(2) Sample injection; 25?g of purified mAb diluted 1:1 with mobile phase A. treatment of fentanyl toxicity. KEYWORDS: mAb, opioid use disorder, fentanyl, synthetic opioids, humanized antibody, antibody characterization, antibody engineering, PTM mitigation, manufacturability, clinical development 4′-Methoxychalcone Introduction Fatal overdoses involving opioids are at an all-time high in IKZF2 antibody the United States,1 and overdose events involving potent synthetic opioids such as fentanyl and its analogs have increased annually since 2013.2C6 Between February 2021 and February 2022, 69,237 overdose deaths associated with synthetic opioids (excluding methadone) were reported, accounting for 88.5% of all opioid-related overdose deaths and 66.6% of all drug-related overdose deaths in the United States.1,7 The current treatment for reversal of opioid-related overdose toxicity is naloxone, a -opioid receptor (MOR) antagonist.8 Naloxone is typically effective for combating opioid overdose; however, for more potent MOR agonists that exhibit a serum half-life longer than that of naloxone (30C90?minutes),9 higher or additional doses of naloxone are often required to reverse overdose and protect against renarcotization. Fentanyl has a serum half-life of?8 hrs;10 consequently, additional dosing of naloxone and extended monitoring for signs of recurring fentanyl toxicity can be needed for 2 or more hours.11,12 Additionally, toxicity related to fentanyl and its analogs is partially mediated by non-MOR signaling such as adrenergic and cholinergic pathways, and therefore refractory to naloxone treatment.13 The drastic and sustained increase in overdose deaths related to synthetic opioids indicates that current methods of intervention are inadequate; therefore, the development of alternative or complementary treatment options is paramount. Monoclonal antibodies (mAb) represent a promising alternative treatment modality for counteracting toxic doses of small-molecule drugs by sequestration of the target molecules in serum, preventing their distribution to drug receptors in the brain. With a typical serum half-life of 21?days in humans,14,15 mAbs offer a 500-fold increase in duration of protection compared to naloxone. This long serum half-life may provide anti-opioid (-opioid) mAb-mediated protection against renarcotization from fentanyl and/or other MOR agonists that persists after naloxone is metabolized, with the potential for extended protection against subsequent fentanyl exposure after mAb administration. Opioid overdose reversal treatment with a MOR antagonist like naloxone can have undesirable side effects, including precipitation of opioid withdrawal symptoms.9,11 Meanwhile, because the mechanism of action of an -opioid mAb is drug sequestration 4′-Methoxychalcone rather than receptor antagonism, precipitated withdrawal is not expected to occur with mAb treatment. Though this has not yet been explored with an -opioid mAb, a nicotine-specific mAb was shown to not precipitate withdrawal in nicotine dependent rats.16 In addition to protection from and reversal of overdose, -opioid mAb may provide utility in the prophylactic treatment of patients with opioid use disorder (OUD), as -opioid vaccines have shown pre-clinical efficacy against various models of OUD.17,18 Several mAbs targeting small molecule drugs of abuse are in various stages of clinical and pre-clinical study. Murine and chimeric anti-fentanyl (-fentanyl) mAb have shown efficacy in rodent models.19C21 A partially humanized 4′-Methoxychalcone anti-cocaine mAb is in late-stage pre-clinical development.22 A chimeric anti-methamphetamine mAb (IXT-m200) that offers protection against methamphetamine toxicity has shown promising safety data in Phase I clinical trials,23C25 and has completed one Phase II study with a second Phase II study (“type”:”clinical-trial”,”attrs”:”text”:”NCT05034874″,”term_id”:”NCT05034874″NCT05034874) in progress. Despite this success with murine and chimeric mAbs (chAb) in pre-clinical and clinical settings, it has been well described that murine mAbs are typically not suitable as a therapeutic treatment in humans due to immunogenicity leading to production of anti-drug antibodies (ADA),26 and chAb with human constant regions and murine variable regions are also susceptible to immunogenicity. 27 Human mAb derived from humanized transgenic mouse or rat models are industry-standard in mAb discovery; however, access to such models is often limited by the costly licensing fees associated with their use or by their proprietary ownership. Thus, humanization of.