Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals.

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells.

Cell engineering strategies typically rely on energy-consuming overexpression of genes or radical gene-knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine-tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation.

N-linked glycosylation is one of the most important post-translational modifications of monoclonal antibodies (mAbs) and is considered to be a critical quality attribute (CQA), as the glycan composition often has immunomodulatory effects. Since terminal galactose residues of mAbs can affect antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC) activation, serum half-life, and antiviral activity it has to be monitored, controlled and modulated to ensure therapeutic effects. The ability of small noncoding microRNAs (miRNAs) to modulate glycosylation in Chinese hamster ovary (CHO) production cells was recently reported establishing miRNAs as engineering tools for modulation of protein glycosylation. In this study, we report the characterization and validation of miRNAs as engineering tools for increased (mmu-miR-452-5p, mmu-miR-193b-3p) or decreased (mmu-miR-7646-5p, mmu-miR-7243-3p, mmu-miR-1668, mmu-let-7c-1-3p, mmu-miR-7665-3p, mmu-miR-6403) degree of galactosylation. Furthermore, the biological mode of action regulating gene expression of the galactosylation pathway was characterized as well as their influence on bioprocess-related parameters. Most important, stable plasmid-based overexpression of these miRNAs represents a versatile tool for engineering N-linked galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production.

Augmented region of interest for untargeted metabolomics mass spectrometry (AriumMS) of multi-platform-based CE-MS and LC-MS data.

In mass spectrometry (MS)-based metabolomics, there is a great need to combine different analytical separation techniques to cover metabolites of different polarities and apply appropriate multi-platform data processing. Here, we introduce AriumMS (augmented region of interest for untargeted metabolomics mass spectrometry) as a reliable toolbox for multi-platform metabolomics. AriumMS offers augmented data analysis of several separation techniques utilizing a region-of-interest algorithm. To demonstrate the capabilities of AriumMS, five datasets were combined. This includes three newly developed capillary electrophoresis (CE)-Orbitrap MS methods using the recently introduced nanoCEasy CE-MS interface and two hydrophilic interaction liquid chromatography (HILIC)-Orbitrap MS methods. AriumMS provides a novel mid-level data fusion approach for multi-platform data analysis to simplify and speed up multi-platform data processing and evaluation. The key feature of AriumMS lies in the optimized data processing strategy, including parallel processing of datasets and flexible parameterization for processing of individual separation methods with different peak characteristics. As a case study, Saccharomyces cerevisiae (yeast) was treated with a growth inhibitor, and AriumMS successfully differentiated the metabolome based on the augmented multi-platform CE-MS and HILIC-MS investigation. As a result, AriumMS is proposed as a powerful tool to improve the accuracy and selectivity of metabolome analysis through the integration of several HILIC-MS/CE-MS techniques.

A novel system for glycosylation engineering by natural and artificial miRNAs.

N-linked glycosylation is a crucial post-translational modification of many biopharmaceuticals, including monoclonal antibodies (mAbs), capable of modifying their biological effect in patients and thus considered as a critical quality attribute (CQA). However, expression of desired and consistent glycosylation patterns remains a constant challenge for the biopharmaceutical industry and constitutes the need for tools to engineer glycosylation. Small non-coding microRNAs (miRNAs) are known regulators of entire gene networks and have therefore the potential of being used as tools for modulation of glycosylation pathways and for glycoengineering. Here, we demonstrate that novel identified natural miRNAs are capable of altering N-linked glycosylation patterns on mAbs expressed in Chinese hamster ovary (CHO) cells. We established a workflow for a functional high-throughput screening of a complete miRNA mimic library and identified 82 miRNA sequences affecting various moieties including galactosylation, sialylation, and α-1,6 linked core-fucosylation, an important glycan feature influencing antibody-dependent cytotoxicity (ADCC). Subsequent validation shed light on the intra-cellular mode of action and the impact on the cellular fucosylation pathway of miRNAs reducing core-fucosylation. While multiplex approaches increased phenotypic effects on the glycan structure, a synthetic biology approach utilizing rational design of artificial miRNAs further enhanced the potential of miRNAs as novel, versatile and tune-able tools for engineering of N-linked glycosylation pathways and expressed glycosylation patterns towards favourable phenotypes.

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10-15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 µg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 µg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low µg/mL-range out of cell supernatant without any sample preparation.

Complexation of daclatasvir by single isomer methylated ß-cyclodextrins studied by capillary electrophoresis, NMR spectroscopy and mass spectrometry.

In capillary electrophoresis an enantioseparation of daclatasvir (DCV) was observed in case of heptakis(2,6-di-O-methyl)-ß-CD, heptakis(2-O-methyl)-ß-CD and ß-CD, while two peaks with a plateau were noted for heptakis(2,3,6-tri-O-methyl)-ß-CD and heptakis(2,3-di-O-methyl)-ß-CD indicating a slow equilibrium. Heptakis(6-O-methyl)-ß-CD and heptakis(3-O-methyl)-ß-CD yielded broad peaks. Nuclear magnetic resonance experiments including nuclear Overhauser effect-based techniques revealed inclusion complex formation for all CDs with the biphenyl ring of DCV within the cavity and the valine-pyrrolidine moieties protruding from the torus. However, in case of heptakis(2,6-di-O-methyl)-ß-CD, heptakis(2-O-methyl)-ß-CD and ß-CD higher order structures with 1:3 stoichiometry were concluded, where the valine moieties enter additional CD molecules via the secondary side. Heptakis(2,3,6-tri-O-methyl)-ß-CD and heptakis(2,3-di-O-methyl)-ß-CD yielded primarily 1:1 complexes. Higher order complexes between DCV and heptakis(2,6-di-O-methyl)-ß-CD were corroborated by mass spectrometry. Complex stoichiometry was not the reason for the slow equilibrium yielding the plateau observed in capillary electrophoresis, but structural characteristics of the CDs especially complete methylation of the secondary rim.

In3SbTe2 as a programmable nanophotonics material platform for the infrared.

The high dielectric optical contrast between the amorphous and crystalline structural phases of non-volatile phase-change materials (PCMs) provides a promising route towards tuneable nanophotonic devices. Here, we employ the next-generation PCM In3SbTe2 (IST) whose optical properties change from dielectric to metallic upon crystallization in the whole infrared spectral range. This distinguishes IST as a switchable infrared plasmonic PCM and enables a programmable nanophotonics material platform. We show how resonant metallic nanostructures can be directly written, modified and erased on and below the meta-atom level in an IST thin film by a pulsed switching laser, facilitating direct laser writing lithography without need for cumbersome multi-step nanofabrication. With this technology, we demonstrate large resonance shifts of nanoantennas of more than 4 µm, a tuneable mid-infrared absorber with nearly 90% absorptance as well as screening and nanoscale "soldering" of metallic nanoantennas. Our concepts can empower improved designs of programmable nanophotonic devices for telecommunications, (bio)sensing and infrared optics, e.g. programmable infrared detectors, emitters and reconfigurable holograms.

Unusual complexation behavior between daclatasvir and γ-Cyclodextrin. A multiplatform study.

During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV-γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV.