scholarly journals Antibody conjugation and formulation

2019 ◽  
Vol 2 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Nathan J Alves
Keyword(s):  
Oral Delivery ◽  
Clinical Implementation ◽  
High Concentration ◽  
Site Specific ◽  
Routes Of Administration ◽  
Drug Conjugates ◽  
Large Size ◽  
Low Concentrations ◽  
Antibody Conjugates ◽  
Antibody Conjugation

ABSTRACT In an era where ultra-high antibody concentrations, high viscosities, low volumes, auto-injectors and long storage requirements are already complex problems with the current unconjugated monoclonal antibodies on the market, the formulation demands for antibody-drug conjugates (ADCs) are significant. Antibodies have historically been administered at relatively low concentrations through intravenous (IV) infusion due to their large size and the inability to formulate for oral delivery. Due to the high demands associated with IV infusion and the development of novel antibody targets and unique antibody conjugates, more accessible routes of administration such as intramuscular and subcutaneous are being explored. This review will summarize various site-specific and non-site-specific antibody conjugation techniques in the context of ADCs and the demands of formulation for high concentration clinical implementation.

scholarly journals Site-specific conjugation of native antibody

2020 ◽  
Vol 3 (4) ◽  
pp. 271-284
Author(s):  
Amissi Sadiki ◽  
Shefali R Vaidya ◽  
Mina Abdollahi ◽  
Gunjan Bhardwaj ◽  
Michael E Dolan ◽  
...  
Keyword(s):  
Selective Reduction ◽  
Stochastic Methods ◽  
Site Specificity ◽  
Specific Chemical ◽  
Site Specific ◽  
Drug Conjugates ◽  
Low Concentrations ◽  
Antibody Conjugates ◽  
Dose Dependent ◽  
Complementarity Determining Region

ABSTRACT Traditionally, non-specific chemical conjugations, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity-determining region may reduce binding affinity and specificity. Conversely, site-specific methods produce well-defined and more homogenous antibody conjugates, ensuring developability and clinical applications. Moreover, several recent side-by-side comparisons of site-specific and stochastic methods have demonstrated that site-specific approaches are more likely to achieve their desired properties and functions, such as increased plasma stability, less variability in dose-dependent studies (particularly at low concentrations), enhanced binding efficiency, as well as increased tumor uptake. Herein, we review several standard and practical site-specific bioconjugation methods for native antibodies, i.e., those without recombinant engineering. First, chemo-enzymatic techniques, namely transglutaminase (TGase)-mediated transamidation of a conserved glutamine residue and glycan remodeling of a conserved asparagine N-glycan (GlyCLICK), both in the Fc region. Second, chemical approaches such as selective reduction of disulfides (ThioBridge) and N-terminal amine modifications. Furthermore, we list site-specific antibody–drug conjugates in clinical trials along with the future perspectives of these site-specific methods.

scholarly journals Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches

2021 ◽  
Vol 14 (4) ◽  
pp. 343
Author(s):  
Ahmad Fawzi Hussain ◽  
Armin Grimm ◽  
Wenjie Sheng ◽  
Chaoyu Zhang ◽  
Marwah Al-Rawe ◽  
...  
Keyword(s):  
Therapeutic Agents ◽  
Amino Acid Residues ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Site Specific ◽  
Drug Conjugates ◽  
Complement Dependent Cytotoxicity ◽  
Antibody Conjugates ◽  
Specific Antigens ◽  
Antibody Conjugate ◽  
Therapeutic Mechanisms

In the last few decades, antibody-based diagnostic and therapeutic applications have been well established in medicine and have revolutionized cancer managements by improving tumor detection and treatment. Antibodies are unique medical elements due to their powerful properties of being able to recognize specific antigens and their therapeutic mechanisms such as blocking specific pathways, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity. Furthermore, modification techniques have paved the way for improving antibody properties and to develop new classes of antibody-conjugate-based diagnostic and therapeutic agents. These techniques allow arming antibodies with various effector molecules. However, these techniques are utilizing the most frequently used amino acid residues for bioconjugation, such as cysteine and lysine. These bioconjugation approaches generate heterogeneous products with different functional and safety profiles. This is mainly due to the abundance of lysine and cysteine side chains. To overcome these limitations, different site-direct conjugation methods have been applied to arm the antibodies with therapeutic or diagnostics molecules to generate unified antibody conjugates with tailored properties. This review summarizes some of the enzyme-based site-specific conjugation approaches.

scholarly journals Synthesis of Fluorescently Labeled Antibodies Using Non-Canonical Amino Acids in Eukaryotic Cell-Free Systems

2021 ◽  
pp. 175-190
Author(s):  
Marlitt Stech ◽  
Nathanaël Rakotoarinoro ◽  
Tamara Teichmann ◽  
Anne Zemella ◽  
Lena Thoring ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Eukaryotic Cell ◽  
Fluorescent Dye ◽  
Site Specific ◽  
Drug Conjugates ◽  
Sds Page ◽  
Antibody Conjugates ◽  
Cell Free Protein Synthesis ◽  
Antibody Drug

AbstractCell-free protein synthesis (CFPS) enables the development of antibody conjugates, such as fluorophore conjugates and antibody-drug conjugates (ADCs), in a rapid and straightforward manner. In the first part, we describe the cell-free synthesis of antibodies containing fluorescent non-canonical amino acids (ncaa) by using pre-charged tRNA. In the second part, we describe the cell-free synthesis of antibodies containing ncaa by using an orthogonal system, followed by the site-specific conjugation of the fluorescent dye DyLight 650-phosphine. The expression of the antibodies containing ncaa was analyzed by SDS-PAGE, followed by autoradiography and the labeling by in-gel fluorescence. Two different fluorescently labeled antibodies could be generated.

scholarly journals Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues

2021 ◽  
Vol 14 (7) ◽  
pp. 672
Author(s):  
Qun Zhou ◽  
Josephine Kyazike ◽  
Ekaterina Boudanova ◽  
Michael Drzyzga ◽  
Denise Honey ◽  
...  
Keyword(s):  
Specific Antibody ◽  
High Selectivity ◽  
Therapeutic Index ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Site Specific ◽  
Drug Conjugates ◽  
High Therapeutic Index ◽  
High Site ◽  
Antibody Conjugation

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

2021 ◽  
Author(s):  
Yasuyoshi Fukuda ◽  
Misako Higashiya ◽  
Takahiro Obata ◽  
Keita Basaki ◽  
Megumi Yano ◽  
...  
Keyword(s):  
Cooling Rate ◽  
Small Volume ◽  
Sucrose Solution ◽  
High Concentration ◽  
Rat Embryos ◽  
Low Concentrations ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Warming Rate ◽  
Very High

Abstract To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

scholarly journals State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Drosophila cell fusion induced by wheat germ agglutinin

1975 ◽  
Vol 18 (1) ◽  
pp. 113-121
Author(s):  
R.M. Rizki ◽  
T.M. Rizki ◽  
C.A. Andrews
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Embryonic Cell ◽  
High Concentration ◽  
Drosophila Cell ◽  
Low Concentrations ◽  
Embryonic Cell Lines ◽  
Scanning Electron

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.

scholarly journals Blooms of bryozoans and epibenthic diatoms in an urbanized sandy Beach (Balneário Camboriú - SC - Brazil): dynamics, possible causes and biomass characterization

2017 ◽  
Vol 65 (4) ◽  
pp. 678-694 ◽  
Author(s):  
Leonardo Rubi Rörig ◽  
Maevi Ottonelli ◽  
Ana Gabriela Itokazu ◽  
Marcelo Maraschin ◽  
João Vitor Heberle Lins ◽  
...  
Keyword(s):  
Saturated Fatty Acids ◽  
Experimental Studies ◽  
Growth Conditions ◽  
Adjacent Area ◽  
High Concentration ◽  
Local Pollution ◽  
Organic Debris ◽  
Quality Of Water ◽  
Low Concentrations ◽  
Environmental Health Problem

Abstract Balneário Camboriu (SC - Brazil) is a touristic city where the disordered growth of the urban population and the implementation of coastal works without proper evaluation generated environmental impacts and affected the sanitary quality of water and sediment of Camboriu River and marine adjacent area. One of the most recent and alarming phenomena observed are the blooms of invasive bryozoans (Arboscuspis bellula and Membraniporopsis tubigera) associated with epibenthic diatoms (Amphitetras antediluviana and Biddulphia biddulphiana). Several clues associate these phenomena, started in 2003, with the excess of nutrients and organic matter in the Camboriú cove and large coastal works such as dredging, landfills and construction of jetties, leading to changes in benthic ecological structure. Being an aesthetic and environmental health problem, the concern of the scientific community and government agencies intensified as the occurrences become more frequent and persistent. This research addresses this issue through environmental and experimental studies. Samplings of the benthic material collected by boat and diving, and blooms monitoring were the environmental approach. The laboratory work included the algal isolation and culture, in addition to growth conditions assessment and chemical biomass analysis. Monitoring data showed a seasonal trend in the blooms, with more conspicuous events in warmer months. Diatoms increase in abundance in colder months and bryozoans in the warmer ones. The diatom A. antediluviana, predominant in the blooms, grew satisfactorily in laboratory cultivation, showing better growth in media with higher concentrations of silicate and phosphate. Bryozoans showed slow growth in laboratory conditions. The deposited material collected in the environment showed low concentrations of saturated fatty acids, but the high biomass suggest a possible use for biofuels production. Biomass samples dominated by bryozoans showed moderate antimicrobial activity against Klebsiella pneumoniae. The explanation for the occurrence of these blooms are still inconclusive, but there is considerable evidence that it is a synergistic effect between the high concentration of bacteria and organic debris in the water related to local pollution and the elimination of natural competitors by coastal works.

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