Small conformational changes in IgG1 detected as acidic charge variants by cation exchange chromatography.

Fully characterizing the post-translational modifications present in charge variants of therapeutic monoclonal antibodies (mAbs), particularly acidic variants, is challenging and remains an open area of investigation. In this study, to test the possibility that chromatographically separated acidic fractions of therapeutic mAbs contain conformational variants, we undertook a mAb refolding approach using as a case study an IgG1 that contains many unidentified acidic peaks with few post-translational modifications, and examined whether different acidic peak fractions could be generated corresponding to these variants. The IgG1 drug substance was denatured by guanidine hydrochloride, without a reducing agent present, and gradually refolded by stepwise dialysis against arginine hydrochloride used as an aggregation suppressor. Each acidic chromatographic peak originally contained in the IgG1 drug substance was markedly increased by this stepwise refolding process, indicating that these acidic variants are conformational variants. However, no conformational changes were detected by small-angle X-ray scattering experiments for the whole IgG1, indicating that the conformational changes are minor. Chromatographic, thermal and fluorescence analyses suggested that the conformational changes are a localized denaturation effect centred around the aromatic amino acid regions. This study provides new insights into the characterization of acidic variants that are currently not fully understood.

Mechanism of Protein-PDMS Visible Particles Formation in Liquid Vial Monoclonal Antibody Formulation.

Visible particles (VPs) formation in liquid monoclonal antibody formulations is a critical quality issue. Formulations that include poloxamer 188 (PX188) as a surfactant are prone to the formation of VPs comprising aggregated complexes of protein and polydimethylsiloxane (PDMS; silicone oil) derived from primary containers. However, the mechanisms through which these VPs form are complicated and remain to be fully elucidated. This study demonstrates for the first time the dominant spot and pathway of protein-PDMS VP formation in a particular liquid vial formulation. Specifically, when a vial sealed with a PDMS-coated stopper is stored in an upright position under conditions whereby the antibody solution has become well-adhered to the stopper and an air phase exists in the vicinity, protein-PDMS aggregates form on the stopper and are then desorbed into the drug solution to be detected as VPs. Here, we evaluated the effects of several factors on VP formation: adhesion of the drug solution to the stopper, storage orientation, silicone coating on the stopper, vial material, and hydrophobicity of PX188. Remarkably, we found that changing any one of the factors could significantly affect VP formation. Our findings are instructive for better understanding the mechanisms of VP formation in vial products and can provide strategies for VP mitigation in biotherapeutics.

Viscosity increase/gelation of therapeutic IgG monoclonal antibodies induced by Zn2+: One possible root cause of clogging of staked-in-needle prefilled syringes.

We investigated the elution of zinc ions (Zn2+) from the elastomer of rigid needle shields (RNS) attached to staked-in-needle prefilled syringes (SIN-PFS) and the physicochemical impacts of Zn2+ on therapeutic IgG monoclonal antibody (mAb) solutions. The elution of metal ions from typical RNS elastomer under realistic buffer and storage conditions was investigated by inductively coupled plasma-mass spectrometry. Among the metal ions examined, only Zn2+ was detected. The elution of Zn2+ from RNS elastomer was found to be buffer-dependent. We investigated the influence of Zn2+ on the viscosity of seven mAb solutions at 180 mg/mL. The effect of Zn2+ clearly depended on antibody type. Drastic increases in viscosity or gelation were observed in four out of the seven mAbs. Dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) showed the effect of Zn2+ on mAb viscosity was explained by the colloidal destabilization of mAb solutions. Thus, Zn2+ leaching from RNS elastomer may possibly increase viscosity or cause gelation, and consequently cause possible needle clogging during long-term storage. DLS and SAXS can predict reactivity of mAbs to Zn2+, and require only small amounts of samples. This makes it possible to predict compatibility with RNS elastomer and evaluate needle clogging risk in SIN-PFSs in the early stages of mAb development.

Impact of Poloxamer 188 Material Attributes on Proteinaceous Visible Particle Formation in Liquid Monoclonal Antibody Formulations.

Surfactants such as Poloxamer 188 (PX188) play an important role in controlling particle formation in biotherapeutic formulations due to interfacial stresses. This study demonstrates for the first time that hydrophobicity of PX188 is a potential critical material attribute (CMA) as far as control of visible particle (VP) formation is concerned. We have found that within PX188 lots satisfying pharmacopeial specifications, there is variability in material attributes such as hydrophobicity, as determined from their reversed-phase high-performance liquid chromatography profiles. However, it currently remains unknown how such variability in hydrophobicity of PX188 affects surfactant function and VP formation. Here, we compared the effect of seven PX188 lots in two monoclonal antibody drug product formulations under various stress conditions. Notably, proteinaceous VP formation was reduced while using a PX188 lot with higher hydrophobicity. Our findings emphasize the importance of monitoring lot-to-lot variability of PX188 and provide insight into potential CMA for improving and controlling material attributes of PX188 for use in liquid biotherapeutic formulations.

[Benedikt Syndrome Associated with Neck Clipping of Ruptured Basilar-Superior Cerebellar Artery Aneurysm:A Case Report].

Benedikt syndrome is caused by a rare type of brain damage to the red nucleus and nearby locations. Clinically, it presents as ipsilateral oculomotor palsy and specific contralateral involuntary movement. These involuntary movements may affect activities of daily living(ADLs)in affected patients. We treated a case of Benedikt syndrome associated with neck clipping of a ruptured basilar-superior cerebellar artery aneurysm. A 66-year-old female patient was admitted to our department with headache. Initial computed tomography imaging and angiography showed subarachnoid hemorrhage and left basilar-superior cerebellar artery aneurysm. We performed neck clipping of the aneurysm. Immediately after the surgery, left oculomotor nerve palsy occurred. Right involuntary movement gradually developed from the fifth day after the surgery. Postoperative magnetic resonance imaging(MRI)showed an infarction around the left red nucleus. This specific involuntary movement significantly impaired the ADLs of the patient and persisted for two months. From three months after the surgery, it gradually improved. One and a half years after the surgery, the ADLs of the patient improved notably. It is important to preserve perforators in the treatment of distal basilar artery aneurysm. However, while ischemia of the red nucleus(e.g., due to perforator damage)may cause specific involuntary movements, they could recover spontaneously after a period of months.

Reconstruction of Near-Total Loss of the Upper and Lower Lips due to Purpura Fulminans with Local Tissue and a Dual-Skin Paddled Anterolateral Thigh Flap.

It is difficult to totally reconstruct the lip, achieving good functional and aesthetic results. There have been few reports of reconstructing complete lip defects. Moreover, upper and lower lip necrosis by purpura fulminans has not been reported. We present a case of a 60-year-old male purpura fulminans patient with upper and lower lip necrosis. Fortunately, our patient had retained his oral commissure function. We reconstructed this defect with an orbicularis oris muscle-skin-mucosal pedicled flap derived from the region between the nasolabial folds for upper lip; a similar bipedicled flap for the lower lip and the donor site was closed with a dual-skin paddled anterolateral thigh flap. Postoperative results were satisfactory, that is, no lip tightness or aperture restriction was seen, and symmetry had been achieved. The new lips exhibited complete sensory recovery. Drooling was minimal during rest and feeding. We could select a method that combined the advantages of local and free flaps. We consider our method for this defect is superior to those described in previous studies, in that the restoration of lip sensation and oral sphincter function can be achieved to some extent in 1 stage while preserving the oral commissure function.

A case of abdominoplasty after removal of giant ovarian cyst.

SUMMARY: When treating a giant ovarian cyst, management of inferior vena cava (IVC) syndrome, bleeding, abdominal competence, and cosmetic appearance are difficult. The details of abdominoplasty for patients with such a cyst have not been discussed. We present a patient requiring 85,000 ml of fluid evacuation before surgery.

[A case of metastatic liver tumor arising from gastric cancer resected after second-line chemotherapy].

A man in his 60s was diagnosed with gastric cancer and underwent distal gastrectomy and D2 lymph node dissection. The histological diagnosis was adenocarcinoma (T3, N1, StageIIB). He was treated with S-1 as adjuvant chemotherapy for 1 year. One year and 2 months after gastrectomy, a computed tomography (CT) scan revealed liver metastasis (S8, 38 mm). The patient was diagnosed with liver metastasis(refractory to S-1), and administered combination chemotherapy with irinotecan and cisplatin. After 4 courses of treatment, a CT scan detected a progressive liver tumor (55 mm). Combination chemotherapy with docetaxel and S-1 was selected as second-line chemotherapy. After 6 courses of treatment, the tumor size reduced to 40 mm in diameter, and no other metastasis was detected. Nine months after the diagnosis of liver metastasis, partial resection of the liver (S8) was performed. A pathological diagnosis of metastatic adenocarcinoma was determined, and the pathological therapeutic effect was rated as Grade 1b. Combination chemotherapy with docetaxel and S-1 was found to be effective, and was thus administrated after the operation. There have been no signs of recurrence for 6 months after the operation. Surgery with perioperative chemotherapy can be an effective treatment for liver metastasis from gastric cancer.

[Salvage gastrectomy and radiotherapy for R-CHOP-refractory gastric malignant lymphoma].

A man in his seventies presented with a stomach abnormality that was revealed upon physical examination.Following workup, he was diagnosed with gastric diffuse large B-cell lymphoma (DLBCL)StageII1 (Lugano staging system for gastrointestinal lymphoma) with low risk as defined by the International Prognostic Index criteria.The entire stomach showed an intense, abnormal FDG uptake by FDG-PET evaluation.He was treated with rituximab plus CHOP (R-CHOP).The patient's body weight decreased by 12 kg during the treatment period.Post -treatment evaluation by gastroscopy and FDG-PET following 5 courses of R-CHOP therapy revealed a residual lesion in the stomach.Total gastrectomy was performed for R-CHOP refractory gastric DLBCL.The pathological diagnosis was DLBCL, and the pathological therapeutic effect was Grade 1a.Lymphoma cells were detected at the duodenal margin of the resected specimen, and an FDG-PET scan showed abnormal FDG uptake in the duodenal stump necessitating salvage chemotherapy (DeVIC therapy)and radiotherapy.The patient's body weight increased by 5 kg after gastrectomy and there were no signs of relapse for 14 months after the operation.Salvage therapy including gastrectomy may be effective for chemotherapy-resistant gastric DLBCL.

[A case of small intestinal lymphoma with ileus requiring surgery during chemotherapy].

Perforation, bleeding, and ileus are known complications of small intestinal lymphoma and can occur either at diagnosis or during the course of treatment. Surgery is an important component in the management of these gastrointestinal complications. However, there is no consensus regarding the indications for and timing of surgery in small intestinal lymphoma. We herein present our experience with a case of small intestinal lymphoma with ileus that required surgery during chemotherapy. A 69-year-old man developed abdominal pain. Computed tomography revealed lower right jaw lymphadenopathy, small intestinal wall thickening, and mesenteric lymphadenopathy. Malignant lymphoma (diffuse large B-cell type) was diagnosed on the basis of a lower jaw lymph node biopsy. The patient was initially administered chemotherapy. After the third cycle of chemotherapy, the patient developed small intestinal obstruction detected upon abdominal computed tomography. Because a stricture persisted despite medical treatment, we performed partial resection of the small intestine. The postoperative course was good, and the patient rapidly resumed chemotherapy. Currently, 6 months after the surgery, the patient is alive without any progression of the lymphoma. A multidisciplinary treatment strategy, including surgery, is desirable to achieve a safe but radical cure for small intestinal lymphoma.

[Short-term safety evaluation of trastuzumab administrated intravenously over 30 minutes with 100 mL saline solution with a focus on infusion reaction].

In 2011, trastuzumab was approved via public knowledge-based application for administration over 30 minutes from the second round if the first infusion is well tolerated. However, cardiovascular strain and the trastuzumab-specific side effect of infusion reaction induced by an increase in the infusion rate on administering trastuzumab with 250 mL saline over 30 minutes need to be considered. To address these concerns, we evaluated trastuzumab administration with a reduced volume of 100 mL saline over 30 minutes. This method was well tolerated without an increase in the frequency of infusion reaction on retrospective comparison of the 2 administration methods. Thus, administration of trastuzumab with 100 mL of saline over 30 minutes considerably reduces infusion time, benefiting both patients and healthcare providers.

[Distribution of toxicity in liver of wild pufferfish Takifugu rubripes].

Livers from wild pufferfish, Takifugu rubripes, can be described as having a smooth frontal side and an upper-region that is attached to the hepatic portal vein. Based on this description, the liver can be divided into 10 parts (L1-5 and R1-5), and in this work, the lethal potency of each part was determined by mouse bioassay. Among the raw livers from 58 individuals, all 10 parts of 16 individuals, and some parts of 4 individuals showed mouse lethality, but no toxicity was detected in any part of the liver from 22 individuals. In the livers of 4 individuals that were partially toxic, the lethal potency of the toxic parts was less than 4 mouse units (MU)/g, and no part showed especially high toxicity. The remaining 16 individuals were considered non-toxic based on the assay of only one part. Among 13 frozen livers, all parts of 9 individuals were toxic, while all parts of 4 individuals were non-toxic. Liquid chromatography-mass spectrometry analysis revealed that all parts of a weakly toxic raw liver and a strongly toxic frozen liver had tetrodotoxin as the major toxin. Regarding the 16 raw and 9 frozen livers, whose parts were all toxic, the relative lethal potency of each part to the mean lethal potency of the individual (8.9-709 MU/g) was calculated, and subjected to a two-way analysis of variance with 2 factors (left/right and top/bottom) for each group of livers (raw or frozen). The analysis indicated non-significance among factors and interactions at a significance level of 5% in the frozen livers. However, in the raw livers, although no interaction between the 2 factors was detected, the right-side and the 4th-portion from the top were significantly higher than the left-side and the other portions, respectively. Therefore, we concluded that individual inspection using R4, which is the region that lies below the right-center location of the liver, is suitable for toxicity evaluation of liver to ensure the safe consumption of pufferfish.

Cavernous lymphangiomas involving bilateral labia minora.

BACKGROUND: The vulva is an extremely rare site for lymphangioma, a benign tumor of the lymphatic vessels. A small number of case reports have described cavernous lymphangioma involving the unilateral labium majus of young women. We report a case of cavernous lymphangiomas involving the bilateral labia minora. CASE: A 14-year-old girl presented with bilateral tumors of the labia minora, which were removed surgically and histologically diagnosed as cavernous lymphangioma. Positive nuclear staining for estrogen and progesterone receptors was demonstrated in the stromal fibroblasts immunohistochemically. CONCLUSION: It is suggested that an increase in estrogen and progesterone levels may have led to, or at least affected, the development of these tumors in this patient. Excision followed by cosmetic repair may be the optimal treatment.

Ommaya reservoir placement followed by Gamma Knife surgery for large cystic metastatic brain tumors.

OBJECT: The combination approach of Ommaya reservoir placement and Gamma Knife surgery (GKS) was evaluated for the treatment of large cystic metastatic brain tumors. METHODS: The medical records of 22 patients harboring 28 tumors, who underwent Ommaya reservoir placement followed by GKS for large cystic metastatic brain tumors were retrospectively reviewed. The patients' ages ranged 26 to 77 years (mean 57.1 years). The most common locations of primary malignancy were the breast (11 patients) followed by the lung (seven patients). The mean maximum diameter of the tumor was 40.1 mm before Ommaya reservoir placement and 31.2 mm at GKS (mean reduction of 19.9%). The mean calculated tumor volume at GKS was 13.4 cm3. The mean tumor margin dose was 16 Gy in 17 patients treated by GKS only and 11 Gy in five patients treated using both GKS and external radiotherapy. The mean follow-up period was 11.5 months. Nineteen (67.9%) of the 28 tumors were controlled. The median patient survival time was 7 months. Asymptomatic intracystic hemorrhage associated with Ommaya reservoir placement was seen in two patients with four tumors, but no serious complication occurred. CONCLUSIONS: Ommaya reservoir placement followed by GKS is relatively effective and safe for large cystic metastatic brain tumors. Gamma Knife surgery should be performed within a few days of Ommaya reservoir placement. Reaccumulation and high viscosity of cystic content must be considered.