Identification of a highly conserved neutralizing epitope within the RBD region of diverse SARS-CoV-2 variants.

The constant emergence of SARS-CoV-2 variants continues to impair the efficacy of existing neutralizing antibodies, especially XBB.1.5 and EG.5, which showed exceptional immune evasion properties. Here, we identify a highly conserved neutralizing epitope targeted by a broad-spectrum neutralizing antibody BA7535, which demonstrates high neutralization potency against not only previous variants, such as Alpha, Beta, Gamma, Delta and Omicron BA.1-BA.5, but also more recently emerged Omicron subvariants, including BF.7, CH.1.1, XBB.1, XBB.1.5, XBB.1.9.1, EG.5. Structural analysis of the Omicron Spike trimer with BA7535-Fab using cryo-EM indicates that BA7535 recognizes a highly conserved cryptic receptor-binding domain (RBD) epitope, avoiding most of the mutational hot spots in RBD. Furthermore, structural simulation based on the interaction of BA7535-Fab/RBD complexes dissects the broadly neutralizing effect of BA7535 against latest variants. Therapeutic and prophylactic treatment with BA7535 alone or in combination with BA7208 protected female mice from the circulating Omicron BA.5 and XBB.1 variant infection, suggesting the highly conserved neutralizing epitope serves as a potential target for developing highly potent therapeutic antibodies and vaccines.

Biparatopic antibody BA7208/7125 effectively neutralizes SARS-CoV-2 variants including Omicron BA.1-BA.5.

SARS-CoV-2 Omicron subvariants have demonstrated extensive evasion from monoclonal antibodies (mAbs) developed for clinical use, which raises an urgent need to develop new broad-spectrum mAbs. Here, we report the isolation and analysis of two anti-RBD neutralizing antibodies BA7208 and BA7125 from mice engineered to produce human antibodies. While BA7125 showed broadly neutralizing activity against all variants except the Omicron sublineages, BA7208 was potently neutralizing against all tested SARS-CoV-2 variants (including Omicron BA.1-BA.5) except Mu. By combining BA7208 and BA7125 through the knobs-into-holes technology, we generated a biparatopic antibody BA7208/7125 that was able to neutralize all tested circulating SARS-CoV-2 variants. Cryo-electron microscopy structure of these broad-spectrum antibodies in complex with trimeric Delta and Omicron spike indicated that the contact residues are highly conserved and had minimal interactions with mutational residues in RBD of current variants. In addition, we showed that administration of BA7208/7125 via the intraperitoneal, intranasal, or aerosol inhalation route showed potent therapeutic efficacy against Omicron BA.1 and BA.2 in hACE2-transgenic and wild-type mice and, separately, effective prophylaxis. BA7208/7125 thus has the potential to be an effective candidate as an intervention against COVID-19.

Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic, which has resulted in more than two million deaths at 2021 February . There is currently no approved therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target SARS-CoV-2 Spike protein. One human antibody, CA521FALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521FALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. CA521FALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521FALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521FALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 RBD in the Spike protein. CA521FALA blocks the interaction by binding all three RBDs of one SARS-CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions against COVID-19 and identifies CA521FALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.

Analytical comparability assessment on glycosylation of ziv-aflibercept and the biosimilar candidate.

Ziv-aflibercept (aflibercept) is a recombinant fusion protein which combines the portions of human vascular endothelial growth factor receptors extracellular domains fused to the Fc portion of human IgG1. It is a highly sialylated glycoprotein with 5 N-glycosylation sites. In this study, a comprehensive strategy for comparability study of the complex glycosylation was developed between aflibercept and the biosimilar candidate including the investigations on N-glycosylation sites, site occupancy, site-specific glycoforms, released glycans and sialic acids. The results indicated that same N-glycosylation sites were identified, site occupancy were 100% except N68 site, site-specific glycoforms and released glycans showed similar glycan species, contents of NANA were at a same level for two products. Minor differences were found between two products. The biosimilar candidate presented lower level of aglycosylation, lower level of glycans containing one terminal sialic acid, higher level of glycans containing two terminal sialic acids, higher level of G0F and Man5, lower level of G1F and G2F compared with aflibercept. However, further studies exhibited no differences were observed in the cell-based biological potency and Fc effector function. Moreover, the biosimilar candidate showed a similar pharmacokinetics curve and bioequivalence compared with aflibercept.

A comprehensive approach for evaluating charge heterogeneity in biosimilars.

Charge heterogeneity is often evaluated during biosimilar development as it is a universal feature of monoclonal antibodies (mAbs). A common approach in the industry is to develop a biosimilar product with a similar overall charge profile as the reference product. However, uncertainty remains with this approach as the same charge profile in two different products may be caused by different mechanisms. In this work, we present a comprehensive investigation of the charge variants of a therapeutic monoclonal antibody and its biosimilar candidate. Not only did the candidate show a similar charge profile as the reference product, our studies revealed that the same factors contributed to the charge variants of the reference product and the biosimilar candidate. We believe our cause-based approach mitigates the risks associated with the profile-based method and is a rational approach for the charge evaluation of biosimilars.

Charge Variants of an Avastin Biosimilar Isolation, Characterization, In Vitro Properties and Pharmacokinetics in Rat.

The similarity between a proposed biosimilar product and the reference product can be affected by many factors. This study is designed to examine whether any subtle difference in the distribution of the charge variants of an Avastin biosimilar can affect its in vitro potency and in vivo PK. Here, the acidic, basic and main peak fractions of a biosimilar product were isolated using high-performance cation-exchange chromatography and were subjected to various studies to compare their in vitro properties and in vivo PK profile. A serial of analytical methods, including size exclusion chromatography (SEC), imaged capillary isoelectric focusing (icIEF) capillary zone electrophoresis (CZE) and cation-exchange chromatography (CEX-HPLC) were also used to characterize the isolated charge variants. The kinetics constant was measured using a Biacore X100 system. The study indicates the biosimilar product has a high similarity with avastin in physicochemical properties. The potency in vitro and PK profile in rat of charge variants and biosimilar product are consistent with avastin.

LC-MS determination and pharmacokinetic study of six phenolic components in rat plasma after taking traditional Chinese medicinal-preparation: Guanxinning lyophilized powder for injection.

A traditional Chinese medicinal preparation (TCMP) named Guanxinning lyophilized powder for injection composed of Salvia miltiorrhiza Bge. (SMB) and Ligusticum chuanxiong Hort. (LCH) was studied. In order to learn the kinetic behaviors of the lyophilized powder and provide proofs for rational administration, we have developed a sensitive and reproducible method for determination and pharmacokinetic study of six main phenolic components {danshensu (DSS), protocatechuic acid (PAC), protocatechuic aldehyde (PAL), chlorogenic acid (CHA), caffeic acid (CAA) and salvianolic acid B (SAB)} of Guanxinning in rat plasma using liquid chromatography-mass spectrometric (LC-MS) method. Sample preparations were carried out by protein precipitation with the addition of methanol followed by liquid-liquid extraction with ethyl acetate-ethyl ether (3:1, v/v) after internal standard (IS, galic acid) spiked. After evaporation to dryness, the resultant residue was reconstituted in methanol and injected onto a Kromasil C(18) column (150 mm x 4.6 mm i.d. with 5 microm particle size). The analytes were analyzed by using negative electrospray ionization (ESI) mass spectrometry in selected ion monitoring (SIM) mode. The method was with good linearity in the range 0.342-85.0 microgmL(-1) for DSS, 0.0647-12.9 microgmL(-1) for PAC, 0.0933-18.7 microgmL(-1) for PAL, 0.0085-3.40 microgmL(-1) for CHA, 0.0138-2.75 microgmL(-1) for CAA and 0.0272-810 microgmL(-1) for SAB (r>0.99). The average extract recoveries of the six analytes from rat plasma were all no less than 75%, the precision and accuracy determined were all within the required limits. This LC-MS method was successfully applied to pharmacokinetic study of the six phenolic components of Guanxinning lyophilized powder for injection in rats.

Pharmacokinetic study of matrine, oxymatrine and oxysophocarpine in rat plasma after oral administration of Sophora flavescens Ait. extract by liquid chromatography tandem mass spectrometry.

A rapid, sensitive and selective high-performance liquid chromatography tandem mass spectrometric method (HPLC-MS) has been developed and validated for the simultaneous determination of matrine (MT), oxymatrine (OMT) and oxysophocarpine (OSP) in rat plasma after oral administration of Sophora flavescens Ait. extract using pseudoephedrine hydrochloride as an internal standard (I.S.). The three analytes were extracted from the plasma samples by liquid-liquid extraction with chloroform. The chromatographic separation was accomplished on a Kromasil C18 column (150 mm x 4.6 mm). Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The total run time was 12 min between injections. The assay had a lower limit of quantification of 1.0 ng/ml for MT, 2.0 ng/ml for OMT and 2.0 ng/ml for OSP using 200 microl of plasma. The calibration curves were linear in the measured range. The overall precision and accuracy for all concentrations of quality controls and standards was better than 15%. The proposed method enables unambiguous identification and quantification of MT, OMT and OSP in vivo. This was the first report on determination of the major quinolizidine alkaloids in rat plasma after oral administration of Sophora flavescens Ait. extract. The results provided a meaningful basis for evaluating the clinical applications of the herbal medicine.

A sensitive liquid chromatography-mass spectrometry method for simultaneous determination of two active chromones from Saposhnikovia root in rat plasma and urine.

A sensitive and efficient liquid chromatography-mass spectrometry method was developed and validated for the simultaneous determination of two active chromones (prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol) from Saposhnikovia root in rat plasma and urine. The plasma or urine samples were prepared by protein precipitation. Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C(18) column with a mobile phase consisted of methanol, water and 0.1% formic acid. Puerarin was added as the internal standard. The method was validated with the concentration range 1.0-100 ng/mL in rat plasma and 10-1000 ng/mL in urine for prim-O-glucosylcimifugin, 1.5-150 ng/mL in plasma and 15-1500 ng/mL in urine for 4'-O-D-glucosyl-5-O-methylvisamminol. The lower limit of quantitation (LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL in plasma, 10 and 15 ng/mL in urine, respectively. The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0% as terms of relative standard deviation (R.S.D.). Accuracy determined at three quality control concentrations (2.0, 25 and 75 ng/mL for prim-O-glucosylcimifugin; 3.0, 37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9% as terms of relative error (R.E.). The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Fangfeng extract to rats. Practical utility of this new LC-MS method was confirmed in pilot pharmacokinetic studies in rats following oral administration.

Determination of deoxyschizandrin in rat plasma by LC-MS.

A simple, rapid and sensitive LC-MS method was developed for quantification of deoxyschizandrin in rat plasma. A 50 miccrol plasma sample was extracted by ether and performed on Elite Hypersil C(18) column (200 mm x 4.6 mm, 5 microm) with the mobile phase of methanol-water (84:16, v/v) in a run time of 6.5 min. The analyte was monitored with positive atmospheric pressure chemical ionization (APCI) by selected ion monitoring (SIM) mode. A good linear relationship was obtained over the range of 1.0-50.0 ng/ml and the validated method was successfully applied for the pharmacokinetic studies of deoxyschizandrin in rat. After oral administration of 4 mg/kg deoxyschizandrin and Schisandra extract which contained the same dose of deoxyschizandrin to male rats, the C(max) of deoxyschizandrin were 15.8+/-3.1 and 34.3+/-16.8 ng/ml, T(max) were 0.51+/-0.13 and 3.83+/-1.83 h, T(1/2) were 5.3+/-2.2 and 6.5+/-3.4h.

Determination and pharmacokinetic study of syringin and chlorogenic acid in rat plasma after administration of Aidi lyophilizer.

A high-performance liquid chromatographic (HPLC) method was developed for the first time to simultaneously quantify syringin and chlorogenic acid in rat plasma using wavelength-transfer technology. The analysis was performed on a Diamonsil C(18) column (200 x 4.6 mm i.d., 5 microm particle size) with isocratic mobile phase consisting of acetonitrile-0.05% phosphoric acid (12:88, v/v). The linear ranges were 0.20-10 and 0.25-30 microg/mL, respectively. The lower limits of quantification were 0.20 and 0.25 microg/mL, respectively. The method was shown to be reproducible and reliable with intraday precision below 8.5 and 6.1%, interday precision below 7.1 and 5.5%, accuracy within +/-7.1 and +/-8.6%, and mean extraction recovery excess of 92.1 and 80.9%, respectively, which were all calculated from the blank plasma sample spiked with syringin and chlorogenic acid at three concentrations of 0.20, 1.0 and 6.0 microg/mL for syringin and 0.25, 2.0 and 20 microg/mL for chlorogenic acid. This method was validated for specificity, accuracy and precision and was successfully applied to the pharmacokinetic study of syringin and chlorogenic acid in rat plasma after intravenous administration of Aidi lyophilizer.

Simultaneous determination of protocatechuic acid, syringin, chlorogenic acid, caffeic acid, liriodendrin and isofraxidin in Acanthopanax senticosus Harms by HPLC-DAD.

A high performance liquid chromatography (HPLC) method was developed for the first time to quantify simultaneously the six major active ingredients in Acanthopanax senticosus (Rupr. et Maxim.) Harms, namely protocatechuic acid, syringin, chlorogenic acid, caffeic acid, liriodendrin and isofraxidin. The analysis was performed by a reverse phase gradient elution with an aqueous mobile phase (containing 0.05% phosphoric acid) modified by acetonitrile and diode-array multiple-wavelength UV detector (DAD). Six regression equations showed good linear relationships between the peak area of each marker and concentration. The recoveries of the markers listed above were 92.3%, 93.9%, 90.3%, 93.1%, 94.3% and 90.7%, respectively. The relative standard deviation of intra-day and inter-day were less than 2.7% and 3.1%, respectively. This method was validated for specificity, accuracy, precision and limits of quantification. Medicinal materials of ten commercial brands were analyzed and found to contain different amounts of the six bioactive markers. The method developed can be used for the quality control of Acanthopanax senticosus (Rupr. et Maxim.) Harms.