A label-free electrochemical immunosensor was developed to rapidly detect tilmicosin (TMC) residues in pork and milk. The immunosensor was constructed by immobilizing a high-affinity monoclonal antibody against TMC on an rGO-PEI-Ag nanocomposite-modified electrode. The rGO-PEI-Ag nanocomposites were prepared by mixing polyethyleneimine (PEI) modified reduced graphene oxide (rGO) with AgNO3 solution. The prepared rGO-PEI-Ag nanocomposites showed good redox activity and conductivity, as characterized by ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), and X-ray diffraction (XRD). During the preparation process, staphylococcal protein A (SPA) was added to targetedly bind the Fc segment of the monoclonal antibody. The immunosensor showed a low detection limit (LOD) of 0.0013 ng/mL, a linear range of 0.01-100 ng/mL, and recoveries ranging from 92.77 to 100.02% in pork and 92.26-101.23% in milk. Furthermore, the immunosensor exhibited good stability, reproducibility, and specificity in detecting TMC in pork and milk real samples.

This study investigates the impact of 2-methyl imidazolium dihydrogen phosphate (2-MIDHP) on monoclonal antibody (mAb) aggregation during the Protein A purification stage, at a low pH (pH 3.0), and the viral inactivation phase. Size-exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) were used to assess the mAb aggregation. Additionally, the influence of 2-MIDHP on mAb recovery, host cell protein (HCP) clearance, and Protein A leaching was investigated. Thermal stability of mAb, eluted in buffers containing 5 % to 25 % 2-MIDHP was analysed, using differential scanning calorimetry (DSC). Structural insights were obtained via circular dichroism (CD) and fluorescence spectroscopy. Our findings indicated that 2-MIDHP exerted a concentration-dependent protective effect against mAb aggregation, at the pH of 3.0. As the concentration of 2-MIDHP was increased from 0 % to 25 %, the aggregation was significantly reduced from 3.8 ± 0.01 % to 0.56 ± 0.002 %, as analysed by SE-HPLC. Addition of 2-MIDHP did not significantly impact the mAb recovery, HCP clearance, or Protein A leaching. DSC data supported these results, with higher 2-MIDHP concentrations leading to increased melting temperatures of mAb. CD and fluorescence spectroscopy revealed no significant changes in the secondary structure or aromatic residue environment in 2-MIDHP-treated samples, despite the observed reduction in aggregation. The results suggested that 2-MIDHP mitigated mAb aggregation during Protein A purification, possibly by stabilizing the protein structure under acidic stress conditions. These findings offer valuable insights for improving the robustness of mAb purification processes, enhancing product quality and yield.

Protein A affinity membrane adsorbers are a promising alternative to resins to intensify the manufacturing of monoclonal antibodies. This study examined the process performance of convective diffusive membrane adsorbers operated in batch and continuous multi-column mode. Therefore, three different processes were compared regarding membrane utilization, productivity, and buffer consumption: the batch process, the rapid cycling parallel multi-column chromatography process, and the rapid cycling simulated moving bed process. The influence of the monoclonal antibody loading concentration (between 0.5 g L-1 and 5.2 g L-1) and the loading flow rate (between 1.25 MV min-1 and 10 MV min-1) on the monoclonal antibody binding behavior of the membrane adsorber were studied with breakthrough curve experiments. The determined breakthrough curves were used to calculate the monoclonal antibody dynamic binding capacity, the duration of the loading steps for each process, and the number of required membrane adsorbers for the continuous processes rapid cycling parallel multi-column chromatography and rapid cycling simulated moving bed. The highest productivity for the batch (176 g L-1 h-1) and rapid cycling parallel multi-column chromatography process (176 g L-1 h-1) was calculated for high monoclonal antibody loading concentrations and low loading flow rates. In contrast, the rapid cycling simulated moving bed process achieved the highest productivity (217 g L-1 h-1) for high monoclonal antibody loading concentrations and loading flow rates. Furthermore, due to the higher membrane utilization, the buffer consumption of the rapid cycling simulated moving bed process (1.1 L g-1) was up to 1.9 times lower than that of the batch or rapid cycling parallel multi-column chromatography operation (2.1 L g-1).

Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production.

Affinity chromatography is a widely used technique for antibody isolation. This article presents the successful synthesis of a novel affinity resin with a mutant form of protein A (BsrtA) immobilized on it as a ligand. The key aspect of the described process is the biocatalytic immobilization of the ligand onto the matrix using the sortase A enzyme. Moreover, we used a matrix with primary amino groups without modification, which greatly simplifies the synthesis process. The resulting resin shows a high dynamic binding capacity (up to 50 mg IgG per 1 mL of sorbent). It also demonstrates high tolerance to 0.1 M NaOH treatment and maintains its effectiveness even after 100 binding, elution, and sanitization cycles.

The treatment of primary Sjögren\'s syndrome (pSS) coexisting with neuromyelitis optica spectrum disorder (NMOSD) using protein-A immunoadsorption combined with immunosuppressive therapy has rarely been reported. Herein, we present the case of a 35-year-old female diagnosed with pSS concomitant with NMOSD (pSS-NMOSD) who demonstrated a positive response to protein-A immunoadsorption after failing to respond to therapy comprising high-dose intravenous methylprednisolone (IVMP) and intravenous immunoglobulin (IVIG). Within one week of receiving three sessions of immunoadsorption combined with immunosuppressive treatment, the patient\'s clinical symptoms (blurred vision, paraparesis, and dysfunctional proprioception) significantly improved. Additionally, a rapid decrease in the circulating levels of Aquaporin-4 immunoglobulin G antibodies (AQP4-IgG), immunoglobulin (Ig) A, IgG, IgM, erythrocyte sedimentation rate (ESR), and rheumatoid factor (RF) were observed. Magnetic resonance imaging (MRI) further revealed a significant reduction in the lesions associated with longitudinal extensive transverse myelitis. During the follow-up period, prednisolone was gradually tapered to a maintenance dose of 5-10 mg/day, whereas mycophenolate mofetil (MMF) was maintained at 1.0-1.5 g/day. The patient\'s condition has remained stable for four years, with no signs of recurrence or progression observed on imaging examination. Therefore, this case suggests that protein A immunoadsorption may represent a potentially effective therapeutic option for patients with pSS-NMOSD who are refractory to conventional treatments.

Appended bispecific antibody (aBsAb) with two single chain variable fragments (scFv) linked at the c-terminus of its heavy chains is one of the promising formats in bispecific therapeutics. The presence of hydrophobic and flexible scFv fragments render aBsAb molecules higher molecule hydrophobicity and structural flexibility compared to monoclonal antibody (mAb), thus making its purification more challenging. We set out to investigate how the unique molecular properties of aBsAb affect its performance on Protein A chromatography. We showed that aBsAb has a high propensity for chromatography-induced aggregation due to its high molecule hydrophobicity, and this couldn\'t be improved by the addition of common chaotropic salts. Moreover, the presence of chaotropic salts, such as arginine hydrochloride (Arg-HCl), retarded aBsAb elution during Protein A chromatography rather than facilitating which was widely observed in mAb Protein A elution. Nevertheless, we were able to overcome the aggregation issue by optimizing elution condition and improved aBsAb purity from 29 % to 93 % in Protein A eluate with a high molecular weight (HMW) species of less than 5 %. We also showed that the high molecular flexibility of aBsAb leads to different hydrodynamic sizes of the aBsAb molecule post Protein A elution, neutralization, and re-acidification, which are pH dependent. This is different from mAbs where their sizes do not change post neutralization even with re-exposure to acid. The above unique observations of aBsAb in Protein A chromatography were clearly explained from the perspectives of its high molecular hydrophobicity and structural flexibility.

Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.

Objective: To investigate the efficacy and safety of protein A immunoadsorption (PAIA) combined with rituximab (RTX) in highly sensitized patients who underwent haplo-hematopoietic stem cell transplantation (haplo-HSCT) . Methods: The clinical data of 56 highly sensitized patients treated with PAIA and RTX before haplo-HSCT at the First Affiliated Hospital of Soochow University and Soochow Hopes Hematonosis Hospital between March 2021 and June 2023 were retrospectively analyzed. The number of human leukocyte antigen (HLA) antibody types and the mean fluorescence intensity (MFI), humoral immunity, adverse reactions during adsorption, and survival within 100 days before and after adsorption were measured. Results: After receiving the PAIA treatment, the median MFI of patients containing only HLA Ⅰ antibodies decreased from 7 859 (3 209-12 444) to 3 719 (0-8 275) (P<0.001), and the median MFI of HLA Ⅰ+Ⅱ antibodies decreased from 5 476 (1 977-12 382) to 3 714 (0-11 074) (P=0.035). The median MFI of patients with positive anti-donor-specific antibodies decreased from 8 779 (2 697-18 659) to 4 524 (0-15 989) (P<0.001). The number of HLA-A, B, C, DR, and DQ antibodies in all patients decreased after the PAIA treatment, and the differences were statistically significant (A, B, C, DR: P<0.001, DQ: P<0.01). The humoral immune monitoring before and after the PAIA treatment showed a significant decrease in the number of IgG and complement C3 (P<0.001 and P=0.002, respectively). Forty-four patients underwent HLA antibody monitoring after transplantation, and the overall MFI and number of antibody types decreased. However, five patients developed new antibodies with low MFI, and nine patients continued to have high MFI. The overall survival, disease-free survival, non-recurrent mortality, and cumulative recurrence rates at 100 days post-transplantation were 83.8%, 80.2%, 16.1%, and 4.5%, respectively. Conclusions: The combination of PAIA and RTX has a certain therapeutic effect and good safety in the desensitization treatment of highly sensitive patients before haplo-HSCT.
目的： 探究高致敏单倍体造血干细胞移植（haplo-HSCT）患者移植前行蛋白A免疫吸附（PAIA）联合利妥昔单抗（RTX）脱敏治疗的疗效及安全性。 方法： 回顾性分析2021年3月至2023年6月苏州大学附属第一医院和苏州弘慈血液病医院收治的高致敏haplo-HSCT患者56例，移植前行PAIA联合RTX脱敏治疗，吸附前后监测HLA抗体种类数量和平均荧光强度（MFI）、体液免疫和吸附过程的不良反应及100 d内的生存情况。 结果： 仅含HLA Ⅰ类抗体的患者接受PAIA治疗后中位MFI由7 859（3 209～12 444）降至3 719（0～8 275）（P<0.001），HLA Ⅰ+Ⅱ类抗体的中位MFI由5 476（1 977～12 382）降至3 714（0～11 074）（P＝0.035），其中抗供者特异性抗体阳性患者的中位MFI由8 779（2 697～18 659）降至4 524（0～15 989）（P<0.001）。所有患者HLA-A、B、C、DR、DQ抗体种类数量在PAIA治疗后均下降，差异均有统计学意义（A、B、C、DR：P<0.001，DQ：P<0.01）。PAIA治疗前后体液免疫监测显示IgG和补体C3数量显著下降（P值分别为<0.001和0.002）。44例患者接受了移植后HLA抗体监测，总体MFI和抗体种类数量均下降，但有5例患者出现低MFI的新生抗体，有9例持续高MFI，移植后100 d总生存率是83.8%，100 d内无病生存率为80.2%，100 d内非复发死亡率为16.1%，移植后100 d复发的累积发生率为4.5%。 结论： PAIA联合RTX对haplo-HSCT前高敏患者的脱敏治疗有一定的疗效，安全性良好。.

Staphylococcal protein-A affinity chromatography has been optimized for antibody purification, achieving a current capacity of up to 90 mg/ml in packed bed. The morphology of the particles, the number of antibodies bound per ligand and the spatial arrangement of the ligands were assessed by in-situ Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) combined with measurement of adsorption isotherms. We employed SAXS measurements to probe the nanoscale structure of the chromatographic resin. From scanning electron microcopy, the morphology and area of the beads were obtained. The adsorption isotherm revealed a bi-Langmuirian behavior where the association constant varied with the critical bulk concentration, indicating multilayer adsorption. Determining the antibody-ligand stoichiometry was crucial for understanding the adsorption mechanism, which was estimated to be 4 at lower concentrations and 4.5 at higher concentrations, suggestive of reversible protein-protein interactions. The same results were reached from the in-situ small angle X-ray scattering measurements. A stoichiometry of 6 cannot be achieved since the two protein A monomers are anchored to the stationary phase and thus sterically hindered. Normalization through ellipsoids facilitated SAXS analysis, enabling the determination of distances between ligands and antibody-ligand complexes. Density fluctuations were examined by subtracting the elliptical fit, providing insights into ligand density distribution. The dense ligand packing of TOYOPEARL® AF-rProtein A HC was confirmed, making further increases in ligand density impractical. Additionally, SAXS analysis revealed structural rearrangements of the antibody-ligand complex with increasing antibody surface load, suggesting reversible association of antibodies.