Segmented flow microfluidic devices offer an attractive means of studying crystallization processes. However, while they are widely employed for protein crystallization, there are few examples of ...their use for sparingly soluble compounds due to problems with rapid device fouling and irreproducibility over longer run‐times. This article presents a microfluidic device which overcomes these issues, as this is constructed around a novel design of “picoinjector” that facilitates direct injection into flowing droplets. Exploiting a Venturi junction to reduce the pressure within the droplet, it is shown that passive injection of solution from a side‐capillary can be achieved in the absence of an applied electric field. The operation of this device is demonstrated for calcium carbonate, where highly reproducible results are obtained over long run‐times at high supersaturations. This compares with conventional devices that use a Y‐junction to achieve solution loading, where in‐channel precipitation of calcium carbonate occurs even at low supersaturations. This work not only opens the door to the use of microfluidics to study the crystallization of low solubility compounds, but the simple design of a passive picoinjector will find wide utility in areas including multistep reactions and investigation of reaction dynamics.
A microfluidic device is presented that enables highly reliable, on‐chip analysis of the precipitation of insoluble crystals in flowing droplets. At the heart of the device is a novel design of picoinjector which employs a Venturi junction to achieve injection into surfactant‐stabilized droplets. Chip operation is conducted for calcium carbonate, and the many advantages over traditional device designs are demonstrated.
Self-assembling peptide-based biomaterials are being developed for use as 3D tissue engineering scaffolds and for therapeutic drug-release applications. Chemical synthesis provides custom-made ...peptides in small quantities, but production approaches based upon transgenic organisms might be more cost-effective for large-scale peptide production. Long lead times for developing appropriate animal clones or plant lines and potential negative public opinion are obstacles to these routes. Microbes, particularly safe organisms used in the food industry, offer a more rapid route to the large-scale production of recombinant self-assembling biomaterials. In this review, recent advances and challenges in the recombinant production of collagen, elastin and de novo designed self-assembling peptides are discussed.
Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies ...remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.
Approaches for antibody discovery have seen substantial improvement and success in recent years. Yet, advancing antibodies into the clinic remains difficult because therapeutic developability ...concerns are challenging to predict. We developed a computational model to simplify antibody developability assessment and enable accelerated early-stage screening. To this end, we quantified the ability of hundreds of sequence- and structure-based descriptors to differentiate clinical antibodies that have undergone rigorous screening and characterization for drug-like properties from antibodies in the human repertoire that are not natively paired. This analysis identified 144 descriptors capable of distinguishing clinical from repertoire antibodies. Five descriptors were selected and combined based on performance and orthogonality into a single model referred to as the Therapeutic Antibody Developability Analysis (TA-DA). On a hold-out test set, this tool separated clinical antibodies from repertoire antibodies with an AUC = 0.8, demonstrating the ability to identify developability attributes unique to clinical antibodies. Based on our results, the TA-DA score may serve as an approach for selecting lead antibodies for further development.
Abbreviations:
Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS), Area Under the Curve (AUC), Complementary-Determining Region (CDR), Clinical-Stage Therapeutics (CST), Framework (FR), Monoclonal Antibodies (mAbs), Observed Antibody Space (OAS), Receiver Operating Characteristic (ROC), Size-Exclusion Chromatography (SEC), Structural Aggregation Propensity (SAP), Therapeutic Antibody Developability Analysis (TA-DA), Therapeutic Antibody Profiler (TAP), Therapeutic Structural Antibody Database (Thera-SAbDab), Variable Heavy (VH), Variable Light (VL).
The majority of ADCs in preclinical and clinical development are for oncology indications where cytotoxic payloads are targeted to antigen-expressing cancer cells. However, the modulation of ...pathogenic cellular activity via ADC-mediated delivery of bioactive small molecules is also an attractive concept for non-oncology indications leading to an expanded application of the technology. Here we summarize those ADCs that have been described so far for non-oncology applications and which cover a variety of payload mechanisms beyond cell killing, from early in vitro proof-of-concept experiments to clinical trials. As our understanding of ADC technology continues to grow, it is anticipated that the development of ADCs as therapeutics for disease areas outside of oncology will also increase.
Some 30 years after its discovery, phage display remains one of the most widely used methods of in vitro selection. Initially developed to revolutionize the generation of therapeutic antibodies, ...phage display is now the first choice for screening artificial binding proteins. Artificial binding proteins can be used as reagents to study protein-protein interactions, target posttranslational modifications, and distinguish between homologous proteins. They can also be used as research and affinity reagents, for diagnostic purposes, and as therapeutics. However, the ability to identify isoform-specific reagents remains highly challenging. We describe an adapted phage display protocol using an artificial binding protein (Affimer) for the selection of isoform-selective binding proteins.
PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They ...are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepTSo, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepTSo represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.
This study presents the crystal structure of a prokaryotic oligopeptide–proton symporter, homologous to mammalian peptide transporters, giving insights into the molecular mechanism by which this important family of proteins act.
Response to Robins et al Ankcorn, Michael J.; Ijaz, Samreen; McPherson, Stuart
Journal of viral hepatitis,
January 2023, 2023-01-00, 20230101, Letnik:
30, Številka:
1
Journal Article
Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling ...in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.
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