitter functional elements. The system was used with a time-of-flight mass spectrometer for comprehensive online LC-CE-MS of proteolytic digests. Analysis of a complex mixture of peptides yielded a peak capacity of approximately 1400 in 50 min. Three replicate runs demonstrated mean reproducibility for LC retention and CE migration times of 0.32% and 0.75% relative standard deviation (RSD), respectively. The same LC-CE-MS method was also used to characterize the N-linked glycosylation of a monoclonal antibody. Glycopeptides from two different N-linked glycosylation sites were separated from all other tryptic peptides and identified using MS data. The relative amounts of each glycoform and total site occupancy were quantified using LC-CE-MS data.

Quantitative chiral analysis of salsolinol in different brain regions of rats genetically predisposed to alcoholism.

A method to determine the catecholamine content in putamen (CPU) and midbrain (MB) regions of the brain of alcohol-preferring rats (P) is presented with a focus on the low-level detection of S,R-salsolinol, a metabolite of dopamine and a putative alcoholism marker. The developed strategy allows both quantitative profiling of related catecholamines and the enantiomeric separation and quantification of the S- and R-salsolinol isomers and their ratios. The described LC/MS strategy simplifies the current methodology that typically employs GC-MS by eliminating the need for derivatization. The data also suggest an increase in the non-enzymatic formation of salsolinol as a consequence of ethanol exposure.

Electrophoretic analysis of N-glycans on microfluidic devices.

We report rapid and efficient electrophoretic separations of N-glycans on microfluidic devices. Using a separation length of 22 cm and an electric field strength of 750 V/cm, analysis times were less than 3 min, and separation efficiencies were between 400,000 and 655,000 plates for the N-glycans and up to 960,000 plates for other sample components. These high efficiencies were necessary to separate N-glycan positional isomers derived from ribonuclease B and linkage isomers from asialofetuin. Structural isomers of N-glycans derived from a blood serum sample of a cancer patient were also analyzed to demonstrate that clinically relevant, complex samples could be separated on-chip with efficiencies similar to those derived from model glycoproteins. In addition, we compared microchip and capillary electrophoresis under similar separation conditions, and the microchips performed as well as the capillaries. These results confirmed that the noncircular cross section of the microchannel did not hamper separation performance. For all experiments, the glycan samples were derivatized with 8-aminopyrene-1,3,6-trisulfonic acid to impart needed charge for electrophoresis and a fluorescent label for detection.

Dominant-negative histone H3 lysine 27 mutant derepresses silenced tumor suppressor genes and reverses the drug-resistant phenotype in cancer cells.

Histone modifications and DNA methylation are epigenetic phenomena that play a critical role in many neoplastic processes, including silencing of tumor suppressor genes. One such histone modification, particularly at H3 and H4, is methylation at specific lysine (K) residues. Whereas histone methylation of H3-K9 has been linked to DNA methylation and aberrant gene silencing in cancer cells, no such studies of H3-K27 have been reported. Here, we generated a stable cell line overexpressing a dominant-negative point mutant, H3-K27R, to examine the role of that specific lysine in ovarian cancer. Expression of this construct resulted in loss of methylation at H3-K27, global reduction of DNA methylation, and increased expression of tumor suppressor genes. One of the affected genes, RASSF1, was shown to be a direct target of H3-K27 methylation-mediated silencing. By increasing DNA-platinum adduct formation, indicating increased access of the drug to target DNA sequences, removal of H3-K27 methylation resensitized drug-resistant ovarian cancer cells to the chemotherapeutic agent cisplatin. This increased platinum-DNA access was likely due to relaxation of condensed chromatin. Our results show that overexpression of mutant H3-K27 in mammalian cells represents a novel tool for studying epigenetic mechanisms and the Histone Code Hypothesis in human cancer. Such findings show the significance of H3-K27 methylation as a promising target for epigenetic-based cancer therapies.

Determination of salsolinol and related catecholamines through on-line preconcentration and liquid chromatography/atmospheric pressure photoionization mass spectrometry.

A new analytical approach has been developed for simultaneous measurements of endogenous salsolinol and major catecholamines in brain tissue of experimental animals. This procedure involves a combination of on-line phenyl boronate affinity preconcentration and microcolumn liquid chromatography, followed by mass spectrometry equipped with an atmospheric pressure photoionization (APPI) source. Flow conditions of the APPI source were optimized for detection sensitivity while different dopants were evaluated. The on-line preconcentration was found essential for the sensitivity requirements of salsolinol measurements in the brain tissue from alcohol-preferring rats subjected to different levels of alcohol exposure.

Determination of trace isoflavone phytoestrogens in biological materials by capillary electrochromatography.

Capillary electrochromatography using a specialty monolithic matrix was utilized in developing a rapid and highly efficient separation of isoflavones in biological materials. Without a preconcentration technique, it is relatively easy to reach ppm-ppb concentrations of these compounds in soy-based foods and verify them structurally using a photodiode array detector. With on-column preconcentration, we were able to measure low-ppb levels in human serum. Using blood samples from human volunteers, whose diet was supplemented by a soy-based product, the method has been validated for high-throughput screening of isoflavones in clinical studies.