Preface

Ion Exchange Chromatography (IEC) is a crucial biochemical separation technique. Based on the interaction between charged molecules and the stationary phase, it is widely applied in protein analysis. This application enables the deduction of protein-related degradation pathways through acid-base variants and provides guiding data for relevant quality control and molecular optimization. However, method development for IEC in protein analysis is extremely challenging. This difficulty stems from the distinct spatial structures and amino acid sequences of each protein—these dramatic variations result in different isoelectric points, sialic acid levels, and glycosylation degrees for each protein, significantly increasing the complexity of method development. The key steps in this development process include multiple stages: selecting appropriate ion exchange media, optimizing buffer systems, determining elution conditions, and adjusting flow rates and column temperatures, among others. These steps ensure the effectiveness and reliability of IEC in protein analysis, yet the cumbersome method development process imposes substantial time costs on experiments, indirectly raising investment costs and entailing the risk of method development failure. This has become an urgent issue in the field of protein drug analysis. Zhoufan Bio has developed a universal mobile phase for Ion Exchange Chromatography and achieved encouraging progress.

Various properties of components in the sample, such as charge characteristics, size, shape, and hydrophobicity, affect the process and results of ion exchange chromatography. Therefore, selecting suitable ion exchange media and buffer systems is particularly important. The optimization of chromatographic conditions (including ionic strength, pH value, temperature, and flow rate) must be determined through experiments. Moreover, even minor changes in ionic strength and pH value during IEC can lead to significant differences in separation performance. Precise control of these parameters when preparing mobile phases for IEC in daily operations is the key to achieving stable and reproducible separation. However, in practical operations, errors may be introduced during the preparation and use of buffers, often causing problems such as the migration of chromatographic peak retention times. To address the aforementioned issues, ZF Bio has independently developed and optimized a universal mobile phase for Ion Exchange Chromatography. This product comprehensively considers analysis costs and time efficiency, balancing separation efficiency, analysis time, and cost during the development of ion chromatography methods. It provides an efficient, cost-effective, and universal option for ion exchange chromatography mobile phases.

Materials and Methods

Instruments and Chromatographic Columns

· Agilent 1260 High Performance Liquid Chromatography (HPLC) System

· ThermoProPac? WCX-10 HPLC Column (10μm, 4×250mm)

Samples

The samples used in the experiment included 8 protein drug samples, all provided by the cooperative partner: the Biopharmaceutical Formulation and Analysis Team of the School of Pharmacy, Zhejiang University.

IEC-HPLC Parameters

The following table lists the chromatographic parameters for IEC-HPLC analysis using ZF-LC-0002 Mobile Phase A&B (10×):

Experimental Parameters

Eight types of proteins were diluted to a concentration of 1 mg/mL and digested with CpB enzyme. After digestion, the samples were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected for injection and detection.

Results

See Figure 1.

Hangzhou ZF Biotechnology Co., Ltd. was founded in 2018. With a highly efficient R&D team, it is a company focusing on the field of biotechnology. The company boasts extensive experience and technological advantages in areas such as protein drug process development and optimization, and molecular diagnostics. Our product range includes commercial capillary gel electrophoresis (CGE) test kits, high-performance liquid chromatography (HPLC) ion exchange test kits, cell culture reagents, and molecular diagnostic reagents. These products are widely applied in fields like life science research and drug development. We have established close cooperative relationships with numerous well-known research institutions and enterprises to jointly promote the development and application of biotechnology.

Beijing XMJ Technology Co., Ltd. is the general agent of ZF Bio. Adhering to a professional and rigorous attitude, it consistently provides customers with high-quality products and services. If you are interested in the aforementioned products, please feel free to send email to info@xmjsci.com or visit the website rmb365.cn to learn more about the products.