Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product ...quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.
The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. ...Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide-alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher's expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.
A strained aza-dibenzocyclooctyne was prepared via a high-yielding synthetic route. Copper-free, strain-promoted click reaction with azides showed excellent kinetics, and a functionalised ...aza-cyclooctyne was applied in fast and efficient PEGylation of enzymes.
Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess ...advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.
Methods for site-selective chemistry on proteins are in high demand for the synthesis of chemically modified biopharmaceuticals, as well as for applications in chemical biology, biosensors and more. ...Inadvertent N-terminal gluconoylation has been reported during expression of proteins with an N-terminal His tag. Here we report the development of this side-reaction into a general method for highly selective N-terminal acylation of proteins to introduce functional groups. We identify an optimized N-terminal sequence, GHHH
- for the reaction with gluconolactone and 4-methoxyphenyl esters as acylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acylation with the 4-methoxyphenyl esters. This Gly-His
tag adds the unique capability for highly selective N-terminal chemical acylation of expressed proteins. We anticipate that it can find wide application in chemical biology and for biopharmaceuticals.
Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious ...diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.
Improved therapies are needed against snakebite envenoming, which kills and permanently disables thousands of people each year. Recently developed neutralizing monoclonal antibodies against several ...snake toxins have shown promise in preclinical rodent models. Here, we use phage display technology to discover a human monoclonal antibody and show that this antibody causes antibody-dependent enhancement of toxicity (ADET) of myotoxin II from the venomous pit viper, Bothrops asper, in a mouse model of envenoming that mimics a snakebite. While clinical ADET related to snake venom has not yet been reported in humans, this report of ADET of a toxin from the animal kingdom highlights the necessity of assessing even well-known antibody formats in representative preclinical models to evaluate their therapeutic utility against toxins or venoms. This is essential to avoid potential deleterious effects as exemplified in the present study.
A series of stimulus-responsive elastin-like polypeptide-poly(ethylene glycol) (ELP-PEG) block copolymers was synthesized. The polymeric building blocks were conjugated via the efficient and specific ...strain-promoted alkyne-azide cycloaddition (SPAAC). For this purpose, ELP and PEG blocks were functionalized with azide and cyclooctyne moieties, respectively. Azides were introduced by applying a recently developed pH-controlled diazotransfer reaction on the primary amines present in ELP (N-terminus and lysine side chains). By varying pH, ELP-blocks with one or two azides were obtained, which subsequently allowed us to synthesize both ELP-PEG diblock copolymers and miktoarm star polymers. Triggering the phase transition of the ELP-block resulted in the formation of an amphiphilic block copolymer, which self-assembled into micelles. This is the first example of an ELP-containing hybrid block copolymer in which PEG as the hydrophilic corona-forming domain is combined with a stimulus-responsive ELP-block. The encapsulation of a hydrophobic fluorescent dye was shown to exemplify the potential of the micelles to serve as nanocarriers for hydrophobic drugs, with the PEG corona providing stealth and steric protection of encapsulated materials.
Sensitivity of biosensors depends on the orientation of bio-receptors on the sensor surface. The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding ...site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups) to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics.
•An SPR biosensor for foot-and-mouth disease virus was built using llama antibody fragments.•Antibodies were provided with one or five azide groups by protein engineering.•Click-chemistry was used to achieve uniform or random orientation of the antibodies.•The detection limit of the uniformly-oriented biosensor was 800 fold lower compared to the randomly oriented biosensor.
Semiconductor nanowires (NWs) are gaining significant importance in various biological applications, such as biosensing and drug delivery. Efficient and controlled immobilization of biomolecules on ...the NW surface is crucial for many of these applications. Here, we present for the first time the use of the CuI‐catalyzed alkyne–azide cycloaddition and its strain‐promoted variant for the covalent functionalization of vertical NWs with peptides and proteins. The potential of the approach was demonstrated in two complementary applications of measuring enzyme activity and protein binding, which is of general interest for biological studies. The attachment of a peptide substrate provided NW arrays for the detection of protease activity. In addition, green fluorescent protein was immobilized in a site‐specific manner and recognized by antibody binding to demonstrate the proof‐of‐concept for the use of covalently modified NWs for diagnostic purposes using minute amounts of material.
Click on nanowires: A method for highly reproducible, covalent functionalization of oxidized semiconductor nanowires with peptides and proteins is reported. The method combines silanization with the CuI‐catalyzed and strain‐promoted alkyne–azide cycloaddition (CuAAC and SPAAC) reactions. A protease FRET substrate and green fluorescent protein were site‐specifically immobilized on GaAs nanowires.
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