Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids.

Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins.

Quantification of SARS-CoV-2 spike and nucleocapsid proteins using isotope dilution tandem mass spectrometry.

The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.

N-linked Glycan Release Efficiency: A Quantitative Comparison between NaOCl and PNGase F Release Protocols.

There are several methods, both chemical and enzymatic, to release N-linked glycans for structural characterization. One of the most common enzymatic release methods is the use of peptide:N-glycosidase F (PNGase F). A less expensive and quicker alternative has been reported for the release of N-linked glycans chemically using sodium hypochlorite (NaOCl), which hydrolyzes the peptide-glycan bond, yielding the intact glycan with a free reducing terminus. Here, we quantitatively analyzed the efficiency of the NaOCl release protocol compared with the PNGaseF release protocol for small-scale analysis (300 µg) using liquid chromatography-single reaction monitoring-mass spectrometry. We determined that the relative glycan composition of released N-linked glycans from the NaOCl protocol is similar to a typical PNGase F protocol, but the absolute recovery of N-linked glycans is significantly lower with the chemical procedure.

Respiratory symptoms and physiologic assessment of ironworkers at the World Trade Center disaster site.

STUDY OBJECTIVES: To characterize respiratory abnormalities in a convenience sample of ironworkers exposed at the World Trade Center (WTC) disaster site for varying lengths of time between September 11, 2001, and February 8, 2002. DESIGN: Cross-sectional study. SETTING: The Mount Sinai Medical Center, a large tertiary hospital. PARTICIPANTS: Ninety-six ironworkers engaged in rescue and recovery with exposure onset between September 11, 2001, and September 15, 2001, who responded to an invitation to undergo respiratory evaluation. MEASUREMENTS: Medical and exposure history, physical examination, spirometry, forced oscillation (FO), and chest radiographs. The relationships of prevalence of respiratory symptoms and presence of obstructive physiology to smoking, exposure on September 11, duration of exposure, and type of respiratory protection were examined using univariate and linear and logistic regression analyses. RESULTS: Seventy-four of 96 workers (77%) had one or more respiratory symptoms (similar in smokers [49 of 63 subjects, 78%] and nonsmokers [25 of 33 subjects, 76%]). Cough was the most common symptom (62 of 96 subjects, 65%), and was associated with exposure on September 11. Chest examination and radiograph findings were abnormal in 10 subjects (10%) and 19 subjects (20%), respectively. FO revealed dysfunction in 34 of 64 subjects tested (53%), while spirometry suggested obstruction in only 11 subjects (17%). Lack of a respirator with canister was a risk factor for large airway dysfunction, and cigarette smoking was a risk factor for small airway dysfunction. No other relationships reached statistical significance. CONCLUSIONS: Respiratory symptoms occurred in the majority of ironworkers at the WTC disaster site and were not attributable to smoking. Exposure on September 11 was associated with a greater prevalence of cough. Objective evidence of lung disease was less common. Spirometry underestimated the prevalence of lung function abnormalities in comparison to FO. Continuing evaluation of symptoms, chest radiographs, and airway dysfunction should determine whether long-term clinical sequelae will exist.