IVD PCR tests, Molecular Genomics Plamsmids and purification
Tuning Monoclonal Antibody Galactosylation using Raman Spectroscopy
HannahJune 12, 20210 Comments
Tuning Monoclonal Antibody Galactosylation using Raman Spectroscopy-Controlled Lactic Acid Feeding
A key aspect of large-scale production of biotherapeutics is a well-designed and consistently-executed upstream cell culture process. Process Analytical Technology (PAT) tools provide enhanced monitoring and control capabilities to support consistent process execution, and also have potential to aid in maintenance of product quality at desired levels.
One such tool, Raman spectroscopy, has matured as a useful technique to achieve real-time monitoring and control of key cell culture process attributes. We developed a Raman spectroscopy-based nutrient control strategy to enable dual control of lactate and glucose levels for a fed-batch CHO cell culture process for monoclonal antibody (mAb) production.
To achieve this, PLS-based chemometric models for real-time prediction of glucose and lactate concentrations were developed and deployed in feedback control loops. In particular, feeding of lactic acid post-metabolic shift was investigated based on previous work that has shown the impact of lactate levels on ammonium as well as mAb product quality.
Three feeding strategies were assessed for impact on cell metabolism, productivity, and product quality: bolus-fed glucose, glucose control at 4 g/L, or simultaneous glucose control at 4 g/L and lactate control at 2 g/L. The third feeding strategy resulted in a significant reduction in ammonium levels (68%) while increasing mAb galactosylation levels by approximately 50%.
This work demonstrated that when deployed in a cell culture process, Raman spectroscopy is an effective technique for simultaneous control of multiple nutrient feeds, and that lactic acid feeding can have a positive impact on both cell metabolism and mAb product quality. This article is protected by copyright. All rights reserved.
Description: A sandwich quantitative ELISA assay kit for detection of Human Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A sandwich quantitative ELISA assay kit for detection of Human Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A sandwich quantitative ELISA assay kit for detection of Rat Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A sandwich quantitative ELISA assay kit for detection of Rat Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A Monoclonal antibody against Human Functional BAFF-R (mouse), mAb . The antibodies are raised in Purified From Concentrated Hybridoma Tissue Culture Supernatant. and are from clone 9B9. This antibody is applicable in FC
Description: The enzyme encoded by this gene is an arylesterase that mainly hydrolyzes paroxon to produce p-nitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene are a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7q21.3.
Description: The enzyme encoded by this gene is an arylesterase that mainly hydrolyzes paroxon to produce p-nitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene are a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7q21.3.
Description: The enzyme encoded by this gene is an arylesterase that mainly hydrolyzes paroxon to produce p-nitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene are a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7q21.3.
Description: A Monoclonal antibody against Human Functional Rab1-GTP, mAb (recombinant). The antibodies are raised in Purified From HEK 293 Cell culture Supernatant. and are from clone ROF7. This antibody is applicable in ICC, IP
Monoclonal Functional Rab6-GTP Antibody, mAb (recombinant), Clone: AA2
Description: A Monoclonal antibody against Human Functional Rab6-GTP, mAb (recombinant). The antibodies are raised in Purified From HEK 293 Cell culture Supernatant. and are from clone AA2. This antibody is applicable in ICC
Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action
Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor ‘invisible’ to the host’s immune system.
Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferationin vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations.
To elucidate C0021158’s inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158’s epitope was consequently mapped to an area some distance from the enzyme’s substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes.
Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2’s epitope translates into more subtle changes within the enzyme’s active site.
An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2’s enzymatic function.
In silicomolecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic.
Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
Development of a physiologically-based pharmacokinetic model for ocular disposition of monoclonalantibodies in rabbits
Development of protein therapeutics for ocular disorders, particularly age-related macular degeneration (AMD), is a highly competitive and expanding therapeutic area.
However, the application of a predictive and translatable ocular PK model to better understand ocular disposition of protein therapeutics, such as a physiologically-based pharmacokinetic (PBPK) model, is missing from the literature.
Here, we present an expansion of an antibody platform PBPK model towards rabbit and incorporate a novel anatomical and physiologically relevant ocular component.
Parameters describing all tissues, flows, and binding events were obtained from existing literature and fixed a priori. First, translation of the platform PBPK model to rabbit was confirmed by evaluating the model’s ability to predict plasma PK of a systemically administered exogenous antibody.
Then, the PBPK model with the new ocular component was validated by estimation of serum and ocular (i.e. aqueous humor, retina, and vitreous humor) PK of two intravitreally administered monoclonal antibodies.
We show that the proposed PBPK model is capable of accurately (i.e. within twofold) predicting ocular exposure of antibody-based drugs. The proposed PBPK model can be used for preclinical-to-clinical translation of antibodies developed for ocular disorders, and assessment of ocular toxicity for systemically administered antibody-based therapeutics.
Development of High Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil
In this study, a set of duplex reverse transcription (RT)-PCR-mediated high resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f.sp. subterranea (Sss), was developed.
The infestation of soil by PMTV was detected by using a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel-electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, -CP, and -TGB) were analyzed together with the existing Sss primers using real-time RT-PCR.
The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by high resolution DNA melting (HRM) analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV and/or Sss was found in 63-100% samples collected from fields in which PMTV-infected tubers had been observed.
In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Between 63%-83% and 100% of the soil samples collected from PMTV-infested fields led to PMTV and Sss infections in the bait tobacco plants, respectively; whereas no PMTV or Sss infected plants were obtained from soil samples collected from PMTV/Sss-free fields.
Description: A sandwich quantitative ELISA assay kit for detection of Human Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A sandwich quantitative ELISA assay kit for detection of Human Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A sandwich quantitative ELISA assay kit for detection of Rat Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A sandwich quantitative ELISA assay kit for detection of Rat Paraoxonase 1 (PON1) in samples from serum, plasma, tissue homogenates, cell lysates, cell culture supernates or other biological fluids.
Description: A Monoclonal antibody against Human Functional BAFF-R (mouse), mAb . The antibodies are raised in Purified From Concentrated Hybridoma Tissue Culture Supernatant. and are from clone 9B9. This antibody is applicable in FC
Description: The enzyme encoded by this gene is an arylesterase that mainly hydrolyzes paroxon to produce p-nitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene are a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7q21.3.
Description: The enzyme encoded by this gene is an arylesterase that mainly hydrolyzes paroxon to produce p-nitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene are a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7q21.3.
Description: The enzyme encoded by this gene is an arylesterase that mainly hydrolyzes paroxon to produce p-nitrophenol. Paroxon is an organophosphorus anticholinesterase compound that is produced in vivo by oxidation of the insecticide parathion. Polymorphisms in this gene are a risk factor in coronary artery disease. The gene is found in a cluster of three related paraoxonase genes at 7q21.3.
Description: A Monoclonal antibody against Human Functional Rab1-GTP, mAb (recombinant). The antibodies are raised in Purified From HEK 293 Cell culture Supernatant. and are from clone ROF7. This antibody is applicable in ICC, IP
Monoclonal Functional Rab6-GTP Antibody, mAb (recombinant), Clone: AA2
Description: A Monoclonal antibody against Human Functional Rab6-GTP, mAb (recombinant). The antibodies are raised in Purified From HEK 293 Cell culture Supernatant. and are from clone AA2. This antibody is applicable in ICC
