Neutralizing Antibody Sample Testing and Report Harmonization.

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication.

Contact transscleral cyclophotocoagulation in the treatment of neovascular glaucoma: a five-year follow-up.

BACKGROUND: The purpose of the study was to evaluate the long-term efficacy of contact transscleral cyclophotocoagulation (TSCPC) performed after panretinal photocoagulation and cryoapplication in patients with angle closure neovascular glaucoma. MATERIAL/METHODS: Thirty eyes of 30 patients with angle closure neovascular glaucoma were enrolled (15 women and 15 men, mean age: 55+/-21 years). Diode TSCPC was performed in all patients after panretinal photocoagulation and cryotherapy. Follow-up was 60 months. RESULTS: The mean pretreatment and postoperative intraocular pressures (IOPs) were 39.8+/-12 and 20.6+/-3.6 mmHg, respectively (p<0.00001). The mean number of treatment sessions was 2.4+/-1.5 (range: 1+/-7), with 18 eyes receiving only one treatment. There was a decrease in visual acuity in 3, hyphema in 4, uveitis in 2, cataract in 3, and phthisis in 1 eye. CONCLUSIONS: 1. TSCPC provides long-term control of IOP in patients with angle closure neovascular glaucoma. 2. Laser panretinal photocoagulation and cryoapplication should be considered before TSCPC. 3. TSCPC is associated with numerous complications, hyphema being the most frequent.

A non-radioactive method for detecting neutralizing antibodies against therapeutic proteins in serum.

The presence of neutralizing antibodies against protein therapeutics continues to cause concern in the biomedical field. These antibodies not only reduce the efficacy of the protein therapeutics, but may also block the normal function of their endogenous counterparts, which can result in serious health risks to the patient. To date, a limited number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. However, many of the existing assays involve the use of radioactive materials. We have established a novel and non-radioactive bioassay system for detecting neutralizing antibodies in patient serum samples. Our assay measures the cell metabolic activities that are closely associated with cell proliferation and apoptosis. The biologic effect of the therapeutic protein and the capability of the antibodies to neutralize the therapeutics are reflected by changes of the cellular metabolic activities triggered by the administration of the therapeutics or presence of the anti-therapeutic protein antibodies. Compared with existing assays, this new assay is equally or more sensitive, and completely eliminates the use of radioactive materials.

Detection of neutralizing anti-therapeutic protein antibodies in serum or plasma samples containing high levels of the therapeutic protein.

Neutralizing antibodies against therapeutic proteins can be potentially harmful if the antibody blocks not only the therapeutic activities of the therapeutic protein but also the normal functions of the endogenous counterpart. Detection of the neutralizing anti-therapeutic protein antibodies generally relies on bioassays measuring changes in the biologic activity of the therapeutic protein triggered by the presence of the antibody. Most of the bioassays, particularly the cell-based in vitro assays, fail to detect neutralizing anti-therapeutic protein antibodies when the remaining therapeutic protein level in the assay samples is high. The remaining therapeutic protein, either a free molecule or an immune complex with anti-therapeutic protein antibodies, can inhibit the neutralizing activity of the antibody and prevent detection. We describe the development of a procedure that uses acid dissociation and affinity adsorption to remove therapeutic protein from assay samples. With this procedure, we can detect the presence of neutralizing anti-therapeutic protein antibodies from samples containing high levels of therapeutic protein.

[The application of the confocal microscopy for the diagnosis of corneal inflammation]. / Zastosowanie mikroskopii konfokalnej do diagnostyki rozinicowej zapalen rogówki.

In the course of keratitis, the early diagnosis is very important to start with the effective treatment. Clinical state and slit lamp biomicroscopic examination not always allows to establish etiologic factor. In this paper, usefulness of the confocal microscopy in vivo examination to diagnose cases of not clear etiology in corneal inflammation, was presented. Authors presented the correlation between the clinical signs and the confocal images basis in five examined patients.

Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.

[Epithelial ingrowth after LASIK--personal experience]. / Wrastanie nablonka rogówki po zabiegach LASIK--doswiadczenia wlasne.

The current incidence of epithelial ingrowth after LASIK varies from 1% to 12%. After second procedure, it increases up to 32%. Known risk factors for this complication include epithelial defects at the time of surgery, or a history of recurrent corneal erosions, corneal basement membrane epithelial dystrophy, history of ingrowth in the other eye, hyperopic LASIK correction, flap instability, repeated LASIK surgeries. We performed 200 LASIK procedures. In 12 patients we found epithelial ingrowth. The follow up period was 2 years. We applied treatment: lifting and manual removal in 7 cases, phototherapeutic keratectomy in 3 cases. The rest of cases were under observation. Epithelial ingrowth is a relatively rare complication, following LASIK.

[Vital changes in corneal structure after LASEK during the early postoperative period]. / Przyzyciowe zmiany struktury rogówki po LASEK-u we wczesnym okresie pooperacyjnym.

UNLABELLED: LASEK (Laser Epithelial Keratomileusis) is a new refractive surgery technique, with corneal epithelial flap formation, Excimer Laser photoablation, and subsequent flap reposition. Confocal microscopy facilitates in vivo evaluation of the corneal structure, i.e., a morphological analysis of its layers, and the monitoring of postoperative alterations. PURPOSE: The purpose of the present study is to assess the corneal structure in vivo morphological after LASEK procedure with the use of confocal microscopy in the early postoperative period. MATERIAL AND METHOD: The analyzed group of patients consisted of 50 eyes in 30 patients who underwent correction of myopia and myopic astigmatism using the LASEK procedure. The epithelial flap formation was carried out with the use of a 20% alcohol solution. The photoablation was performed with the Excimer Laser MEL 70 G-Scan Aesculap Meditec. Postoperative observations were made using a Confoscan 2 (Fortune Technologies). The evaluations were performed on the first postoperative day, and then at 1, 2, 4 and 12 weeks following surgery. RESULTS: Confocal microscopy revealed some changes in the epithelium and corneal stroma. At the site of the photoablation the structure of collagen fibers was blurred, with increased reflectivity. No scar tissue was found. The pattern of keratoctic nuclei in the anterior corneal stroma was typical for the postphotoablation state during the whole observation period. Bowman's membrane and the subepithelial nerve plexus were absent in the central part of the cornea. The findings kept changing in the course of the follow up time. CONCLUSIONS: Confocal microscopy enables the monitoring of changes, which occur in the corneal structure after LASEK refractive procedures; this facilitates the evaluation of corneal healing. LASEK allows for prompt stabilization of the epithelial structure and the anterior corneal stroma. No overproduction of collagen fibers was observed at the photoablation area during the healing process.

[Use of amniotic membrane transplantation for treating bullous keratopathy]. / Zastosowanie przeszczepów blony owodniowej w leczeniu zwyrodnienia pecherzowego rogówki.

UNLABELLED: One of the complications of cataract surgery is bullous keratopathy, which is the result of decompensation of the corneal endothelium. The clinical signs are manifested in stromal and epithelial edema with bullae producing. The amniotic membrane, which is taken from the placenta of young mothers during the caesarean section, may have beneficial influence on the process of healing the corneal structure. The study aimed to evaluate effectiveness of bullous keratopathy treatment using amniotic membrane transplantation, as an alternative method of treatment after cataract surgery. MATERIAL AND METHODS: The amniotic membrane transplantation was performed in 18 eyes of 18 patients (10 females and 8 males), aged 62 to 85 with the bullous keratopathy. After removing the pathologically changed epithelium, the amniotic membrane was covered on cornea in local anesthesia, using interrupted sutures 10.0 nylon. A therapeutic contact lens were applied. Control examinations were performed 3, 6 and 12 weeks after the operation. The best corrected visual acuity was evaluated. The local state was investigated using slitbiomocroscopy and confocal microscopy. RESULTS: In all the patients' regression of subjective complaints was observed (pain, light sensitivity and tearing). In 12 cases improvement of visual acuity was achieved. The evaluation of corneas in slit biomicroscopy revealed greater clarity and less corneal edema. On confocal microscopy, decrease in blurred collagen fibers and background illumination were observed. The epithelium contained cystic structures (blisters) in 6 cases only. The endothelial cells were pleomorphic and polymegathic. CONCLUSION: The amniotic membrane transplantation is an effective method of treatment for bullous keratopathy and has beneficial influence on the process of corneal healing and the improvement of visual acuity and diminish subjective symptoms.

[LASEK--new laser refractive surgical technique. Personal experience]. / LASEK--nowa technika laserowej chirurgii refrakcyjnej. Doswiadczenia wlasne.

PURPOSE: The purpose of this study was to evaluate the clinical results of refractive error correction LASEK. MATERIAL AND METHODS: 115 eyes were analyzed after LASEK. The eyes were divided into 4 groups (myopia, myopic astigmatism, hyperopia, hyperopic astigmatism). The follow-up has taken 3 months. The effectiveness of this method was evaluated on the basis of the best visual acuity without correction after LASEK procedure, compared to the best visual acuity with correction before LASEK. The course of the healing process, the state of cornea and subjective symptoms were also evaluated. RESULTS: After 2 weeks in whole group the mean UCVA was in the same range of values compared with the BCVA before the operation. There were no postoperative complications observed. CONCLUSION: On the basis of our own observations we can say that LASEK is an effective and safe method for correction of refractive errors.

[In vivo evaluation of corneal structure changes after refractive procedures]. / Przyzyciowe zmiany struktury rogówki po zabiegach chirurgii refrakcyjnej.

PURPOSE: The aim of the study was to evaluate and compare in vivo the corneal structure changes after refractive procedures (PRK, LASIK, LASEK). MATERIAL AND METHOD: The analysed group of patients consisted of 226 corneas in 126 patients, who underwent correction of myopia and myopic astigmatism, using the procedures: PRK (120 eyes), LASIK (56 eyes) and LASEK (50 eyes). The photoablation of the corneas was performed with the Excimer Laser MEL 60 and MEL 70 G-Scan Aesculap Meditec. Postoperative observations were made using a confocal microscopes Confoscan P4 (Tomey) and ConoScan 2 (Fortune Technologies). The evaluations were performed in the early (up to 3 months) and late postoperative period (after PRK and LASIK-up to 2 years; after LASEK-up to 6 months). RESULTS: The confocal microscopy revealed some changes within the corneal epithelium and anterior part of stroma after PRK, LASIK and LASEK. After PRK, there was increased desquamation of superficial epithelial cells in early postoperative period. These cells were elongated after LASIK and LASEK procedures. After PRK and LASEK, the Bowman's membrane was absent in the central part of the cornea, during the whole observation period. After all these procedures, the anterior part of the corneal stroma in the ablation zone, showed increased background illumination of collagen fibres and an irregular pattern of elongated keratocytic nuclei, in the early postoperative period. No scar tissue--"haze" was found in cases after LASEK, what may occur after PRK procedure. The findings kept changing in the course of the follow up time. CONCLUSIONS: Confocal microscopy enables in vivo monitoring of changes, which occur in the corneal structure after refractive procedures; this facilitates the evaluation of corneal healing. LASEK is the least invasive refractive procedure, which allows prompt stabilization of the corneal structure.

[Macular corneal dystrophy--clinical state, histopathologic, immunohistochemical examinations and genetical dependence]. / Dystrofia plamkowata--obraz kliniczny, histopatologiczny, immunohistochemiczny i zaleznosci genetyczne.

Macular corneal dystrophy is a rare corneal disease, with autosomal recessive inheritance, in which characteristic diffuse corneal clouding and reduction of corneal thickness are present. Authors in this article presented the clinical state and molecular pathology underlying it's importance in diagnosis of the macular corneal dystrophy. As in other inherited diseases, the latest achievement of molecular biology, concerning the new mutation of the CHST6 gene, changed the classification of the macular corneal dystrophy, and can have the significant influence on genetic therapy of this disease.

[Correction of myopia by PRK method with the use of MEL 80 excimer laser--initial report]. / Korekcja krótkowzrocznosci metoda PRK z uzyciem lasera excimerowego MEL 80--doniesienie wstepne.

PURPOSE: The aim of this study was to evaluate the effectiveness of PRK procedure by estimation: changes of visual acuity, refractive errors, IOP, pachymetry, high order aberrations before and after surgery. MATERIAL AND METHODS: In the First Department and Clinic of Ophthalmology Medical School 22 patients went through ophthalmological examination for PRK procedure. In this study we used MEL 80 excimer laser. There were 13 women (15 eyes) and 5 men (7 eyes). The myopia was corrected from -1.75D till -6.5D. The patients were from 22 to 41 years of age. RESULTS: The correction for myopia by PRK method with MEL 80 excimer laser is effective and safe laser procedure. Stabilization of the refraction post photorefractive keratectomy was after 2 months and there was no increase of intraocular pressure in all cases. The procedure increased the high order aberrations.

Nonpenetrating very deep sclerectomy with reticulated hyaluronic acid implant in glaucoma treatment.

BACKGROUND: The purpose of our study was to assess intraocular pressure control (IOP) and postoperative complications in nonpenetrating very deep sclerectomy (NPVDS) with reticulated hyaluronic acid implant (SKGEL) and Mitomycin C (MMC). MATERIAL/METHODS: Fifty eyes from fifty patients with medically uncontrolled glaucoma were randomized to either the NPVDS or NPDS group. The NPVDS procedure was similar to traditional NPDS (control group); however, excision of sclera and exposure of ciliary body were also performed, and only a narrow scleral flap was retained at a distance of 0.5 mm from Schlemm's canal. Mitomycin-C 0.2 mg/ml was applied on and under the superficial flap of the sclera during both NPVDS and NPDS. Follow-up examinations were carried out at 1 week, and then at 1, 3, 6, and 12 months after surgery. Success was defined as IOP <22 mmHg with or without glaucoma medication and laser procedures. RESULTS: The 12-month success rate in the NPVDS group was 96%, not significantly higher than in the control group (88%, p=0.88). There was no statistically significant difference in IOP between the NPVDS (15.9+/-2.5 mmHg) and NPDS (16.3+/-3.6 mmHg) groups (p=0.57). Complications included four cases of hyphema, three of choroidal detachment (myopic eyes), and one of filtering bleb fibrosis in the NPVDS group, and three cases of hyphema, two of choroidal detachment (myopic eyes), and three of filtering bleb fibrosis in the NPDS group. CONCLUSIONS: NPVDS is an effective surgical modality for patients with glaucoma. The safety of NPVDS is comparable to that of NPDS.