Cation-exchange Chromatography Research Articles

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Removal of signal peptide variants by cation exchange chromatography: A case study

Signal peptide (SP) is required for secretion of recombinant proteins and typically cleaved by signal peptidase at its C-region to generate the mature proteins. Miscleavage of the SP is reported occasionally, resulting in a truncated- or elongated-terminal sequence. In the present work, we demonstrated that cation exchange (CEX) chromatography is an effective means for removing SP variants with a case study. With the selected resin/conditions, the chromatographic performance is comparable between runs performed at the low end and high end of load density and elution range. The procedure described in this work can be used as a general approach for resin selection and optimization of chromatographic conditions to remove byproducts that bind more strongly than the product to the selected resin.

Coupling cation and anion exchange chromatography for fast separation of monoclonal antibody charge variants

A design procedure for the separation of charge variants of a monoclonal antibody (mAb) was developed, which was based on the coupling of cation-exchange chromatography (CEX) and anion-exchange chromatography (AEX) under high loading conditions. The design of the coupled process was supported by a dynamic model. The model was calibrated on the basis of band profiles of variants determined experimentally for the mAb materials of different variant compositions. The numerical simulations were used to select the coupling configuration and the loading conditions that allowed for efficient separation of the mAb materials into three products enriched with each individual variant: the acidic (av), main (mv) and basic (bv) one. In the CEX section, a two-step pH gradient was used to split the loaded mass of mAb into a weakly bound fraction enriched with av and mv, and a strongly bound fraction containing the bv-rich product. The weakly bound fraction was further processed in the AEX section, where the mv-rich product was eluted in flowthrough, while the av-rich product was collected by a step change in pH. The choice of flow distribution and the number of columns in the CEX and AEX sections depended on the variant composition of the mAb material. For the selected configurations, the optimized mAb loading density in the CEX columns ranged from 10 to 26 mg mL-1, while in the AEX columns it was as high as 300 or 600 mg mL-1, depending on the variant composition of the mAb material. By proper selection of the loading condition, a trade-off between yield and purity of the products could be reached.

Utilization of Matrix Effect for Enhancing Resolution in Cation Exchange Chromatography.

In ion chromatography studies, the matrix effect of other inorganic ions present in the sample is a well-known phenomenon. In this work, the behavior of inorganic and organic ions was studied in a system overloaded with ammonium ions. The ammonium ions came from a solution of ammonium hydroxide in various concentrations (0.25-1.25%). In this system, which was significantly overloaded with ammonium ions, the behavior of three ions were tested (lithium, tris, and sodium cations). The measurements were performed at different eluent concentrations (6-17 mM), chromatographic column temperatures (25-40 °C), and injected volumes (15-40 µL). The retention times of sodium and lithium ions increased with increasing amounts of injected ammonium, while tris remained essentially unchanged, indicating that the resolution of these ions can be influenced by varying the concentration of the matrix. The results suggested that the observed effect was due to a combination of the pH change caused by the injected matrix, the dissociation of tris ions, the dissociation of the carbocylic ion-exchange groups of stationary phase, the change in buffer capacity, and the amount of ammonium ion introduced. It has been shown that in a well-designed experiment, the addition of ammonium hydroxide to the sample at concentrations greater than 1% can improve the efficiency of organic and inorganic cation separation. It was found that 8 mM methanesulfonic acid eluent, 30 °C, 1% ammonium hydroxide matrix concentration, and 25 µL injection were optimal for the baseline separation of tris and sodium ions on the high-capacity Dionex CS16 column. These ions could not be separated on this column without the presence of the ammonium matrix.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

One-step N-Terminomics Based on Isolation of Protein N-Terminal Peptides From LysargiNase Digests by Tip-Based Strong Cation Exchange Chromatography

We have developed a one-step isolation method for protein N-terminal peptides from LysargiNase digests by pipette tip-based strong cation exchange (SCX) chromatography. This CHAMP-N (CHromatographic AMplification of Protein N-terminal peptides) method using disposable and parallel-processable SCX tips instead of conventional HPLC SCX columns facilitates simple, sensitive, reproducible and high-throughput N-terminomic profiling without sacrificing the high identification numbers and selectivity achieved by the HPLC-based method. By applying the CHAMP-N method to HEK293T cells, we identified novel cleavage sites for signal and transit peptides, and non-canonical translation initiation sites. Finally, for proteome-wide terminomics, we present a simple and comprehensive N- and C-terminomics platform employing three different tip-based approaches, including CHAMP-N, in which protease digestion and one-step isolation by tip LC are commonly used to achieve complementary terminome coverages.

Characterization of Egg White Lysozyme from Local Duck Mojosari and Alabio

Lysozyme, a natural antibacterial, has potential use in several applications. Generally, it is obtained from chicken egg whites. However, the idea of using local duck eggs as a source of lysozyme is not wellknown. This study aimed to isolate and purify the egg white of Mojosari and Alabio ducks to characterize lysozyme. Additionally, the study analyzed the effect of temperature treatment on lysozyme enzymatic activity. Three different temperature treatments were used: Low Temperature Long Time (63 ℃, 30’), High Temperature Short Time (72 ℃, 15”), and sterilization (121 ℃, 15’). The Amberlite resin cation exchange chromatography method was successfully used to isolate and purify Mojosari and Alabio duck egg whites, achieving purity levels of 94.92-96.09%. The presence of lysozyme was confirmed by the existence of bands that were primarily composed of lysozyme. The results showed that each aspect of the characteristics of lysozyme and enzymatic activity were significantly affected (p<0.05) by the type of egg white. Enzymatic activity indicated that Alabio duck lysozyme was thermosensitive and could only survive at LTLT temperature, while Mojosari duck lysozyme could survive at LTLT and HTST pasteurization temperatures. However, both lysozymes did not survive the sterilization temperature.

Role of Charge Heterogeneity on Physical Stability of Monoclonal Antibody Biotherapeutic Products.

Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated. The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions. Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants. Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.

Efficient experimental method for purifying allergens from aqueous extracts

Studying the molecular and immunological basis of allergic diseases often requires purified native allergens. The methodologies for protein purification are usually difficult and may not be completely successful. The objective of this work was to describe a methodology to purify allergens from their natural source, while maintaining their native form. The purification strategy consists of a three-step protocol and was used for purifying five specific allergens, Ole e 1, Amb a 1, Alt a 1, Bet v 1 and Cup a 1.Total proteins were extracted in PBS (pH 7.2). Then, the target allergens were pre-purified and enriched by salting-out using increasing concentrations of ammonium sulfate. The allergens were further purified by anion exchange chromatography. Purification of Amb a 1 required an extra step of cation exchange chromatography. The detection of the allergens in the fractions obtained were screened by SDS-PAGE, and Western blot when needed. Further characterization of purified Amb a 1 was performed by mass spectrometry. Ole e 1, Alt a 1, Bet v 1 and Cup a 1 were obtained at > 90 % purity. Amb a 1 was obtained at > 85 % purity.Overall, we propose an easy-to-perform purification approach that allows obtaining highly pure allergens. Since it does not involve neither chaotropic nor organic reagents, we anticipate that the structural and biological functions of the purified molecule remain intact. This method provides a basis for native allergen purification that can be tailored according to specific needs.

A Cyber-Physical Production System for the Integrated Operation and Monitoring of a Continuous Manufacturing Train for the Production of Monoclonal Antibodies.

The continuous manufacturing of biologics offers significant advantages in terms of reducing manufacturing costs and increasing capacity, but it is not yet widely implemented by the industry due to major challenges in the automation, scheduling, process monitoring, continued process verification, and real-time control of multiple interconnected processing steps, which must be tightly controlled to produce a safe and efficacious product. The process produces a large amount of data from different sensors, analytical instruments, and offline analyses, requiring organization, storage, and analyses for process monitoring and control without compromising accuracy. We present a case study of a cyber-physical production system (CPPS) for the continuous manufacturing of mAbs that provides an automation infrastructure for data collection and storage in a data historian, along with data management tools that enable real-time analysis of the ongoing process using multivariate algorithms. The CPPS also facilitates process control and provides support in handling deviations at the process level by allowing the continuous train to re-adjust itself via a series of interconnected surge tanks and by recommending corrective actions to the operator. Successful steady-state operation is demonstrated for 55 h with end-to-end process automation and data collection via a range of in-line and at-line sensors. Following this, a series of deviations in the downstream unit operations, including affinity capture chromatography, cation exchange chromatography, and ultrafiltration, are monitored and tracked using multivariate approaches and in-process controls. The system is in line with Industry 4.0 and smart manufacturing concepts and is the first end-to-end CPPS for the continuous manufacturing of mAbs.

Implementation of a LC-MS based multi-attribute method (MAM) and intact multi-attribute method (iMAM) workflow for the characterisation of a GLP-Fc fusion protein

Over the past few years, the implementation of mass spectrometry (MS) in QC laboratories has become a more common occurrence. The multi-attribute method (MAM), and emerging intact multi-attribute method (iMAM), are powerful analytical tools utilising liquid chromatography−mass spectrometry (LC-MS) methods that enable the monitoring of critical quality attributes (CQAs) in biotherapeutic proteins in compliant settings. Both MAM and iMAM are intended to replace or supplement several conventional assays with a single LC-MS method utilising MS data in combination with robust, semi-automated data processing workflows. MAM and iMAM workflows can also be implemented into current Good Manufacturing Practices environments due to the availability of CFR 11 compliant chromatography data system software. In this study, MAM and iMAM are employed for the analysis of 4 batches of a glucagon-like peptide-Fc fusion protein. MAM approach involved a first the discovery phase for the identification of CQAs and second, the target monitoring phase of the selected CQAs in other samples. New peak detection was performed on the data set to determine the appearance, absence or change of any peak. For native iMAM workflow both size exclusion and strong cation exchange chromatography were optimized for the identification and monitoring of CQAs at the intact level.

Production and separation of 7Be for use in an ion source

Beryllium-7 (7Be) was created by proton irradiation of natural (natB) and enriched (10B) boron targets. The targets were dissolved in nitric acid, and the 7Be was separated from the bulk boron target material by cation-exchange chromatography. An average recovery of (99.4 ± 3.7)% was obtained for 6 separations. The purified 7Be sample was placed into a batch-mode ion source to create a 7Be beam that was delivered at an average rate of 5 × 105 pps to end users at the National Superconducting Cyclotron Laboratory.

Preparation of carboxyl-functionalized silica core-shell microspheres and their applications in weak cation exchange chromatography, heavy metal removal, and lysozyme enrichment.

Chromatography is a technique of separation based on adsorption and/or interaction of target molecules with stationary phases. Herein, we report the design and fabrication of BTDA@SiO2 core-shell microspheres as a new class of stationary phase and demonstrate its impressive performance for chromatographic separations. The silica microspheres of BTDA@SiO2 were synthesized by in situ method with 1,3,5-benzenetricarboxaldehyde and 3,5-diaminobenzoic to separate peptides and proteins on high-performance liquid chromatography. The BTDA@SiO2 core-shell structure has a high specific surface area and retention factor of 4.27 and 8.31 for anionic and cationic peptides, respectively. The separation factor and resolution were high as well. A typical chromatogram illustrated nearly baseline resolution of the two peptides in less than 3 min. The BTDA@SiO2 was also highly stable in the pH range of 1 to 14. Furthermore, the prepared BTDA@SiO2 core-shell material not only be used for chromatographic separation but also as heavy metal removal from water. Using a BTDA@SiO2, we also achieved a lysozyme enrichment with a maximum saturated adsorption capacity reaching 714mg/g. In summary, BTDA@SiO2 has great application prospects and significance in separation and purification systems.

Analytical performance evaluation of the Mindray enzymatic assay for hemoglobin A1c measurement

Hemoglobin A1c (HbA1c) plays a crucial role in diabetes management. We aimed to evaluate the analytical performance of a new enzymatic method kit for HbA1c measurement. The performance of the enzymatic method, including precision, accuracy, and linearity, was evaluated. Moreover, the interference effect from conventional interferents, Hb derivatives, Hb variants, and common drugs were assessed. In addition, the agreement of HbA1c results was compared between enzymatic methods, cation-exchange high-performance liquid chromatography (HPLC), and immunoassays. The intra-assay, between-assay, and total precision of HbA1c were all lower than 2%. HbA1c showed good linearity within the range of 3.96–20.23%. The enzymatic assay yielded results consistent with the external quality control samples, with a bias of less than ± 6% from the target values. The enzymatic method showed no interference from bilirubin, intralipid, vitamin C, Hb derivatives, common Hb variants, as well as antipyretic analgesics and hypoglycemic drugs. The HbA1c results of the enzymatic assay showed good agreement and accuracy compared to those obtained from the HPLC method and the immunoassay. The enzymatic method kit performed on the BS-600M chemistry analyzer is a reliable and robust method for measuring HbA1c. It is suitable for routine practice in clinical chemistry laboratories.

Interference of hemoglobin variants with HbA1c measurements: comparison of 6 commonly used HbA1c methods with the IFCC reference method.

Glycated hemoglobin, or hemoglobin A1c (HbA1c), serves as a crucial marker for diagnosing diabetes and monitoring its progression. We aimed to assess the interference posed by common Hb variants on popular HbA1c measurement systems. A total of 63 variant and nonvariant samples with target values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method were included. We assessed 6 methods for measuring HbA1c in the presence of HbS, HbC, HbD, HbE, and fetal hemoglobin (HbF): 2 cation-exchange high-performance liquid chromatography (HPLC) methods (Bio-Rad D-100 and HLC-723 G8), a capillary electrophoresis (CE) method (Sebia Capillarys 3 TERA), an immunoassay (Roche c501), an enzyme assay system (Mindray BS-600M), and a boronate affinity method (Primus Premier Hb9210). The HbA1c results for nonvariant samples from the 6 methods were in good agreement with the IFCC reference method results. The Bio-Rad D-100, Capillarys 3, Mindray BS-600M, Premier Hb9210, and Roche c501 showed no interference from HbS, HbC, HbD, and HbE. Clinically significant interference was observed for the HLC-723 G8 standard mode. Elevated HbF levels caused significant negative biases for all 6 methods, which increased with increasing HbF concentration. Elevated levels of HbF can severely affect HbA1c measurements by borate affinity, immunoassays, and enzyme assays.

Preparation of Lactobacillus paracei TK1501 postbiotic and its inhibitory effect against Helicobacter pylori infection in mice

To prepare a postbiotic using soybean fermentation product of Lactobacillus paracasei TK1501 and evaluate its inhibitory effect against Helicobacter pylori (Hp) infection in mice. L. paracasei TK1501 was cultured for 32 h at 37 ℃ in an anaerobic condition for solid substrate fermentation with a solid to water ratio of 1:1.5 in the substrate and an inoculation density of 5×107 CFU/mL. The postbiotic was isolated and purified using macroporous resin XAD-16N adsorption, cation exchange chromatography and HPLC, and its stability and antibacterial activity were assessed. The inhibitory effect of this postbiotic against Hp infection was evaluated in a mouse model with gastric mucosal Hp infection, which were treated with the postbiotic via gavage for 4 weeks at the dose of 0.02 or 0.1 mL. Serum levels of TNF-α and IL-1β of the mice were analyzed after the treatments, and gastric tissues of the mice were collected for HE staining. L. paracasei TK1501 postbiotic could be easily degraded by protease and had good thermal stability and tolerance to exposures to acid, base, and organic solvents. In the in vitro experiment, the postbiotic showed strong inhibitory effects in bacterial cultures of Staphylococcus aureus, Hp and other common pathogenic bacteria without obviously affecting the resident bacteria in the digestive tract. In the mouse models, treatment with the postbiotic at the dose of 0.1 mL significantly alleviated Hp infection and lowered the serum levels of TNF-α and IL-1β of the mice. L. paracasei TK1501 postbiotic has strong inhibitory effects on Hp and Staphylococcus aureus but not on normal intestinal flora in mice.

First report of antioxidant potential of peptide fraction derived from Colaconema formosanum (Rhodophyta) protein hydrolysates

Foods that contain natural antioxidants can improve people's health and performance and make them more resistant to environmental stressors like diseases, lousy living conditions, and heat stress. Marine algae are well known for their function as an antioxidant. Colaconema formosanum is a new rhodophyte discovered in Taiwan and its bioactive activities are rarely studied. This work aims to prepare the protein hydrolysate fraction with antioxidant properties in C. formosanum using single enzyme or complex enzyme hydrolysis. Afterward, a 3 kDa molecular weight cut-off (MWCO) was used for centrifugation, and then fractionation was carried out using Strong Cation Exchange (SCX) Chromatography. The quantification of antioxidant activity was performed using the DPPH test. The results showed that the thermolysin hydrolysate (IC50 1.48 ± 0.92 mg/mL) and the combination of trypsin-thermolysin hydrolysates (IC50 1.37 ± 0.84 mg/mL) had the highest antioxidant activity. The fractionation procedure with strong cation exchange (SCX) yielded fraction C from thermolysin hydrolysates, which exhibited the most significant antioxidant activity with an IC50 value of 0.47 ± 0.02 mg/mL. The fraction 4 derived from trypsin-thermolysin hydrolysates exhibited the most potent antioxidant activity (IC50: 0.34 ± 0.002 mg/mL). No previous reports have been made about the antioxidant properties of the hydrolysate fractions derived from C. formosanum proteins. Consequently, these findings could serve as a foundation for exploring peptides that have potential therapeutic sources for reducing oxidative stress.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60–90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 μg to 160 μg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

Isolation and characterization of rabies monoclonal antibody charge variants.

Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.

Chemical composition and amino acids of Locusta migratoria (Orthoptera Acrididae) fed wheat seedlings (Triticum aestivum) or rape leaves (Brassica napus)

Abstract Locusts have high nutritional value and are considered alternative protein sources for humans and animals. Diet can strongly affect the nutritional composition of insects. In this study, the influences of diets – wheat (Triticum aestivum) seedlings and rape (Brassica napus) leaves – on the chemical composition and amino acids of the migratory locust (Locusta migratoria) were assessed. The crude protein, soluble carbohydrate and triglyceride were analyzed using the Bradford method, anthrone colorimetry, and glycerol kinase chromogenic method, respectively. Additionally, amino acids were determined using cation exchange chromatography. The study revealed that wheat seedlings had lower water content than rape leaves. Although the crude protein content was comparable, the soluble carbohydrates of wheat seedlings were approximately 2.5 times higher than those of rape leaves. The body weight (fresh weight and dry weight), crude protein and triglyceride of locusts significantly increased when fed wheat seedlings compared with when fed rape leaves, with values of 48.08%, 31.58%, 43.83% and 25.00%, respectively. The high water content of rape leaves resulted in the high water content of rape leaf-fed locusts. Plants also affect the amino acid composition of locusts. The eight amino acids (four essential amino acids and four non-essential amino acids) and the total amino acids of rape leaf-fed locusts were significantly enhanced compared with those of wheat seedlings. The essential amino acid index and biological value indicate that the protein quality of locusts fed rape leaves has been significantly improved. These results demonstrate that the nutritional composition of L. migratoria can be altered by manipulating the diet. Insects with controllable nutrients can be provided by insect farms and further studies will improve the development of insects as food or feed.