Current monoclonal antibody (mAb) biomanufacturing typically involves expression in mammalian cells during upstream processing (USP), followed by purification during downstream processing (DSP). DSP is a major bottleneck, primarily caused by the prevalent Protein A affinity chromatography capture step. Key drawbacks include the extensive clarification required to prevent clogging of the chromatography column, limited productivity due to diffusional mass transport, and the risk of mAb denaturation under acidic elution conditions. To address these challenges, we introduce the novel concept of calcium-dependent magnetic separation using iron oxide nanoparticles with covalent immobilization of an engineered calcium-dependent affinity ligand (ZCa). The ZCa ligand is employed for mild elution conditions, while the dispersed particle adsorbent promises fast mAb interaction and processing of non-clarified feeds. Using Trastuzumab as a model mAb, we confirmed the intended rapid capture directly from Chinese hamster ovary (CHO) culture and achieved high mAb purities, with reductions in DNA and host cell protein (HCP) comparable to state-of-the-art Protein A chromatography. The demonstrated potential of integrating the clarification and the capture steps is promising for improving productivity and simultaneously reducing costs. Additionally, we developed a mild and sustainable elution protocol based on citrate/NaCl buffers, reaching recoveries above 90% at pH 6.0. The functionality of Trastuzumab after the novel separation approach was proven by high physical integrity and binding functionality to the human epidermal growth factor receptor 2 (HER2). Our findings underline the high potential of calcium-dependent magnetic separation for integrated, gentle, and sustainable bioseparation of mAbs.

Protein secretion is an essential process of mammalian cells. In biomanufacturing, this process can be optimized to enhance production yields and biotherapeutic quality. While cell line engineering and bioprocess optimization have yielded high protein titers for some recombinant proteins, many remain difficult to express. Here, we investigated factors influencing protein expression in Chinese hamster ovary (CHO) cells, expressing 2,135 Human Secretome Project proteins. While the abundance of mRNA from recombinant proteins explained less than 1% of observed variation in secretion titers, analysis of 218 biochemical and biophysical descriptors uncovered intrinsic protein features that account for ~15% of secretion variability, pinpointing key drivers such as molecular weight, cysteine content, and N-linked glycosylation, and establishing a roadmap for rational design of difficult-to-express proteins. We subsequently analyzed RNA-Seq data from 95 CHO cell cultures, each expressing a distinct recombinant protein, spanning a wide range of titers. Host cell transcriptomic signatures showed strong correlations with titer, thereby providing insights into cellular processes that covary with expression. Cells failing to produce proteins exhibited increased ubiquitin-mediated proteasomal degradation, including ER-associated degradation; whereas high-producing cells demonstrated enhanced lipid metabolism and a stronger response to oxidative stress, suggesting these factors may support successful recombinant protein productions. Together, using this resource, we quantified the contributions of various protein and cellular factors that correlate with the expression of diverse recombinant human proteins in a heterologous host, thereby providing insights for next-generation CHO cell engineering.

In vitro methods for developing binding domains have been well-established for many years, owing to the cost-efficient synthesis of DNA and high-throughput selection and screening technologies. However, generating high-affinity binding domains often requires the development of focused maturation libraries for a second selection, which typically demands a detailed understanding of the binding surfaces from the initial selection, a process that can be time-consuming. In this study, we accelerated this process by using deep sequencing data from the first selection to guide the design of the maturation library. Additionally, we employed a high-throughput screening system using flow cytometry based on Escherichia coli display to identify conditional binding domains from the selection output. This approach enabled the development of a high-affinity binder targeting the cancer biomarker HER3, with a binding affinity of 3.3 nM at extracellular pH 7.4, 100 times higher than the first-generation binding domain. Notably, the binding domain features a pH-dependent release mechanism, enabling rapid release in slightly acidic environments (pH ≈6), which resemble endosomal conditions. When conjugated to the cytotoxin mertansine (DM1), the binding domain demonstrated specific cytotoxic activity against HER3-expressing cell lines, with an IC50 of 2–5 nM. The presented approach enables the efficient development of conditional binding domains which hold promise for therapeutic applications.

Current downstream processing of monoclonal antibodies (mAbs) is limited in throughput and requires harsh pH conditions for mAb elution from Protein A affinity ligands. The use of an engineered calcium-dependent ligand (ZCa) in magnetic separation applications promises improvements due to mild elution conditions, fast processability, and process integration prospects. In this work, we synthesized and evaluated three magnetic nanoparticle types immobilized with the cysteine-tagged ligand ZCa-cys. Ligand homodimers were physically immobilized onto bare iron oxide nanoparticles (MNP) and MNP coated with tetraethyl orthosilicate (MNP@TEOS). In contrast, ZCa-cys was covalently and more site-directedly immobilized onto MNP coated with (3-glycidyloxypropyl)trimethoxysilane (MNP@GPTMS) via a preferential cysteine-mediated epoxy ring opening reaction. Both coated MNP showed suitable characteristics, with MNP@TEOS@ZCa-cys demonstrating larger immunoglobulin G (IgG) capacity (196 mg g−1) and the GPTMS-coated particles showing faster magnetic attraction and higher IgG recovery (88 %). The particles pave the way for the development of calcium-dependent magnetic separation processes.

This review outlines the historical development and versatile applications of one of the most well-studied bacterial proteins, namely the immunoglobulin (Ig)-binding staphylococcal protein A (SpA) of Staphylococcus aureus. Each segment of the SpA operon, from the 5’ promoter region and signal peptide to the 3’ cell wall anchoring region, has been exploited for various innovative applications in areas such as immunology and biotechnology. We provide an overview of selected applications and concepts that have had a significant impact on life science research, and some that have also led to significant commercial implications. In the 1980s, the SpA promoter and signal sequence were utilized in Escherichia coli for recombinant production of various proteins, yielding product secretion to the culture medium and thereby simplifying product recovery. The five homologous Ig-binding domains of SpA gained tremendous interest in the late 1980s, largely due to the rise of monoclonal antibodies (mAbs) for therapeutic use, prompting a growing demand for effective affinity ligands to facilitate their purification. Over the years, these Ig-binding domains have been extensively investigated and re-engineered to bind proteins other than antibodies, leading in the mid-1990s to the development of the affibody affinity protein technology. Today, affibody molecules are being investigated in late-stage clinical trials as potential protein therapeutics for various indications. Finally, the cell wall anchoring regions of SpA inspired the development of a surface display system for Staphylococcus carnosus, which has emerged as a technology platform in combinatorial protein engineering for work with large peptide, antibody and affibody libraries.

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody binding. Hence, these variants contains molecular “on/off” switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

Due to its small size and high affinity binding, the engineered scaffold protein ADAPT6 is a promising targeting probe for radionuclide imaging of human epidermal growth factor receptor type 2 (HER2). In a Phase I clinical trial, [99mTc]Tc-ADAPT6 demonstrated safety, tolerability and capacity to visualize HER2 expression in primary breast cancer. In this study, we aimed to select the optimal parameters for distinguishing between breast cancers with high and low expression of HER2 using [99mTc]Tc-ADAPT6 in a planned Phase II study. HER2 expression was evaluated in primary tumours and metastatic axillary lymph nodes (mALNs). SPECT/CT imaging of twenty treatment-naive breast cancer patients was performed 2 h after injection of [99mTc]Tc-ADAPT6. The imaging data were compared with the data concerning HER2 expression obtained by immunohistochemical evaluation of samples obtained by core biopsy. Maximum Standard Uptake Values (SUVmax) afforded the best performance for both primary tumours and mALNs (areas under the receiver operating characteristic curve (ROC AUC) of 1.0 and 0.97, respectively). Lesion-to-spleen ratios provided somewhat lower performance. However, the ROC AUCs were still over 0.90 for both primary tumours and mALNs. Thus, lesion-to-spleen ratios should be further evaluated to find if these could be applied to imaging using stand-alone SPECT cameras that do not permit SUV calculations.

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.