The structural characterization of glycoproteins both as biotherapeutic products and as endogenous biomarkers in plasma and tissues remains one of the most analytically demanding tasks in modern bioanalysis. Glycosylation is a highly complex, heterogeneous, and context-dependent post-translational modification that influences protein folding, function, pharmacokinetics, and immunogenicity. High throughput (HTP) glycomics and glycoproteomics methods have recently been developed as essential tools to dissect this complexity, and their implementation into routine workflows for industry has been tested. Accurate glycoprotein analysis requires resolving site-specific glycoform variations across batches and navigating complex plasma and tissue samples with high dynamic range and structural isomers. Tissue analysis adds challenges like limited material and extracellular matrix interference. Accurate interpretation depends on advanced bioinformatics tools and machine learning algorithms, which must navigate an enormous search space of potential glycan structures and account for variable ionization efficiencies, incomplete fragmentation, and co-eluting species. Despite these hurdles, precise glycoprotein characterization is essential for therapeutic safety and for leveraging glycans as biomarkers in diseases such as cancer and neurodegeneration. Ongoing innovation is key to advancing high-throughput glycoanalysis in clinical and biopharmaceutical settings. We have developed HTP glycomics and glycoproteomics mass spectrometry-based methods to address these challenges. We have now optimized technologies ensuring the retention of labile residues such as sialic acids and fucose, and non-carbohydrate substituents like acetylation and sulfation. We have worked out advanced workflows that combine enrichment techniques, specialized digestion and derivatization protocols, and orthogonal mass spectrometry platforms (such as LC-MS/MS with EThcD, stepped HCD and UVPD) to structurally elucidate and preserve native glycosylation patterns. These developments are a continuation of our ongoing efforts of using state-of-the-art MS instrumentation to address newly arising difficulties in glycoprotein characterization and applying these tools to assist the industry in characterizing new biologics products.

Ensuring consistent product quality in cell culture-based vaccine manufacturing requires a thorough understanding of the process parameters that affect titers, yields, and impurity levels. This study applies Quality by Design (QbD) principles to an influenza A virus (IAV) production process operated in batch mode using two monoclonal suspension MDCK cell lines, C59 and C113 (Sartorius, Germany), with distinct charcteristics, focusing on process robustness and optimization. Based on knowledge from previous process development [1], a quantitative risk assessment including biological and technical parameters was performed to identify Critical Process Parameters (CPPs). Using a Design of Experiments (DoE) approach in an Ambr® 15 scale-down system, four key CPPs (pH value, dissolved oxygen concentration, viable cell concentration at time of infection, and multiplicity of infection) were investigated at three levels. After data analysis and modeling, we obtained dedicated design spaces for each cell clone characterized by high process robustness with a less than 1% risk of failure and even some indications for virus titer and yield improvement, while keeping process-related impurities such as DNA and total protein concentration low. Scale-up experiments in a 2 L single-use stirred tank bioreactor confirmed the validity of these conditions. Total virus titers of 2.95±0.06 log10(HAU/100 µL) and 3.13±0.12 log10(HAU/100 µL) were obtained for C59 and C113 cells, respectively [2]. By applying QbD principles, this study not only improves IAV production but also demonstrates a framework applicable to manufacturing of other cell culture-based vaccines. The results provide valuable insights for optimizing manufacturing processes, reducing batch failure risks, and supporting regulatory approval through data-driven process characterization. [1] Zinnecker et al., 2024, Eng. Life Sci., https://doi.org/10.1002/elsc.202300245 [2] Zinnecker et al., 2025, Eng. Life Sci., https://doi.org/10.1002/elsc.70027

The rise in cancer, autoimmune, inflammatory, and infectious diseases in recent decades has led to a surge in the development of monoclonal antibodies (mAbs) therapies, now the most widely used family of biologics. To meet the growing global demand, biopharmaceutical industries are intensifying their production processes. One approach to achieve more efficient production of effective mAbs is to develop tools for real-time quality monitoring. Specifically, the glycosylation profile of mAbs must be closely monitored, since it greatly impacts their therapeutic efficacy and innocuity, making it a critical quality attribute. In this study, we developed a surface plasmon resonance-based integrated assay allowing for the simultaneous quantification and glycosylation characterization of mAbs in crude samples, hence permitting the at-line analysis of bioreactor cell cultures. Thanks to the high specificity of the interaction between biosensor surface-bound protein A and the Fc region of mAbs, we quantified crude IgG samples under mass transport limitations. Next, by flowing running buffer on the surface, impurities contained in the mAbs samples were washed away from the biosensor surface, allowing subsequent recording of the kinetics between the captured mAbs and injected FcγRII receptors. Of interest, with this strategy, we were able to quantify terminal galactosylation and core fucosylation of IgG lots, two important glycan modifications for mAb efficacy.

A novel monoclonal antibody (mAb) (anti-sST2) (U. Chile) with therapeutic potential for autoimmune disease Ulcerative colitis and other similar diseases with high levels of sST2 soluble protein was designed. For the production of this mAb, a possible secretory bottle neck is hyphotesized, observed at a previous analysis. Unfolded/misfolded proteins could cause reticulum stress, activating the unfolded protein response and possible apoptosis; also an increase on mAb demand could raise reactive oxygen species (ROS) leading to an accumulation of protein in the endoplasmic reticulum. To improve this scenario, process engineering approaches were studied, supplementing the culture medium with three different molecules: a novel and promissory antioxidant, a recognized antioxidant and a chemical additive, to decrease folding and aggregation problems, and with it is expected to enhance production of the novel mAb. Independent supplementation improved specific production (qP), with a value of 47%, 43.5% and 46.8% more than control for novel antioxidant, recognized antioxidant and chemical additive, respectively. Also, CHO cells were able to grow at optimal conditions on a studied range of the novel antioxidant, maintaining cell viability over 85% in culture, and improving the specific cell growth rate (µ) (0.55 1/d), a 30% higher than control, which also improved qP. As expected, supplementation of both a known antioxidant and a novel antioxidant were able to reduce intracellular ROS.

Over the past decade, cell culture media (CCM) optimization has been a key strategy for obtaining high yields and improving productivity, while ensuring product quality in biopharmaceutical production. Conventional fed-batch media formulations for CHO cell cultivation typically consist of a basal medium, a pH-neutral main feed (feed A) as well as an alkaline feed (feed B). The separate, alkaline feed B is mainly needed to dissolve the key amino acids L-cystine and L-tyrosine, which are otherwise hardly soluble around pH 7 in aqueous solutions. However, these conventional media formulations come with inherent challenges such as high process complexity based on separate preparation as well as certain quality risks resulting from potential pH spikes and operator safety concerns. The transition from dual-feed to single-feed systems in CHO cell culture is a promising strategy to streamline biopharmaceutical production. It can reduce bioprocessing complexity by decreasing the number of vessels needed for the operation and even enhance monoclonal antibody titers. This study explores how the use of the chemically defined (di)peptides N,N’-di-L-lysyl-L-cystine [(Lys-Cys)2] and glycyl-L-tyrosine (Gly-Tyr) enables a simplified system comprising only the basal medium and one feed at neutral pH.

Inducible transcriptional circuits are useful tools to fine-tune gene expression and enable real-time control over desirable cellular phenotypes. In contrast to conventional inducible systems that function in an on/off manner, Linearizer circuits enable gradual gene expression in response to increasing inducer molecule concentrations, somewhat akin to dimmer light switches. Linearizer circuits have extensive potential applications across biopharmaceutical cell culture processes, for example: (i) controlled and coordinated expression of peptide chains that form multimeric therapeutic proteins to enhance the yield of bi- and multi-specifics, (ii) inducible and tuneable expression of adeno-associated viral vector (AAV) components to maximise gene therapy vector yields, and (iii) real-time glycosylation control to enable robust quality assurance of therapeutic glycoproteins. A key challenge in deploying Linearizer circuits is that the stoichiometry of their components (repressor operon sites, repressor protein, and gene of interest) must be ensured for optimal performance (minimal basal GOI expression, fold induction, and linear dose response to inducer concentrations). This study evaluates the performance of two negatively autoregulated Linearizer circuits (TetR_Lin and PhlF_Lin) in Chinese hamster ovary (CHO) cells using two different genomic integration strategies, targeted integration (TI) and random integration (RI). Each circuit consists of a fluorescent protein reporter (eGFP or mCherry) and a repressor gene (TetR or PhlF). For RI, Linearizers were deployed using transfection and antibiotic resistance selection. For TI, orthogonal landing pads with the Cre/lox and Bxb1/att recombinase mediated cassette exchange systems were used. Our results show that RI results in high basal expression, low fold induction, and a nonlinear response across inducer concentrations. In contrast, TI achieves lower basal expression, a strong linear response to inducer molecule concentration, and a broader induction range. Our results suggest that the suboptimal performance observed with RI is likely due to stoichiometric imbalances of Linearizer circuit components, and that the issues are resolved with targeted integration. By optimising Linearizer circuit performance in CHO cells, this project paves the way towards real-time control and optimisation – at the cellular level – of biopharmaceutical cell culture processes.