Accurate determination of drug-to-antibody ratio of interchain cysteine-linked antibody-drug conjugates by LC-HRMS.

Accurate determination of the drug-to-antibody ratio (DAR) of interchain cysteine-linked antibody-drug conjugates (ADCs) is challenging. High-resolution mass spectrometry (HRMS) analysis of the ADCs at the intact or subunit level provides a feasible way to measure the DAR. However, the measured DAR is usually lower than the true DAR because of the variation in ionization efficiency between different DAR species. In this work, we developed a novel standard-free HRMS method involving isotope-labeled payload conjugation, protease digestion, and liquid chromatography-HRMS (LC-HRMS) analysis for accurate determination of the DAR of the interchain cysteine-linked ADCs with cleavable or non-cleavable linkers. Isotope-labeled payload conjugations eliminated the structural and chemical differences between different DAR species and ensured that the drugs or payload-containing peptides could be separated from each other in the mass spectrometer. A papain digestion strategy for ADCs with cleavable linkers showed a DAR of 3.79, with a relative standard deviation (RSD) of 0.48 (n = 3). Similarly, the trypsin and chymotrypsin digestion strategy that is applicable to ADCs with non-cleavable linkers showed a DAR of 3.77 and an RSD of 0.86 (n = 3). The DAR determined by this method was consistent with the DAR of the ADCs that was measured by the UV/Vis method. This method will be very useful to researchers working in the field of ADC discovery and development. Graphical abstract.

Characterization of Positional Isomers of Interchain Cysteine Linked Antibody-Drug Conjugates by High-Resolution Mass Spectrometry.

Interchain cysteine linked antibody-drug conjugates (ADCs) are emerging therapeutic products that antagonize cancers. The toxic payloads are selectively linked to the interchain cysteines and generate heterogeneous mixtures of positional isomers. These positional isomers might contribute differently to the therapeutic efficacy because of the variation in conjugation stability, and thus they need to be well characterized. However, the characterization of the positional isomers of interchain cysteine linked ADCs is very challenging, mainly because of the high similarity between those isomers. In this research, we developed a novel mass spectrometry method for the characterization of positional isomers of interchain cysteine linked ADCs. The subunit analysis and the bottom-up analysis provided abundant information about the drug numbers and drug linking positions on each chain. Because the method can provide accurate data on drug linking numbers and positions on each chain, it will be very useful for researchers in cancer drug development and cancer treatment.

Development and validation of an LC-MS/MS Method for the quantitation of heparan sulfate in human urine.

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC-MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC-MS/MS method for the quantitation of heparan sulfate in human urine using selected high-abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid-phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.

De Novo Transcriptome Sequencing and the Hypothetical Cold Response Mode of Saussurea involucrata in Extreme Cold Environments.

Saussurea involucrata grows in high mountain areas covered by snow throughout the year. The temperature of this habitat can change drastically in one day. To gain a better understanding of the cold response signaling pathways and molecular metabolic reactions involved in cold stress tolerance, genome-wide transcriptional analyses were performed using RNA-Seq technologies. A total of 199,758 transcripts were assembled, producing 138,540 unigenes with 46.8 Gb clean data. Overall, 184,416 (92.32%) transcripts were successfully annotated. The 365 transcription factors identified (292 unigenes) belonged to 49 transcription factor families associated with cold stress responses. A total of 343 transcripts on the signal transduction (132 upregulated and 212 downregulated in at least any one of the conditions) were strongly affected by cold temperature, such as the CBL-interacting serine/threonine-protein kinase (CIPKs), receptor-like protein kinases, and protein kinases. The circadian rhythm pathway was activated by cold adaptation, which was necessary to endure the severe temperature changes within a day. There were 346 differentially expressed genes (DEGs) related to transport, of which 138 were upregulated and 22 were downregulated in at least any one of the conditions. Under cold stress conditions, transcriptional regulation, molecular transport, and signal transduction were involved in the adaptation to low temperature in S. involucrata. These findings contribute to our understanding of the adaptation of plants to harsh environments and the survival traits of S. involucrata. In addition, the present study provides insight into the molecular mechanisms of chilling and freezing tolerance.

Construction and Quality Analysis of Transgenic Rehmannia glutinosa Containing TMV and CMV Coat Protein.

Plant viruses, especially tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are serious threats to Rehmannia glutinosa which is a "top grade" herb in China. In the present study, TMV- and CMV-resistant Rehmannia glutinosa Libosch. plants were constructed by transforming the protein (CP) genes of TMV and CMV into Rehmannia glutinosa via a modified procedure of Agrobacterium tumefaciens-mediated transformation. Integration and expression of TMV CP and CMV CP transgenes in 2 lines, LBA-1 and LBA-2, were confirmed by PCR, Southern blot and RT-PCR. Both LBA-1 and LBA-2 were resistant to infection of homologous TMV and CMV strains. The quality of transgenic Rehmanniae Radix was evaluated based on fingerprint analysis and components quantitative analysis comparing with control root tubes. These results showed that chemical composition of transgenic Rehmanniae Radix were similar to non-transgenic ones, which demonstrated that the medical quality and biosafety of transgenic Rehmanniae Radix were equivalent to non-transgenic material when consumed as traditional Chinese medicinal (TCM).

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity.

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down-regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9-hydroperoxyoctadecadienoic acid (9-HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild-type seeds by measuring the ultraviolet absorption of 9-HPOD at 234 nm and by high-performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS-91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.

Quantitation of P450 3A4 endogenous biomarker - 4ß-hydroxycholesterol - in human plasma using LC/ESI-MS/MS.

4ß-Hydroxycholesterol (4ß-HC) has been proposed as a new endogenous biomarker for cytochrome P450 3A4/5 activity. Therefore, it is important to have a robust method for its accurate determination in human plasma. Here a liquid chromatography-tandem mass spectrometry with electrospray ionization (LC/ESI-MS/MS) assay for the quantitation of 4ß-HC in human plasma is described. While the calibration standards were prepared in a surrogate matrix for human plasma, the quality control samples were prepared in human plasma to mimic the incurred study samples. In order to achieve accurate determination of 4ß-HC, the chromatographic separation of 4ß-HC from its isomers, especially 4α-hydroxycholesterol (4α-HC), was crucial. In the absence of an authentic 4α-HC standard at the time of this study, an alternative selectivity test strategy was developed to confirm the separation. After being alkalized with potassium hydroxide, the human plasma sample (50 µL) was extracted with hexane, derivatized into picolinyl esters using picolinic acid, extracted again with hexane, and then analyzed by LC/ESI-MS/MS. The calibration curve range was 5-500 ng/mL and the chromatographic separation was achieved on a 50 × 2.1 mm Thermal Hypersil Gold column with a gradient elution. The assay accuracy, precision, linearity, selectivity and analyte stability throughout the analysis were established. The validated assay was successfully applied to a Phase I clinical study for the measurement of 4ß-HC in human plasma.

Pilot and pivotal study to evaluate the bioequivalence of two paroxetine 40 mg tablet formulations in healthy Chinese subjects.

The purpose of this study was to conduct a pilot study in order to obtain reliable results for further planning of a well-designed pivotal trial comparing the bioequivalence (BE) of two paroxetine tablet formulations in healthy Chinese subjects. Before conducting the pivotal trial, the pilot trial enrolled 14 subjects to help in study design, establishing the recruitment period, determining pharmacokinetics (PK) time points and sample size, and assessing BE of the two formulations. The single-center, randomized, open-label, single-dose, two period crossover study with a 7-day washout interval was conducted after obtaining information from the fasted pilot trial in 72 healthy volunteers for a pivotal study under fed and fasted conditions, respectively. There were 19 PK sample collection time points employed in both the pilot and pivotal trials. A sensitive and specific liquid chromatography- tandem mass spectrometry (LC-MS/ MS) method was developed and validated for determining paroxetine in human plasma. BE between two articles was determined by calculating 90% confidence intervals (CIs) for the ratio of Cmax 91.38 - 110.39% for the pilot trial, 99.81 - 114.08% for pivotal trial under fasted condition, and 94.06 - 110.41% for pivotal trial under fed condition, AUC(0-t) 96.06 - 110.52% for pilot study, 100.88 - 113.05% for the pivotal trial under fasted condition, and 97.08 - 106.06% for pivotal study under fed condition, and AUC(0-∞) 96.17 - 110.42% for the pilot study, 100.85 - 112.81% for the pivotal trial under fasted condition and 97.22 - 106.14% for the pivotal study under fed condition, respectively. These values for the test and reference products are within the 80 - 125% interval proposed by FDA and EMEA. It was concluded that the proposed method was successfully applied to a PK study in healthy Chinese volunteers, and results showed from both the pilot and pivotal studies that the two paroxetine formulations are bioequivalent in their rates and extent of absorption.

Determination of metformin in rat plasma by HILIC-MS/MS combined with Tecan automation and direct injection.

Metformin is a well-known oral antihyperglycemic drug used in treatment of type II diabetes. Analysis of metformin in biological fluids is a challenge owing to its high polarity and small molecular size, which lead to poor retention of metformin on reversed-phase liquid chromatographic columns. A high-throughput method was developed and validated for the determination of metformin in rat plasma in support of preclinical toxicology studies, using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) and Tecan automated sample preparation. Extracted samples were directly injected onto the unbounded silica column with an aqueous-organic mobile phase. This HILIC-MS/MS method was validated for accuracy, precision, sensitivity, stability, matrix effect, recovery and calibration range. Acceptable intra-run and inter-run assay precision (coefficient of variation ≤ 3.9%) and accuracy (99.0-101.8%) were achieved over a linear range of 50-50,000 ng/mL. Metformin is stable in rat plasma for at least 6 h at room temperature, 147 days at -70°C and through three freeze (-70°C) and thaw cycles. Metformin is also stable in rat whole blood for at least 2 h at room temperature and in an ice-water bath. The validated method was successfully used in support of several preclinical studies where metformin is dosed together with an investigational drug substance. The ruggedness of the validated method was demonstrated by the incurred sample reproducibility test.

Development and validation of an LC-MS/MS method for the quantification of fascin proteins in human serum.

Background: Fascin is an actin-bundling protein that has been linked to tumor cell migration, invasion, metastasis, disease progression and mortality, thus serving as a novel cancer biomarker. Bioanalytical methods to measure fascin in biological matrices are sparsely reported, while accurate quantitation of fascin levels may lend support for fascin as a promising therapeutic target. Method: An LC-MS/MS-based method involving protein precipitation, enzymatic digestion and solid phase extraction was developed and validated for the quantitation of fascin in human serum. Linearity over a calibration range of 5-500 ng/ml with a LLOQ of 5 ng/ml, great accuracy and precision, excellent parallelism as well as high extraction recovery were achieved. Conclusion: This method provides a valuable tool for anticancer drug development and cancer treatment.

[Isolation and identification of endophytic fungi from different swollen root of Rehmannia glutinosa].

The swollen root of Rehmannia glutinosa is used as one kind of important Chinese traditional medicine. The root of R. glutinosa usually swelled in rotational cropping but not in continuous cropping. The rhizosphere microorganisms of R. glutinosa under different farming condition were thought related to that. In this study, the endophytic fungi in the root of R. glutinosa growing in various soil conditions were isolated for the study of the relationship between the microorganisms and the root enlargement of their host plants. The dominant endophytes, Verticillium spp., Fusarium oxysporum, F. redolens and Ceratobasidium spp. were identified by morphological observation and 18S rDNA and ITS sequence analysis. The preliminary investigation showed that the excessive growth of Verticillium and Fusarium genus fungi is unfavorable for the R. glutinosa root swelling, but Ceratobasidium fungi has no effects on the root enlargement.

An Ultrasensitive LC-APPI-MS/MS Method for Simultaneous Determination of Ciclesonide and Active Metabolite Desisobutyryl-Ciclesonide in Human Serum and Its Application to a Clinical Study.

BACKGROUND: The development of more efficient drug delivery devices for ciclesonide inhalation products requires an ultrasensitive bioanalytical method to measure systematic exposure of ciclesonide (CIC) and its active metabolite desisobutyryl-ciclesonide (des-CIC) in humans. METHOD: Serum sample was extracted with 1-chlorobutane. A reversed-phase liquid chromatography coupled with atmospheric pressure photoionization-tandem mass spectrometry (LC-APPI-MS/MS) method was used for quantification of 1-500 pg/mL for both analytes in a 0.500-mL serum. The analysis time was 4.7 min/injection. CIC-d11 and des-CIC-d11 were used as the internal standards. RESULTS: Calibration curves showed good linearity (r2 > 0.99) for both analytes. This novel method was precise and accurate with interassay precision and accuracy of all within 9.6% CV and ± 4.0% bias for regular QC samples. Extraction recovery was approximately 85% for both analytes. Serum samples are stable for 3 freeze-thaw cycles, 24 h at bench top, and up to 706 days at both -20 °C and -70 °C. This method was successfully used to support a pharmacokinetic (PK) comparison between the inhalation suspensions and an inhalation aerosol of ciclesonide in healthy participants. The method robustness was also supported by the good incurred sample reanalysis reproducibility. CONCLUSION: APPI, a highly selective and sensitive ionization source, made possible for quantifying CIC and des-CIC with a lower limit of quantification (LLOQ) of 1 pg/mL in human serum by LC-MS/MS. A 10-fold sensitivity improvement from the most sensitive reported method (LLOQ, 10 pg/mL) is essential to fully characterize the PK profiles of CIC and des-CIC in support of the clinical development of the ciclesonide-related medications for patients.

Isolation and characterization of a gene encoding cinnamate 4-hydroxylase from Parthenocissus henryana.

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) plays an important role in the phenylpropanoid pathway, which produces many economically important secondary metabolites. A gene coding for C4H, designated as PhC4H (GenBank accession no. DQ211885) was isolated from Parthenocissus henryana. The full-length PhC4H cDNA is 1,747 bp long with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 40-bp 5' non-coding region and a 189-bp 3'-untranslated region. Secondary structure of the deduced PhC4H protein consists of 41.78% alpha helix, 15.64% extended strand and 42.57% random coil. The genomic DNA of PhC4H is 2,895 bp long and contains two introns; intron I is 205-bp and intron II is 1,172-bp (GenBank accession no. EU440734). DNA gel blot analysis revealed that there might be a single copy of PhC4H in Parthenocissus henryana genome. By using anchored PCR, a 963-bp promoter sequence was isolated and it contains many responsive elements conserved in the upstream region of PAL, C4H and 4CL including the P-, A-, L- and H-boxes.

Automation With Intelligence in Drug Research.

The industry has adopted Clinical Data Interchange Standards Consortium standards for clinical trial data and the Food and Drug Administration electronic common technical document standard for documents for many years but still faces many challenges. The solutions based on these standards enable integration among solo systems, but the integration needs to be based on business requirements and provides the end-to-end intelligence for the business. The more standards are adopted, the more meaningful and timely metadata are needed to manage the change of the standards and need to be applied in the process. Automation that uses artificial intelligence and machine learning will be the next game changer in the industry to provide data with higher quality and more efficiency. This article discusses the challenges in managing standards adoption, potential approaches for automation through using robotic processes, artificial intelligence, and the maturity model for metadata-driven automation in clinical research.

Simultaneous quantification of total antibody and antibody-conjugated drug for XMT-1522 in human plasma using immunocapture-liquid chromatography/mass spectrometry.

XMT-1522, an antibody-drug conjugate (ADC) currently in Phase I clinical development, represents the first Dolaflexin®-based, cleavable ADC with a high drug-antibody ratio (DAR). In this work, a novel immunocapture LC-MS/MS method was successfully developed for the simultaneous quantification of both total antibody and cleavable antibody-conjugated drug auristatin F-hydroxypropylamide (AF-HPA) in human plasma. This method utilized microwave-assisted enzymatic digestion for the total antibody and chemical release of the drug from ADC on a 96-well based immunocapture sample preparation platform. The total antibody and the conjugated drug AF-HPA were separated and subsequently quantified concurrently by LC-MS/MS. The linear range of the standard curve for total antibody was from 50 to 5000 ng/mL and for AF-HPA was from 3.3 to 330 ng/mL. The linearities showed R2 ≥ 0.993 for total antibody and R2 ≥ 0.996 for AF-HPA, respectively. The intra- and inter-day precision and accuracy were well within 15%. The validated method, with the characteristics of high efficiency, great selectivity, free of carryover, short LC-MS/MS time (˜3.5 min) and low sample volume (20 µl), was successfully applied for analyzing Phase 1 cancer patient samples.

Transcriptome sequencing of Verticillium dahliae from a cotton farm reveals positive correlation between virulence and tolerance of sugar-induced hyperosmosis.

Verticillium dahliae causes disease symptoms in its host plants; however, due to its rapid variability, V. dahliae is difficult to control. To analyze the reason for this pathogenic differentiation, 22 V. dahliae strains with different virulence were isolated from a cotton farm. The genetic diversity of cotton varieties make cotton cultivars have different Verticillium wilt resistance, so the Xinluzao 7 (susceptible to V. dahliae), Zhongmian 35 (tolerant), and Xinluzao 33 (resistant) were used to investigate the pathogenicity of the strains in a green house. Vegetative compatibility groups (VCGs) assays, Internal Transcribed Spacer (ITS) PCR, and pathogenicity analysis showed that SHZ-4, SHZ-5, and SHZ-9 had close kinship and significantly different pathogenicity. Transcriptome sequencing of the three strains identified 19 of 146 unigenes in SHZ-4_vs_ SHZ-5, SHZ-5_vs_ SHZ-9, and SHZ-4_vs_ SHZ-9. In these unigenes, three proteinase and four polysaccharide degrading hydrolases were found to be associated with the pathogenicity. However, due to a number of differentially expressed genes in the transport, these unigenes not only played a role in nutrition absorption but might also contribute to the resistance of sugar-induced hyperosmosis. Moreover, the tolerance ability was positively related to the pathogenicity of V. dahliae. This resistance to sugar-induced hyperosmosis might help V. dahliae to access the nutrition of the host. The pathogenicity of V. dahliae correlated with the resistance of sugar-induced-hyperosmosis, which provides clues for the cultivation of V. dahliae resistant varieties.

Development and validation of LC-MS/MS methods for the quantification of the novel anticancer agent guadecitabine and its active metabolite ß­decitabine in human plasma, whole blood and urine.

Guadecitabine (SGI-110), a dinucleotide of ߭decitabine and deoxyguanosine, is currently being evaluated in phase II/III clinical trials for the treatment of hematological malignancies and solid tumors. This article describes the development and validation of bioanalytical assays to quantify guadecitabine and its active metabolite ߭decitabine in human plasma, whole blood and urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Since ߭decitabine is rapidly metabolized further by cytidine deaminase, plasma and whole blood samples were kept on ice-water after collection and stabilized with tetrahydrouridine (THU) directly upon sample collection. Sample preparation consisted of protein precipitation for plasma and whole blood and dilution for urine samples and was further optimized for each matrix and analyte separately. Final extracts were injected onto a C6-phenyl column for guadecitabine analysis, or a Nova-Pak Silica column for ߭decitabine analysis. Gradient elution was applied for both analytes using the same eluents for each assay and detection was performed on triple quadrupole mass spectrometers operating in the positive ion mode (Sciex QTRAP 5500 and QTRAP 6500). The assay for guadecitabine was linear over a range of 1.0-200 ng/mL (plasma, whole blood) and 10-2000 ng/mL (urine). For ߭decitabine the assay was linear over a range of 0.5-100 ng/mL (plasma, whole blood) and 5-1000 ng/mL (urine). The presented methods were successfully validated according to the latest FDA and EMA guidelines for bioanalytical method validation and applied in a guadecitabine clinical mass balance trial in patients with advanced cancer.

Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum.

A RING zinc finger ankyrin protein gene,designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4- type RING finger domain was found at the C-terminal region of the AdZFP1 protein,and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root,stem and leaf of the plant.Semi-quantitative reverse- transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity,cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

[Isolation and identification of surfactin producing Bacillus subtilis strain and its effect of surfactin on crude oil].

Strains producing biological surfactants were isolated from formation water of Daqing oil field, Heilongjiang Province, China. From which a lipopeptide producing strain ZW-3 was screened out by PCR of the sfp gene. The morphology, cultural characteristics, physiological, biochemical properties and chemotaxonomy of strain ZW-3 were studied. The strain is rod shaped (0.7-0.8 microm x 2-3 microm), gram-positive, spore-forming and aerobic bacteria. Its (G+C) content was determined to be 42.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to the genus Bacillus subtilis, and the metabolites of strain ZW-3 was analyzed by thin layer chromatography and high performance liquid chromatography, the result indicated that the biosurfactant strain ZW-3 produced was surfactin. It could reduce surface tension of bacterial fermentation culture medium and water/oil- interfacial tension from 68.82 mN/m to 24.88 mN/m and from 23.53 mN/m to 4.57 mN/m, respectively, and its mixture with 1.8% NaOH could reduce water/oil- interfacial tension to an ultra low level (1.2 x 10(-3) mN/m), Its critical micelle concentration (CMC) was tested to be 33.3 mg/L (3.24 x 10(-5) mol/L)at 25 degrees C, and it had excellent emulsifying (2.89U) and foaming property. All these results showed that this biosurfactant had great potential in pharmaceutics, environmental protection, cosmetic, oil recovery and many other application fields.

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid.

BACKGROUND: Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types. RESULTS: The model presented in this paper is as effective as SGF digestion experiments, but more stable and reproducible. On the basis of this model, food allergens are satisfactorily classified as ACS and NACS types by their pathways for sensitization; the former are relatively resistant to gastric fluid digestion while the later are relatively labile. CONCLUSION: The results suggest that it is better to classify allergens into ACS and NACS types when understanding the relationship between their digestibility and allergenicity and the digestibility of a target foreign protein is a parameter for evaluating its allergenicity during safety assessments of GM food.