Characterization workflow for fragments detected in capillary electrophoresis sodium dodecyl sulfate analysis of therapeutic monoclonal antibodies.

Product-related fragments in monoclonal antibodies (mAbs) can have a significant impact on the efficacy and safety of the product. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is a commonly used method for fragment quantification, but it has challenges in peak identification due to the inability to enrich components and the incompatibility of SDS with mass spectrometry (MS). This article presents a workflow for identifying peaks in CE-SDS analysis. The workflow involves comparing the migration time of peaks with that of standards and utilizing MS analysis to identify fragments. By employing this innovative systematic workflow, we successfully identified the CE-SDS impurity peaks of seven antibody products. Among them, four products exhibited characteristic fragments associated with disulfide bonds (light chain [LC], heavy-light [HL] chain, heavy-heavy [HH] chain, and HH-LC) and a glycosylation-related fragment non-glycosylated heavy chain. Additionally, one product showed a fragment formed by the connection of HC_C130 and HC_C130 , which is associated with a thioether bond. Furthermore, two other products displayed amino acid backbone breakage, with one product showing clipping at the HC region of A233 -G285 and the other product showing clipping at the HC regions of A97 -S158 and N342 -T366 . This workflow can be applied in early drug research, process development, or during the biologics license application stage to characterize fragments in therapeutic mAbs analyzed by CE-SDS.

Systematically amino acid analysis: Advancements in extinction coefficient determination for therapeutic proteins.

Biologicals developers often face challenges in accurately determining the extinction coefficient (EC) measurement. We have successfully improved the precision and robustness of the widely recognized amino acid analysis method for EC determination, through a stepwise optimization process. Extensive analyses based on 114 observations, covering eight proteins over three years were performed, with a maximum relative standard deviation of 1.5% among multiple analysts, and a maximum deviation of 2.8% from the theoretical EC across the eight given proteins examined.

In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry.

In-depth characterization of charge heterogeneity is a pivotal step desired in the therapeutics antibody development. To this end, a novel on-line multidimensional liquid chromatography-mass spectrometry (MDLC-MS) method for charge variant characterization was developed to dig out potential risks on safety and efficacy. This method implemented 96-well plate fractionation and on-column preconcentration by multi-injection, thereby facilitating detection of charged species at low abundance. Eleven charge variants of mAb-A were preliminarily characterized by 2DLC(CEX × RP-C4)-MS. TRVHS and RVHS signal peptide variants of mAb-A were found in basic peaks of the CEX profile. The results supported process development in a timely manner, and the signal peptide-containing variants with potential immunogenicity were successfully removed by an optimized purification process. The retained seven charge variants of mAb-A were further characterized by 4DLC(CEX × RP-C4 × Trypsin×RP-C18)-MS. Post-translational modifications including deamidation, cyclization of N-terminal glutamine, C-terminal lysine truncation as well as proline amidation, and methionine oxidation were identified, and their potential risks were evaluated. Biological activity of the seven charge variants was evaluated by 2DLC (CEX × FcγRIIIa). Increased FcγRIIIa receptor binding affinity was observed in the acidic variants. The MDLC-MS detection can be completed in 72 h with 1.25 mg of mAb, demonstrating to be sample-economic, time-effective, and labor-saving. It provided a powerful and timely tool for charge variant characterization and met the aggressive timeline desired for antibody development.

Demonstrating analytical similarity of a biosimilar HLX04 to bevacizumab with a panel of state-of-the-art methods and tiering of quality attributes.

Therapeutical monoclonal antibodies are structurally and functionally complex, whereas the innovator's manufacturing processes are proprietary. With respect to the similarity assessment, a proposed biosimilar product needs to demonstrate a side-by-side comparison between the reference product (RP) and candidate product in terms of physicochemical properties and biological activities, as well as nonclinical and clinical outcomes. Here, a comprehensive analytical similarity assessment was performed for in-depth comparison of HLX04, China-sourced Avastin® (CN-Avastin®), and Europe-sourced Avastin® (EU-Avastin®) following a tier-based quality attribute (QA) evaluation. A series of orthogonal and state-of-the-art analytical techniques were developed for the assessment. Ten lots of HLX04 were compared with 29 lots bevacizumab RP. Referred to the characterization results, HLX04 is highly similar to the RPs with respect to physicochemical properties and biological functions. In addition, HLX04 was found with similar stability and degradation behaviors upon multiple stressed conditions to bevacizumab. Minor differences were observed in glycosylation, aggregates, FcγRIIIa(F), and FcγRIIIa(V) binding activities; nevertheless, they were evaluated and demonstrated not to impact clinical outcomes. According to the reported clinical results, the totality of evidence, including the pharmacokinetic, efficacy, safety, and immunogenicity, further shows that HLX04 is similar to CN-/EU-Avastin®.

CCR2 downregulation attenuates spinal cord injury by suppressing inflammatory monocytes.

Specific elimination of blood-derived macrophages/monocytes following spinal cord injury (SCI) may suppress neurotoxicity without affecting the neuroprotective microglia at the injury sites. We aimed to deplete hematogenous monocytes by downregulating CCR2 through siCCR2-loaded nanoparticles and investigated its outcome in the recovery of locomotor function of SCI mice. We induced SCI in mice and examined the influx of blood-derived monocytes into the injury site. We constructed nanoparticles loaded with siRNA targeting CCR2 and examined its efficiency in downregulating the CCR2 expression in cultured RAW264.7 cells and monocytes in vivo. Finally, we assessed the effects of CCR2 downregulation in pro-inflammatory cytokine production, axon regeneration, and locomotor recovery of the SCI mice. We found that SCI significantly increased the CCL2 expression and number of blood-derived macrophages/monocytes in the lesion area. Nanoparticles loaded with siCCR2 significantly suppressed the CCR2 expression in hematogenous macrophages/monocytes, reduced the number of hematogenous macrophages/monocytes, and reduced pro-inflammatory cytokine production at the injury site. Finally, CCR2 downregulation promoted axon regeneration and improved locomotor recovery in SCI mice. Our study suggests that siCCR2 loading nanoparticles are efficient and specific in downregulating hematogenous macrophages/monocytes without affecting the neuroprotective microglia and its efficacy in promoting locomotor recovery in SCI mice warrants further investigation for its clinical application in SCI.

Design, synthesis, biological evaluation, homology modeling and docking studies of (E)-3-(benzo[d][1,3]dioxol-5-ylmethylene) pyrrolidin-2-one derivatives as potent anticonvulsant agents.

A series of (E)-3-(benzo[d][1,3]dioxol-5-ylmethylene)pyrrolidin-2-one derivatives were designed, synthesized, and evaluated for their anticonvulsant activities. In the preliminary screening, compounds 5, 6a-6f and 6h-6i showed promising anticonvulsant activities in MES model, while 6f and 6g represented protection against seizures at doses of 100 mg/kg and 0.5 h in scPTZ model. The most active compound 6d had a high-degree protection against the MES-induced seizures with ED50 value of 4.3 mg/kg and TD50 value of 160.9 mg/kg after intraperitoneal (i.p.) injection in mice, which provided 6d in a high protective index (TD50/ED50) of 37.4 comparable to the reference drugs. Beyond that, 6d has been selected and evaluated in vitro experiment to estimate the activation impact. Apparently, 6d clearly inhibits the Nav1.1 channel. Our preliminary results provide new insights for the development of small-molecule activators targeting specifically Nav1.1 channels to design potential drugs for treating epilepsy. The computational parameters, such as homology modeling, docking study, and ADME prediction, were made to exploit the results.

Lack of efficacy in a randomised trial of a brief intervention to reduce drug use and increase drug treatment services utilisation among adult emergency department patients over a 12-month period.

OBJECTIVES: Assess the 12-month efficacy of a brief intervention (BI) on reducing drug use and increasing drug treatment services utilisation among adult emergency department (ED) patients. METHODS: This randomised, controlled trial enrolled 18-64-year-old ED patients needing a drug use intervention. Treatment arm participants received a tailored BI while control arm participants only completed the study questionnaires. Self-reported past 3-month drug use and engagement in drug treatment services were compared by study arm at 3-month intervals over 1 year. Multiple imputations were performed to overcome loss-to-follow-up. RESULTS: Of the 1030 participants, follow-up completion ranged 55%-64% over the four follow-ups. At 12 months, the two study arms were similar in regards to mean: (1) proportion reporting any drug use (treatment: 67.1% (61.6 to 72.6), control: 74.4% (69.4 to 79.4)); (2) drug use frequency on a five-point scale (treatment: 3.7 (3.3 to 4.2), control: 4.6 (4.0 to 5.2)); (3) total days of drug use (treatment: 28.3 (23.2 to 33.4), control: 33.4 (28.5 to 38.2)); (4) most number of times drugs used/day (treatment: 4.6 (3.6 to 5.5), control: 6.1 (4.8 to 7.3)) and (5) typical number of times drugs used/day (treatment: 3.3 (2.5 to 4.1), control: 5.1 (3.9 to 6.2)). Utilisation of drug treatment services also was similar by study arm. In multivariable regression analyses, patients who were homeless or had higher drug use at baseline continued to have greater drug use in follow-up. CONCLUSIONS: Among adult ED patients requiring a drug use intervention, this BI did not decrease drug use or increase drug treatment services utilisation over a 12-month period more than the control condition. TRIAL REGISTRATION NUMBER: NCT01124591; Pre-trial.

Synthesis and Evaluation of 5-(o-Tolyl)-1H-tetrazole Derivatives as Potent Anticonvulsant Agents.

A series of 5-(o-tolyl)-1H-tetrazole derivatives were synthesized and evaluated for their anticonvulsant activities. 1-(2-Methylbenzyl)-5-(o-tolyl)-1H-tetrazole (3h) showed important anticonvulsant activity against the MES-induced seizures, as well as lower neurotoxicity with an ED50 value of 12.7 mg/kg and a TD50 value of over 500 mg/kg after intraperitoneal injection into mice, providing 3h with a high protective index (TD50 /ED50 ) of over 39.4. The achieved results prove that the distinctive compounds could be valuable as a model for future development, adaptation, and investigation to construct more active analogues.

[Study on Chemical Components of Tripterospermum chinense ( â ¡)].

Objective: To investigate the chemical components of Tripterospermum chinense. Methods: Various column chromatography were used in the isolation and purification,and physiochemical constant determination and spectral analysis were adopted to determine the chemical structures. Results: A novel monoterpenoid and seven known compounds were isolated and identified as tripterospermum acid ester A( 1),strychnovoline( 2),p-hydroxybenzaldehyde( 3),isovitexin( 4),7-O-rhamnopyransoyl-isoorientin( 5),trifoliside( 6),2″-O-rhamnopyransoyl-trifoliside( 7) and sweroside( 8). Conclusion: Compound 1 is a new monoterpenoid,compounds 2,3 are isolated from this plant for the first time.

[Chemical Constituents from Roots and Rhizome of Trillium tschonoskii( â £)].

Objective: To investigate the chemical constituents from the roots and rhizome of Trillium tschonoskii. Methods: The85% methanol elution parts from the roots and rhizome of Trillium tschonoskii were separated and purified by polyamide,silica gel,RPC18,Sephadex LH-20 column chromatography and the preparation of high performance liquid. Chemical structures were identified by MS,1D and 2D-NMR experiment. Results: Ten steroidal saponins compounds were isolated and identified as diosgenin-3-O-α-L-rhamnopyranosyl-( 1→4)-α-L-rhamnopyranosyl-( 1→4)-[α-L-rhamnopyranosyl( 1 →2) ]-ß-D-glucopyranoside( 1),trikamsteroside E( 2),deoxytrikamsteroside E( 3),( 23 S,24S,25S)-spirostan-5-ene-1ß,3ß,21,23,24-pentahydroxy-1ß-O-α-L-rhamnopyranosyl-( 1 →2)-[O-ß-D-xylopyranosyl( 1→3) ]-O-α-L-pyranarabinoside( 4),trikamsteroside B( 5),27-hydroxy-trikamsteroside B( 6),3-acetyl-pennogenin( 7),dioscoreanoside I( 8),sileneoside G( 9) and intergristerone B( 10). Conclusion: Compounds 8 ~ 10 are isolated from this genus for the first time,compounds 1,3,4,6,7 are isolated from this plant for the first time.

[Chemical Constituents from Saururus chinensis].

OBJECTIVE: To investigate the chemical constituents of active components of Saururus chinensis on anti-nicotine withdrawal symptoms. METHODS: Various column chromatography were used in the isolation and purification, and physiochemical constant determination and spectral analysis were adopted to determine the chemical structures. RESULTS: Six chemical compounds were isolated from the active part of anti-withdrawal symptoms, and were identified as 4'-hydroxyl-3,3',4,5,5'-pentamethoxy-7,7'-epoxylignan (1) ,3-(2-nitroethyl)-1-methoxyindole(2), elemicin (3), erythro-(7R, 8S) - (-) - (3,4,5-trimethoxy-7-hydroxy-1'-allyl-3', 5'-dimethoxy)-8-O-4'-neolignan (4), 3,4,5-trimethoxy-phenylacrylaldehyde (5) and dibutyl phthalate (6). CONCLUSION: Compound 1 is a novel lignan, compounds 2 - 6 are firstly isolated from this plant.

[Study on Phenylpropanoids of Gardenia jasminoides].

OBJECTIVE: To investigate the phenylpropanoids constituents of Gardenia jasminoides. METHODS: Various column chromatography were used in the isolation and purification, physiochemical constant determination and spectral analysis were adopted to determine the chemical structures. RESULTS: Eight compounds were isolated from Gardenia jasminoides, including 4-hydroxy-cinnamic acid methylester (1), 3, 5-dimethoxy-4-hydroxy-cinnamic acid methylester (2), pisoninol II (3), 7-hydroxy-orebiusin A (4), (E) -3-(4'-hydroxyphenyl) -acrylic acid n-butyl ester (5), (E) -3-(4'-methoxyphenyl) -acrylic acid n-butyl ester (6), 4-methoxyl-phenylpropanol n-butyl ether (7) and cycloolivil (8). CONCLUSION: All compounds are firstly isolated from this plant.

Ratjadone C-mediated nuclear accumulation of HDAC4: implications on Runx2-induced osteoblast differentiation of C3H10T1/2 mesenchymal stem cells.

Histone deacetylases (HDACs) are a group of enzymes that deacetylate ε-N-acetyl lysine residues of histone and non-histone proteins and play an important role in gene regulation. HDAC4, a class-IIa HDAC, has been reported to shuttle between nucleus and cytoplasm in response to various cellular stimuli. The nucleo-cytoplasmic shuttling of HDAC4 is critical, and an anomalous nuclear localization might affect the cellular differentiation program. While the subcellular localization of HDAC4 has been reported to be vital for myoblast differentiation and chondrocyte hypertrophy, nuclear accumulation of HDAC4 during Runx2-induced osteoblast differentiation of stem cells has not been characterized. Ratjadone C is a natural compound that inhibits the nuclear export of proteins. Here, we show that Runx2 is a more potent transcription factor than Osterix in inducing osteoblast differentiation. Under the influence of ratjadone C, HDAC4 is retained in the nucleus and co-localizes with Runx2. However, forced nuclear accumulation of HDAC4 by ratjadone C or overexpression of the nuclear resident form of HDAC4 does not inhibit osteoblast differentiation, suggesting that the Runx2- induced osteogenic program of C3H10T1/2 cells is not affected by HDAC4. Even though phosphorylation of HDAC4 affects its compartmentalization and the stemness of progenitor cells, we found that total HDAC4 and phosphorylated HDAC4 remain cytoplasmic under both osteogenic and nonosteogenic conditions. Collectively, this work demonstrates that, regardless of the nucleo-cytoplasmic presence of HDAC4, the Runx2-induced osteoblast differentiation program of C3H10T1/2 cells remains unaffected. Additionally, the ratjadone C-mediated nuclear retention assay can potentially be used as a screening tool to identify novel regulatory mechanisms of HDAC4 and its functional partners in various pathophysiological conditions.

[Study on chemical components of Tripterospermum chinense].

OBJECTIVE: To investigate the chemical components of the whole herb of Tripterospermum chinense. METHODS: Various column chromatography methods were used in the isolation and purification. Physio-chemical constant determination and spectral analysis were adopted to determine the chemical structures. RESULTS: Ten compounds were isolated and identified as 1, 7-dihydroxy-3,8-dimethoxyxanthone(1),1,3-dihydroxy-7,8-dimethoxyxanthone (2) 1,3,6,7-tetrahydroxyxanthone (3),1,8-dihydroxyxanthone(4),2'-deoxythymidine(5), 4-hydroxyphthalide(6),2,4-dihydroxy benzyl alcohol (7),2,5-dihydroxyphenetole (8), saponarin (9) and 4'-methoxysaponarin(10). CONCLUSION: Compounds 2 - 8 and 10 are isolated from this plant for the first time.

[Phenylpropanoids constituents of Eucommia ulmoides leaves].

OBJECTIVE: To investigate phenylpropanoids constituents from Eucommia ulmoides leaves. METHODS: Various column chromatographic methods were used in isolation and purification. Physiochemical constant determination and spectral analysis were adopted to determine the chemical structures of phenylpropanoids. RESULTS: Nine phenylpropanoids were isolated and identified as caffeic acid (1), chlorogenic acid methylester (2), syringin (3), guaiacylglycerol (4), 5-methoxy-guaiacylglycerol (5), 5,9-dimethoxy-guaiacylglycerol (6), 9-n-butyl-guaiacylglycerol (7), 9-n-butyl-isoguaiacylglycerol (8), 8'-methoxy-olivil (9). CONCLUSION: Compounds 5 - 9 are isolated from this plant for the first time.

[Study on chemical constituents of Gardenia jasminoides(III)].

OBJECTIVE: To investigate the chemical constituents of Gardenia jasminoides fruits. METHODS: Various column chromatography were used in the isolation and purification, and physiochemical constant determination and spectral analysis were adopted to determine the chemical structures. RESULTS: Twelve compounds were isolated from Gardenia jasminoides including jasminoside I (1), gardenoside (2), gardaloside (3), 3-hydroxy-urs-12-ene-11-ketone(4), 5, 4'-dihydroxyl-7, 3', 5'-trimethoxyflavone (5), 5, 7, 3', 4', 5'-pentamethoxyflavone(6), 3, 5, 6, 4'-tetrahydroxy-3', 5'-dimethoxyflavone (7), shikimic acid (8), 1, 2, 4-benzenetriol (9), 3, 4-dimethoxy-benzoic acid (10), dibutyl phthalate (11) and diisobutyl phthalate (12). CONCLUSION: Compounds 4 - 7 and 9 -10 were isolated from this plant for the first time.

[Study on chemical constituents of Eucommia ulmoides leaves].

OBJECTIVE: To investigate the chemical constituents of Eucommia ulmoides leaves. METHODS: Various column chromatography were used in the isolation and purification, physiochemical constant determination and spectral analysis were adopted to determine the chemical structures. RESULTS: Ten compounds were isolated and identified as borreriagenin (1), n-butyl-O-ß-D-fructopyranoside (2), α-D-glucopyranosyl-(1-->1')-3'-amino-3'-deoxy-ß-D-glucopyranoside (3), ß-D-fructofuranosyl-α-D-galactopyranoside (4), ß-D-fructose (5), diisobutyl phthalate (6), 5-hydroxy-9-isopropylether-guaiacylglycerol (7), 4-hydroxyphenylethanol-8-O-ß-D-apiofuranosyl(1-->6)-ß-D-glucopyranoside (8), lariciresinol (9), and (3S,5R,6R,9S)-tetrahydroxy-7-ene-megastigmane (10). CONCLUSION: All compounds are isolated from this genus for the first time.

[Study on chemical constituents of iridoids from eucommiae folium].

OBJECTIVE: To investigate the chemical constituents of iridoids from Eucommiae Folium. METHODS: The compounds were isolated and purified by various column chromatography, the structures were identified on the basis of physiochemical properties and spectral analysis. RESULTS: Nine iridoids were isolated and identified as asperuloside (1), daphylloside (2), scandoside methyl ester (3), loganin (4), 8-epi-loganin (5), 7-epi-loganin (6), deacetyl asperulosidic acid methyl ester (7), geniposide (8) and geniposidic acid (9). CONCLUSION: Compounds 2 - 7 are isolated from this plant for the first time, compounds 4 - 6 are isolated from this genus for the first time.

Identification of nonvolatile organic compounds (NVOCs) in biopharmaceuticals through non-target analysis and quantification using complexation-precipitation extraction.

Single-use systems in biopharmaceutical manufacturing can potentially release chemical constituents (leachables) into drug products. Prior to conducting toxicological risk assessments, it is crucial to establish the qualitative and quantitative methods for these leachables. In this study, we conducted a comprehensive screening and structure elucidation of 23 leachables (nonvolatile organic compounds, NVOCs) in two antibody drugs using multiple (self-built and public) databases and mass spectral simulation. We identified 7 compounds that have not been previously reported in medical or medicinal extractables and leachables. The confidence levels for identified compounds were classified based on analytical standards, literature references, and fragment assignments. Most of the identified leachables were found to be plasticizers, antioxidants, slip agents or polymer degradants. Polysorbate (namely Tween) is commonly used as an excipient for protein stabilization in biopharmaceutical formulations, but its ionization in liquid chromatography-electrospray ionization mass spectrometry can interfere with compound quantification. To address this, we employed a complexation-precipitation extraction method to reduce polysorbate content and quantify the analytes. The developed quantitative method for target NVOCs demonstrated high sensitivity (limit of quantification: 20 or 50 µg/L), accuracy (recoveries: 77.2 to 109.5 %) and precision (RSD ≤ 8.2 %). Overall, this established method will facilitate the evaluation of NVOC safety in drug products.

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing.

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.