[Transformation of scientific research results into teaching resources to improve the teaching effect: taking the teaching of the chromatographic method in the pharmaceutical analysis course as an example].

The pharmaceutical analysis course is a three-dimensional knowledge network that connects several courses to form a new comprehensive knowledge node involving a large knowledge system and flexible knowledge structure. In this course, the subject of chromatography covers a wide range of topics. However, because accurate content is challenging to present, the teaching effect of this subject is poor. In this work, we sought to achieve the educational purpose of establishing morality and cultivating talent, as well as the goal of training highly skilled professionals, by taking the teaching of chromatography in the pharmaceutical analysis course as an example of transforming scientific research results into teaching resources. The resources obtained are integrated into the teaching process to provide innovative and scientific research ideas to students with the aim of not only helping them understand and master technical knowledge but also exercise their ability to raise and solve problems. Furthermore, we expound on how to introduce scientific development frontiers and formulate scientific problems through curriculum design. We also describe how our strategy can promote the teaching effect and achieve teaching objectives. Based on the characteristics of rapid knowledge update and equal emphasis on theory and practice in pharmaceutical analysis, the course is designed by introducing new advances in scientific development, formulating scientific problems, and adopting question- and problem-based learning methods for teaching. The teaching effect is then evaluated through diversified assessment, student feedback, and self-evaluation. The results show that the transformation of scientific research results into teaching resources plays a significant role in stimulating students' interest in learning, improving students' ability to solve problems, and achieving curriculum objectives, all of which greatly improve the teaching effect.

Establishment of a graphene oxide-assisted nucleic acid chromatography strip detection technology for Prorocentrum minimum.

In recent decades, the harmful algal blooms (HABs) caused by Prorocentrum minimum have caused serious environmental damage and economic losses. The detection of P. minimum plays an important role in warning the outbreak of P. minimum-forming HABs. By utilizing the powerful absorption of graphene oxide (GO) on short-stranded DNA, a GO-assisted nucleic acid chromatography strip (GO-NACS) was proposed here to achieve a highly sensitive, specific, intuitive, and convenient detection of P. minimum. In particular, this study used our previously reported conventional-NACS (C-NACS) as a control to evaluate the improvement of detection performance with the use of GO. The performance of GO-NACS was evaluated from the perspectives of specificity, sensitivity, stability, and practicality. The specificity test demonstrated that it had a high degree of specificity and did not display cross-reacting with non-target algal species. The sensitivity test with the genomic DNA indicated that it had a detection limit of 1.30 × 10-3 ng µL-1, representing a 10-fold higher sensitivity than C-NACS and a 100-fold higher sensitivity than agarose gel electrophoresis (AGE). The interference test with non-target algal species demonstrated that it had a good detection stability, and the interfering algal species had no obvious effect on the detection of P. minimum. The practicality test with simulated natural water samples showed that the cellular detection limit of GO-NACS was 6.8 cells mL-1, which was 10-fold and 100-fold lower than that of C-NACS and AGE, respectively. In conclusion, the established GO-NACS may offer a novel alternative technique for the detection of P. minimum while guaranteeing specificity and enhancing sensitivity without requiring extensive apparatus.

Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

A Rapid, Equipment-Free DNA Isolation Method for DNA Barcoding.

This rapid, equipment-free DNA isolation procedure using chromatography paper is a simple method that can be performed in less than 30 min and requires no wet lab experience. With minimal expense, it offers an affordable alternative for anyone wanting to explore biodiversity. It also provides an excellent option for use in classrooms or other activities that are time limited. The method works best for plants or lichens, producing stable DNA on Whatman® chromatography paper at room temperature, which can be eluted as needed.

Isolation of Extracellular Outer Membrane Vesicles (OMVs) from Escherichia coli Using EVscore47 Beads.

Outer membrane vesicles (OMVs) are attractive for biomedical applications based on their intrinsic properties in relation to bacteria and vesicles. However, their widespread use is hampered by low yields and purities. In this study, EVscore47 multifunctional chromatography microspheres were synthesized and used to efficiently isolate functional OMVs from Escherichia coli. Through this technology, OMV loss can be kept to a minimum, and OMVs can be harvested using EVscore47 at 11-fold higher yields and ~13-fold higher purity than those achieved by means of ultracentrifugation. Based on the results presented here, we propose a novel EVscore47-based isolation of OMVs that is fast and scalable.

Evaluation of Physicochemical Properties of Ipsapirone Derivatives Based on Chromatographic and Chemometric Approaches.

Drug discovery is a challenging process, with many compounds failing to progress due to unmet pharmacokinetic criteria. Lipophilicity is an important physicochemical parameter that affects various pharmacokinetic processes, including absorption, metabolism, and excretion. This study evaluated the lipophilic properties of a library of ipsapirone derivatives that were previously synthesized to affect dopamine and serotonin receptors. Lipophilicity indices were determined using computational and chromatographic approaches. In addition, the affinity to human serum albumin (HSA) and phospholipids was assessed using biomimetic chromatography protocols. Quantitative Structure-Retention Relationship (QSRR) methodologies were used to determine the impact of theoretical descriptors on experimentally determined properties. A multiple linear regression (MLR) model was calculated to identify the most important features, and genetic algorithms (GAs) were used to assist in the selection of features. The resultant models showed commendable predictive accuracy, minimal error, and good concordance correlation coefficient values of 0.876, 0.149, and 0.930 for the validation group, respectively.

Proceedings of the 2023 Viral Clearance Symposium, Session 3: 2023 VCS New Modalities in Chromatography and Adsorptive Filters.

The session provided an update on the application and mechanistic understanding of intensified unit operations (e.g., mixed mode depth filters, mixed mode AEX) since the last conference in 2019. One of the key gaps identified in the 2019 Viral Clearance Symposium session on the topic was for more investigation required to achieve a clear understanding of the molecular mechanisms of virus removal and the relevance of different moleculés interactions including resin, virus, and product. Further investigation into worst-case conditions for these unit operations is also warranted. One of the key outcomes from that 2019 discussion was also that multimodal anion exchangers can have robust and effective virus removal, depending on process and impurities-an observation that was recapitulated with more specific case studies and evidenced by broader application of these chromatographic resins in late-stage regulatory filings.

Small-Angle X-Ray Scattering for Macromolecular Complexes.

Small angle X-ray scattering (SAXS) is a versatile technique that can provide unique insights in the solution structure of macromolecules and their complexes, covering the size range from small peptides to complete viral assemblies. Technological and conceptual advances in the last two decades have tremendously improved the accessibility of the technique and transformed it into an indispensable tool for structural biology. In this chapter we introduce and discuss several approaches to collecting SAXS data on macromolecular complexes, including several approaches to online chromatography. We include practical advice on experimental design and point out common pitfalls of the technique.

Efecto de las metodologías analíticas en la cuantificación de bitartrato de epinefrina según la USP-43 y USP-44 / Effect of analytical methodologies on the quantification of epinephrine bitartrate according to USP-43 and USP-44

Introducción: El bitartrato de epinefrina, también conocido como epinefrina, es un ingrediente farmacéutico importante en el tratamiento de diversas enfermedades, pero su medición precisa es esencial para garantizar la seguridad del medicamento. La Farmacopea de los Estados Unidos (USP) establece los estándares para su análisis, pero la elección del método afecta la precisión de las mediciones. Este estudio investiga cómo los diferentes métodos afectan la medición del bitartrato de epinefrina según las versiones USP-43 y USP-44, que tienen implicaciones significativas para la calidad y la regulación de los medicamentos en el campo. Método: Se eligieron el método volumétrico y el método cromatográfico para comparación. Se utilizaron muestras de epinefrina bitartrato de alta pureza que cumplían con los estándares de la USP-43 y USP-44.Resultados: Los resultados obtenidos por ambos métodos se comparan entre sí y se evalúan según los límites de especificación definidos por USP-43 y USP-44. Los valores obtenidos para algunos parámetros, como la concentración y la pureza del bitartrato de epinefrina, varían considerablemente entre los distintos métodos analíticos. Conclusiones: Este estudio destaca la importancia de una cuidadosa selección del método analítico al evaluar el bitartrato de epinefrina según las directrices USP-43 y USP-44. La elección de la tecnología afecta a los resultados y, por tanto, a la calidad y seguridad de los productos farmacéuticos que contienen esta sustancia. Se recomienda validar el método en cada laboratorio y comparar los resultados con los estándares USP. (AU)

Introduction: Epinephrine bitartrate, also known as epinephrine, is an important pharmaceutical ingredient in the treatment of various diseases, but its accurate measurement is essential to ensure the safety of the drug. The United States Pharmacopeia (USP) sets the standards for its analysis, but the choice of method affects the precision of the measurements. This study investigates how different methods affect the measurements of epinephrine bitartrate based on USP-43 and USP-44, which have significant implications for drug quality and regulation in the field. Method: The volumetric method and chromatographic method were chosen for comparison. High-purity epineph-rine bitartrate samples that met USP-43 and USP-44 standards were used. Results: The results obtained by both methods are compared with and evaluated according to the specification lim-its defined by USP-43 and USP-44. The values obtained for some parameters, such as the concentration and purity of epinephrine tartrate, vary considerably between the different analytical methods. Conclusions: This study highlights the importance of carefully selecting analytical methods when evaluating epi-nephrine tartrate according to USP-43 and USP-44 guidelines. The choice of technology affects the results and, therefore, the quality and safety of the pharmaceutical products containing this substance. It is recommended to validate the method in each laboratory and compare the results with USP standards. (AU)

[Determination of organic acids and anions in exhaled breath by condensation collection-ion chromatography].

A non-invasive condensation collection-ion chromatography method was established for the determination of organic acids and anions including lactic acid, formic acid, acetic acid, pyruvic acid, chloride, nitrate, nitrite, and sulfate in the exhaled breath of humans. The breath exhaled was condensed and collected using a home-made exhaled breath condensation equipment. This equipment included a disposable mouthpiece as a blow-off port, one-way valve and flow meter, cold trap, disposable condensate collection tube placed in the cold trap, and gas outlet. A standard sampling procedure was used. Before collection, the collection temperature and sampling volume were set on the instrument control panel, and sampling was started when the cold-trap temperature dropped to the set value, while maintaining the balance. Subjects were required to gargle with pure water before sampling. During the sampling process, the subjects were required to inhale deeply until the lungs were full of gas and then exhale evenly through the air outlet. When the set volume was collected, the instrument made a prompt sound; then, the collection was immediately ended, the expiration time was recorded, and the average collection flow was calculated according to the expiration time and sampling volume. After collection, the disposable condensation collection tube was immediately taken out, sealed, and stored in the refrigerator at -20 ℃ away from light, and immediately used for further testing. The organic acids and anions in exhaled breath condensation (EBC) were filtered through a 0.22 µm membrane filter before injection and detected by ion chromatography with conductivity detection. Factors such as collection temperature and collection flow rate during condensation collection were optimized. The optimal cooling temperature was set at -15 ℃, and the optimal exhaled breath flow rate was set at 15 L/min. The mobile phase consisted of a mixture of sodium carbonate (1.5 mmol/L) and sodium bicarbonate (3 mmol/L). The flow rate was 0.8 mL/min, and the injection volume was 100 µL. An IC-SA3 column (250 mm×4.0 mm) was used, and the temperature was set at 45 ℃. An ICDS-40A electrodialysis suppressor was used, and the current was set at 150 mA. The linear ranges of the eight organic acids and anions were 0.1-10.0 mg/L; their correlation coefficients (r) were ≥0.9993. The limits of detection (LODs) for the eight organic acids and anions were 0.0017-0.0150 mg/L based on a signal-to-noise ratio of 3, and the limits of quantification (LOQs) were 0.0057-0.0500 mg/L based on a signal-to-noise ratio of 10. The intra-day precisions were 5.06%-6.33% (n=5), and the inter-day precisions were 5.37%-7.50% (n=5). This method was used to detect organic acids and anions in the exhaled breath of five healthy subjects. The contents of organic acids and anions in the exhaled breath were calculated. The content of lactic acid was relatively high, at 1.13-42.3 ng/L, and the contents of other seven organic acids and anions were 0.18-11.0 ng/L. During a 10 km-long run, the majority of organic acids and anions in the exhaled breath of five subjects first increased and then decreased. However, due to abnormal metabolism, the content changes of lactic acid, acetic acid, pyruvic acid and chloride in one subject were obviously different from others during exercise, showing a continuous rise. This method has the advantages of involving a simple sampling process and exhibiting good precision, few side effects, and no obvious discomfort or risk to the subjects. This study provides experimental ideas and a theoretical basis for future research on human metabolites.

[Ion chromatography for the determination of n-butylamine in the workplace air].

Objective: To establish a method for the determination of n-butylamine in the air of the workplace by ion chromatography. Methods: In February 2022, on-site sampling was carried out using an atmospheric sampler. N-butylamine was adsorbed by a neutral silica gel tube and then performed for qualitative and quantitative determination by ion chromatography after ultrasonic desorption with 10 mmol/L sulfuric acid solution. Results: The linear range of the method was 0.0375-100.0 µg/ml, the linear equation of the standard curve was y=0.0713x-0.0327, the correlation coefficient was 0.9992. The detection limit of the method was 11.25 µg/L, and the lower limit of quantification was 37.50 µg/L, the lowest quantitative concentration was 0.025 mg/m(3) (in term of sampling 7.5 L). The average desorption efficiency of the method was 91.50%-95.38%, the precision was 1.10%-2.30%, the standard recovery was 83.83%-100.02%, sampling efficiency was 100.00%. Conclusion: This method is fast, sensitive and accurate, and can be used for the determination of n-butylamine in the air of workplace.

On the contribution of the top and bottom walls in micro-pillar array columns and related high-aspect ratio chromatography systems.

We report on a steady-state based, and hence highly accurate numerical modelling study of the effect of the top and bottom wall in the current generation of micro-pillar array columns. These have a mesoporous retention layer that not only covers the pillar walls but also the bottom wall. Our results show that the performance of these columns can in general not be improved by also covering the top wall with the same layer, despite the increased column symmetry this approach would offer. The reason for this is that the local species retardation caused by a retentive layer is much stronger than the pure flow arresting effect of an uncovered wall. At least, this has a crucial impact in high aspect-ratio systems such as micro-pillar array columns because these require a small inter-pillar distance to promote mass transfer together with a large channel depth to enable a sufficiently high flow rate. On the other hand, a notable improvement could be made if micro-pillar array would be produced without having a retentive layer at the bottom. At Péclet number Pe = 50 and aspect ratio AR = 5 for flow-channels, this gain amounts up to about 4.5 h-units at a zone retention factor k'' = 2 and 1.75 h-units at k'' = 16 (gain scales almost linearly with Pe). To verify these results, we also considered another high aspect-ratio system with a simplified geometry: the open-tubular channel with a flat-rectangular cross-section. This led to very similar observations, thus confirming the findings for the micro-pillar array. The results produced in the present study also allow us to conclude that the classic modelling paradigm adopted in chromatography, which is based on the independency and hence additivity of the hCm- and hCs-contributions, can lead to large modelling errors in chromatographic systems with a high aspect-ratio, even when their geometry is so simple as that of a straight open-tubular channel with constant cross-section. Indeed, when both zones are treated independently, the analysis misses how the vertical diffusion through the retentive layer helps suppressing the vertical gradients in the mobile zone. The diffusion through this layer occurs in a ratio of k''Ds/Dm (Dm being the diffusion coefficient in mobile phase zone and Ds being the diffusion coefficient in stationary phase zone), such that at high retention factors this diffusion contribution even becomes the dominant one.

Chromatography based profiling of feruloylated arabinans and galactans.

Profiling of pectic arabinans and galactans by analysis of the released oligosaccharides after backbone cleavage provides information on the complexity of the polymer structure. In plants of the family Amaranthaceae, arabinan and galactan substitution with ferulates extends the polysaccharide complexity, changing its chemical properties. Knowledge of the ferulate environment is crucial to understand structure-function-relationships of feruloylated pectins. Here, we present an approach to separate enzymatically generated feruloylated and non-feruloylated arabino- and galactooligosaccharides, followed by deesterification and semiquantitative analysis by HPAEC-PAD using previously reported relative response factors. Application of this approach to sugar beet pectins and insoluble and soluble dietary fiber preparations of amaranth and quinoa suggests that ferulates are preferably incorporated into more complex structures, as nicely demonstrated for feruloylated galactans. Also, ferulate substitution appears to negatively affect enzymatic cleavage by using endo-enzymes. As a consequence, we were able to tentatively identify new feruloylated tri- and tetrasaccharides of galactans isolated from sugar beet pectins.

Nanoconfined Electrokinetic Chromatography (NEC): Gradient Separation and Sensing of Short DNA Fragments at the Single-Molecule Level.

Glass nanopipets have been demonstrated to be a powerful tool for the sensing and discrimination of biomolecules, such as DNA strands with different lengths or configurations. Despite progress made in nanopipet-based sensors, it remains challenging to develop effective strategies that separate and sense in one operation. In this study, we demonstrate an agarose gel-filled nanopipet that enables hyphenated length-dependent separation and electrochemical sensing of short DNA fragments based on the electrokinetic flow of DNA molecules in the nanoconfined channel at the tip of the nanopipet. This nanoconfined electrokinetic chromatography (NEC) method is used to distinguish the mixture of DNA strands without labels, and the ionic current signals measured in real time show that the mixed DNA strands pass through the tip hole in order according to the molecular weight. With NEC, gradient separation and electrochemical measurement of biomolecules can be achieved simultaneously at the single-molecule level, which is further applied for programmable gene delivery into single living cells. Overall, NEC provides a multipurpose platform integrating separation, sensing, single-cell delivery, and manipulation, which may bring new insights into advanced bioapplication.

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates.

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25-120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions.

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids.

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.

Simultaneous prediction of 16 quality attributes during protein A chromatography using machine learning based Raman spectroscopy models.

Several key technologies for advancing biopharmaceutical manufacturing depend on the successful implementation of process analytical technologies that can monitor multiple product quality attributes in a continuous in-line setting. Raman spectroscopy is an emerging technology in the biopharma industry that promises to fit this strategic need, yet its application is not widespread due to limited success for predicting a meaningful number of quality attributes. In this study, we addressed this very problem by demonstrating new capabilities for preprocessing Raman spectra using a series of Butterworth filters. The resulting increase in the number of spectral features is paired with a machine learning algorithm and laboratory automation hardware to drive the automated collection and training of a calibration model that allows for the prediction of 16 different product quality attributes in an in-line mode. The demonstrated ability to generate these Raman-based models for in-process product quality monitoring is the breakthrough to increase process understanding by delivering product quality data in a continuous manner. The implementation of this multiattribute in-line technology will create new workflows within process development, characterization, validation, and control.

Molecular recognition and binding between human plasminogen Kringle 5 and α-chain of human complement component C3b by frontal chromatography and dynamics simulation.

The binding and molecular recognition between α-chain of human complement C3b (α-chain of C3b) and human plasminogen Kringle 5 (Kringle 5) were studied and explored by frontal chromatography and dynamics simulation in the combination of bio-specific technologies. The specific interaction between the α-chain of C3b and Kringle 5 was initially confirmed by ligand blot and ELISA (Kd = 4.243×10-6 L/mol). Furthermore, the binding determination conducted via frontal chromatography showed that the presence of a single binding site between them, with the binding constant of 2.98 × 105 L/mol. Then the molecular recognition by dynamics simulation and molecular docking showed that there were 9 and 13 amino acid residues respective in the Kringle 5 and α-chain of C3b directly implicated in the binding and the main stabilizing forces were electrostatic force (-55.99 ± 11.82 kcal/mol) and Van der Waals forces (-42.70 ± 3.45 kcal/mol). Additionally, a loop structure (65-71) in Kringle 5 underwent a conformational change from a random structure to an α-helix and a loop structure (417-425) in α-chain of C3b was closer to the molecular center, both of them were more conducive to the binding between them. Meanwhile, the involvement of the lysine binding site of Kringle 5 played an important role in the binding process. In addition, the erythrocyte-antibody complement rosette assay substantiated that the presence of Kringle 5 hindered the transportation of α-chain of C3b to antigen-antibody complex in a dose-dependent manner. These findings collectively indicated that the α-chain of C3b is very likely a receptor protein for Kringle 5, which provides a methodology for other similar investigations and valuable insights into expansion of the pharmacological effects and potential application of Kringle 5 in immune-related diseases.