Identification and characterization of a residual host cell protein hexosaminidase B associated with N-glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product.

Host cell proteins (HCPs) are process-related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N-glycan heterogeneity profile. However, significant N-glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N-acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography-mass spectrometry (LC-MS)-based proteomics approach to be enriched in the impacted stability batches from mAb-1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb-1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

Detection of low-molecular-weight mast cell-activating factors in serum from patients with chronic spontaneous urticaria

Background: Functionally active autoantibodies to IgE and to the high-affinity IgE receptor (FcεRI) can be detected in serum in about 40% of patients with chronic spontaneous urticaria (CSU). Recent studies showed that serum from patients with CSU can induce activation of mast cells, irrespective of whether they carry high-affinity IgE receptors. Objective: To evaluate mast cell activation induced by factors in the serum of CSU patients with a molecular weight lower than that of autoantibodies. Methods: Eight CSU patients and 5 healthy controls were evaluated. Whole serum and serum fractionated at 100, 50, and 30 kDa were used to stimulate in vitro LAD2 mast cells. The enzymatic activity of β-hexosaminidase was evaluated in supernatants and cell pellets as a measure of mast cell degranulation. Results: Mean (SEM) release of mast cell β-hexosaminidase induced by whole serum from CSU patients was higher than that induced by serum from the healthy controls (14.4 [2.7%] vs 5.1 [2.4%]; P =.027). In addition, serum fractions below 100 kDa and below 50 kDa from CSU patients induced mast cell degranulation that was significantly higher than that induced by the corresponding fractions in sera from healthy controls (10.2% [1.4%] vs 3.8% [1.9%] [P=.024] and 10.1% [1.2%] vs 3.9% [1.7%] [P=.012], respectively). In 4 CSU patients, we evaluated serum fractions <30 kDa, which retained their capacity to activate mast cells (11.0% [0.7%]). Conclusions: This study shows that sera from CSU patients may contain low-molecular-weight mast cell-activating factors other than autoantibodies. These factors could be an additional mechanism contributing to the pathogenesis of CSU (AU)

Introducción: Los autoanticuerpos IgE funcionalmente activos y los receptores de alta afinidad para la IgE (FcεRI) pueden ser detectados n el suero de aproximadamente un 40% de los pacientes con urticaria crónica espontánea (UCE). Estudios recientes muestran que el uero de estos pacientes puede inducir activación de mastocitos con o sin receptores de alta afinidad para la IgE. Objetivo: El objetivo de este estudio fue evaluar la actividad de los factores séricos de los sueros de pacientes con UCE con un peso molecular inferior al de los autoanticuerpos. Métodos: Para ello se estudiaron 8 pacientes con UCE y 5 controles sanos. El suero completo de cada uno de ellos y el fraccionado a 100,50 y 30 kDA se utilizó para estimular in vitro mastocitos LAD2. La actividad enzimática de la β-hexosaminidasa se determinó en los sobrenadantes y en el botón celular con el fin de cuantificar la degranulación mastocitaria. Resultados: En cuanto a los resultados obtenidos se observó una liberación de β-hexosaminidasa mastocitaria inducida por los sueros completos de los pacientes con UCE (14,4±2,7%, media ± EE de la media) significativamente mayor que la inducida por sueros de controles sanos (5,1±2,4%; p=0,027). Así mismo, las fracciones séricas inferiores a 100 kDa e inferiores a 50 kDa de los pacientes con UCE indujeron degranulación mastocitaria significativamente superior a la inducida por las fracciones correspondientes de sueros controles (10,2±1,4% vs 3,8±1,9% [p=0,024] and 10,1±1,2% vs 3,9±1,7% [p=0,012], respectivamente). En 4 pacientes con UCE observamos que las fracciones inferiores a 30 kDa mantenían la capacidad de activar a los mastocitos (11,0±0,7%). Conclusiones: En conclusión, este estudio muestra que el suero de los pacientes con UCE puede contener factores de bajo peso molecular diferentes a los autoanticuerpos que son capaces de activar a los mastocitos. Este hallazgo podría contribuir a conocer los mecanismos de la patogénesis de la UCE (AU)

N-acetyl-beta-D-hexosaminidase and its isoenzymes A and B in blood serum and urine, as a potential colon cancer markers.

BACKGROUND/AIMS: Evaluation of N-acetyl-beta-D-hexosaminidase (HEX), and its isoenzymes A (HEX A) and B (HEX B) activity in blood serum and urine as potential markers of colorectal cancer. METHODOLOGY: The study was performed in blood serum and urine of 32 patients with adenocarcinoma, 6 with adenocarcinoma mucinosum of the colon, and 20 healthy people. The activity of HEX, HEX A and HEX B was determined in blood serum and urine by spectrophotometric method of Marciniak et al. The concentration of CEA was determined in blood serum by immunoenzymatic method (MEIA). The concentration of protein was assessed by the Lowry method, whereas the concentration of creatinine in urine by the Jaffe method (without deproteinization). RESULTS: A significant increase in the concentration of HEX, HEX A and HEX B activity was proved in serum and urine of patients with colon adenocarcinoma. In patients with colon adenocarcinoma mucinosum, the higher activity of HEX was revealed in blood serum compared to healthy people, and the significantly higher activity of HEX and HEX B expressed as pKat/mg of creatinine, was found in urine. We observe a significant increase in the activity of HEX, HEX A and HEX B expressed in pKat/mg of creatinine was found in urine of patients bearing tumor of diameter 6.0-7.0 cm in comparison to patients with tumor of diameter 4.0-5.0 cm. CONCLUSIONS: The present study results suggest that determination of HEX, HEX A and HEX B activity in blood serum and urine may be used to detect colon cancer in its early stages. However, the use of HEX, HEX A and HEX B activity in oncological diagnostics requires further studies on a larger group of patients.