Tuning Monoclonal Antibody Galactosylation utilizing Raman Spectroscopy-Managed Lactic Acid Feeding
A key facet of large-scale manufacturing of biotherapeutics is a well-designed and consistently-executed upstream cell tradition course of. Course of Analytical Expertise (PAT) instruments present enhanced monitoring and management capabilities to help constant course of execution, and now have potential to help in upkeep of product high quality at desired ranges.
One such device, Raman spectroscopy, has matured as a helpful approach to attain real-time monitoring and management of key cell tradition course of attributes. We developed a Raman spectroscopy-based nutrient management technique to allow twin management of lactate and glucose ranges for a fed-batch CHO cell tradition course of for monoclonal antibody (mAb) manufacturing.
To realize this, PLS-based chemometric fashions for real-time prediction of glucose and lactate concentrations have been developed and deployed in suggestions management loops. Specifically, feeding of lactic acid post-metabolic shift was investigated primarily based on earlier work that has proven the influence of lactate ranges on ammonium in addition to mAb product high quality.
Three feeding methods have been assessed for influence on cell metabolism, productiveness, and product high quality: bolus-fed glucose, glucose management at four g/L, or simultaneous glucose management at four g/L and lactate management at 2 g/L. The third feeding technique resulted in a big discount in ammonium ranges (68%) whereas growing mAb galactosylation ranges by roughly 50%.
This work demonstrated that when deployed in a cell tradition course of, Raman spectroscopy is an efficient approach for simultaneous management of a number of nutrient feeds, and that lactic acid feeding can have a optimistic influence on each cell metabolism and mAb product high quality. This text is protected by copyright. All rights reserved.

Structural and purposeful characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 perform by way of a novel non-competitive mechanism of motion
- Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 inside particular tumor microenvironments generates an immunosuppressive area of interest that successfully renders the tumor ‘invisible’ to the host’s immune system.
- Elevated ARG2 expression results in a concomitant depletion of native L-arginine ranges, which in flip results in suppression of anti-tumor T-cell-mediated immune responses. Right here we describe the isolation and characterization of a excessive affinity antibody (C0021158) that inhibits ARG2 enzymatic perform utterly, successfully restoring T-cell proliferation in vitro. Enzyme kinetic research confirmed that C0021158 reveals a noncompetitive mechanism of motion, inhibiting ARG2 independently of L-arginine concentrations.
- To elucidate C0021158’s inhibitory mechanism at a structural stage, the co-crystal construction of the Fab in complicated with trimeric ARG2 was solved. C0021158’s epitope was consequently mapped to an space a ways from the enzyme’s substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct areas of ARG2 bear main conformational modifications.
- Notably, the spine construction of a surface-exposed loop is totally rearranged, resulting in the formation of a brand new quick helix construction on the Fab-ARG2 interface. Furthermore, this large-scale structural transforming at ARG2’s epitope interprets into extra refined modifications inside the enzyme’s energetic website. An arginine residue at place 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 can also be predicted to change the pOkA of a key catalytic histidine residue at place 160, additional attenuating ARG2’s enzymatic perform.
- In silicomolecular docking simulations predict that L-arginine is unable to bind successfully when antibody is sure, a prediction supported by isothermal calorimetry experiments utilizing an L-arginine mimetic. Particularly, focusing on ARG2 within the tumor microenvironment via the appliance of C0021158, doubtlessly together with customary chemotherapy regimens or alternate immunotherapies, represents a possible new technique to focus on immune chilly tumors.
Growth of a physiologically-based pharmacokinetic mannequin for ocular disposition of monoclonal antibodies in rabbits
- Growth of protein therapeutics for ocular problems, significantly age-related macular degeneration (AMD), is a extremely aggressive and increasing therapeutic space. Nonetheless, the appliance of a predictive and translatable ocular PK mannequin to better perceive ocular disposition of protein therapeutics, corresponding to a physiologically-based pharmacokinetic (PBPK) mannequin, is lacking from the literature. Right here, we current an growth of an antibody platform PBPK mannequin in direction of rabbit and incorporate a novel anatomical and physiologically related ocular element.
- Parameters describing all tissues, flows, and binding occasions have been obtained from current literature and glued a priori. First, translation of the platform PBPK mannequin to rabbit was confirmed by evaluating the mannequin’s skill to foretell plasma PK of a systemically administered exogenous antibody.
- Then, the PBPK mannequin with the brand new ocular element was validated by estimation of serum and ocular (i.e. aqueous humor, retina, and vitreous humor) PK of two intravitreally administered monoclonal antibodies. We present that the proposed PBPK mannequin is able to precisely (i.e. inside twofold) predicting ocular publicity of antibody-based medication. The proposed PBPK mannequin can be utilized for preclinical-to-clinical translation of antibodies developed for ocular problems, and evaluation of ocular toxicity for systemically administered antibody-based therapeutics.
A hybridoma-derived monoclonal antibody with excessive homology to the aberrant myeloma mild chain
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AMM02955G | Leading Biology | 0.1ml | EUR 633.6 |
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0801095 | Zeptometrix | 1 mg | EUR 1985 |
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801095 | Zeptometrix | 1 mg | EUR 1890 |
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Description: Rabbit Anti Human PON1 Monoclonal Clone ACAG-16 |
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E10-30571A | EnoGene | 100ul | EUR 225 |
Description: Available in various conjugation types. |
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E10-30572 | EnoGene | 100ul | EUR 225 |
Description: Available in various conjugation types. |
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AMR09422G | Leading Biology | 0.05ml | EUR 580.8 |
Description: A Monoclonal antibody against Human PON3 (clone 5G11). The antibodies are raised in Mouse and are from clone 5G11. This antibody is applicable in WB and IHC-P |
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Description: Mouse Anti Mouse BDNF Monoclonal Clone |
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MCD160-PE | Alpha Diagnostics | 50 tests | EUR 416.4 |
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AMR09435G | Leading Biology | 0.1ml | EUR 633.6 |
Description: A Monoclonal antibody against Human PON-1. The antibodies are raised in Rabbit and are from clone EPR2892. This antibody is applicable in WB and IHC |
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Description: Mouse Anti Mouse tPA Monoclonal Clone H27B6 |
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Description: Mouse Anti Mouse AIF1 Monoclonal Clone 5G4E7 |
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APR16043G | Leading Biology | 0.1mg | EUR 633.6 |
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 |
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IMSAMLTUBB3C120UL | Innovative research | each | EUR 341 |
Description: Mouse Anti Human TUBB3 Monoclonal Clone |
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IRTAMSFX19050C100UG | Innovative research | each | EUR 281 |
Description: Rat Anti Mouse Factor X Monoclonal Clone 9050 |
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IRTAMSFX19050C500UG | Innovative research | each | EUR 638 |
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IRTAMSFX29051C100UG | Innovative research | each | EUR 281 |
Description: Rat Anti Mouse Factor X Monoclonal Clone 9051 |
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IRTAMSFX29051C500UG | Innovative research | each | EUR 638 |
Description: Rat Anti Mouse Factor X Monoclonal Clone 9051 |
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IRTAMSFVII9031C100UG | Innovative research | each | EUR 281 |
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Description: Mouse Anti Human TTR Monoclonal Clone 4E4 |