RESUMEN
Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell-based ultra scale-down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non-hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (~10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand.
Asunto(s)
Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Biotecnología/métodos , Células CHO , Técnicas de Cultivo de Célula/métodos , Cricetulus , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/genética , Inmunoglobulina G/aislamiento & purificación , Fenómenos Mecánicos , Proteínas Recombinantes de Fusión/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
PURPOSE: To develop a reliable preimplantation genetic diagnosis protocol for couples who both carry a mutant PKHD1 gene wishing to conceive children unaffected with autosomal recessive polycystic kidney disease (ARPKD). METHODS: Development of a unique protocol for preimplantation genetic testing using whole genome amplification of single blastomeres by multiple displacement amplification (MDA), and haplotype analysis with novel short tandem repeat (STR) markers from the PKHD1 gene and flanking sequences, and a case report of successful utilization of the protocol followed by successful IVF resulting in the birth of an infant unaffected with ARPKD. RESULTS: We have developed 20 polymorphic STR markers suitable for linkage analysis of ARPKD. These linked STR markers have enabled unambiguous identification of the PKHD1 haplotypes of embryos produced by at-risk couples. CONCLUSIONS: We have developed a reliable protocol for preimplantation genetic diagnosis of ARPKD using single-cell MDA products for PKHD1 haplotyping.
Asunto(s)
Riñón Poliquístico Autosómico Recesivo/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Preimplantación/métodos , Receptores de Superficie Celular/genética , Femenino , Marcadores Genéticos/genética , Haplotipos , Humanos , Lactante , Repeticiones de Microsatélite/genética , Riñón Poliquístico Autosómico Recesivo/genética , EmbarazoRESUMEN
This article covers the rapidly advancing field of preimplantation genetic diagnosis (PGD), the molecular genetic analysis of cells taken from embryos formed through in vitro fertilization (IVF). The article focuses on current practices in patient management, relevant IVF and PGD procedures, molecular methods used in the genetic analysis, and technical difficulties that can affect test results. It discusses the growing list of indications for PGD including chromosomal disorders, monogenic disorders and human leukocyte antigen typing typing of embryos. The article also examines some of the emerging technologies being introduced into PGD.
Asunto(s)
Fertilización In Vitro , Enfermedades Genéticas Congénitas/diagnóstico , Diagnóstico Preimplantación , Biopsia , Humanos , Hibridación Fluorescente in Situ , Valor Predictivo de las Pruebas , Diagnóstico Preimplantación/métodosRESUMEN
A multi-site study to assess the accuracy and performance of the biplex Invader assay for genotyping five polymorphisms implicated in venous thrombosis was carried out in seven laboratories. Genotyping results obtained using the Invader biplex assay were compared to those obtained from a reference method, either allele-specific polymerase chain reaction (AS-PCR), restriction fragment length polymorphism (PCR-RFLP) or PCR-mass spectrometry. Results were compared for five loci associated with venous thrombosis: Factor V Leiden, Factor II (prothrombin) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, and plasminogen activator inhibitor (PAI-1) 4G/5G. Of a total of 1448 genotypes tested in this study, there were 22 samples that gave different results between the Invader biplex assay and the PCR-based methods. On further testing, 21 were determined to be correctly genotyped by the Invader Assay and only a single discrepancy was resolved in favor of the PCR-based assays. The compiled results demonstrate that the Invader biplex assay provides results more than 99.9% concordant with standard PCR-based techniques and is a rapid and highly accurate alternative to target amplification-based methods.
Asunto(s)
Análisis Mutacional de ADN/métodos , ADN/genética , Polimorfismo de Nucleótido Simple , Trombosis de la Vena/diagnóstico , Trombosis de la Vena/genética , Factor V/genética , Colorantes Fluorescentes , Genotipo , Humanos , Espectrometría de Masas , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Inhibidor 1 de Activador Plasminogénico/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Protrombina/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de FluorescenciaRESUMEN
Identifying an HLA-matched sibling donor for hematopoietic stem cell transplantation (HSCT) is time-consuming and expensive, and often limited by reimbursement caps imposed by insurance providers. To improve the effectiveness and efficiency of screening for HLA-matched siblings, we developed an assay for determining HLA identity using a panel of nine informative short tandem repeat (STR) loci located throughout the HLA complex. The STR panel was assessed for accuracy in identifying HLA-matched siblings in 88 family workups comprising a total of 132 related donor and recipient typing comparisons. All sibling pairs with identical STR alleles were also HLA identical. Of the 48 pairs mismatched at one or more STR alleles, all were genotypically HLA non-identical at one or more loci. The sensitivity and specificity of STR analysis for identifying HLA-matched siblings were 91% and 100%, respectively. Three false negatives occurred due to an STR mutation or possible HLA-DPB1/DQB1 recombination. Additionally, STR genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that can be used to inexpensively screen potential sibling donors for HLA identity.
Asunto(s)
Antígenos HLA/genética , Prueba de Histocompatibilidad/métodos , Donadores Vivos , Repeticiones de Microsatélite/genética , Polimorfismo Genético , Hermanos , Alelos , Salud de la Familia , Femenino , Genotipo , Cadenas beta de HLA-DP/genética , Cadenas beta de HLA-DQ/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Masculino , Mutación , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: To aid couples wishing to conceive children who are HLA matched to a sibling in need of a hematopoietic progenitor cell transplant, we developed a preimplantation HLA haplotype analysis of embryos that utilizes tri-, tetra-, and pentanucleotide STR markers. METHODS: For preimplantation HLA genotyping, we use polymorphic STR markers located across the HLA and flanking regions, selecting exclusively tri-, tetra-, and pentanucleotide repeats. These markers can be resolved using either capillary electrophoresis (CE) or polyacrylamide gels. RESULTS: We have developed 43 reliable STR markers for preimplantation HLA matching. Selected STR markers enabled unambiguous identification of embryos whose HLA haplotypes were matched with the affected patient using polyacrylamide gel or capillary electrophoresis. CONCLUSIONS: The use of tri-, tetra-, and pentanucleotide repeat markers and polyacrylamide gels for STR genotyping in HLA matching is a simple and cost effective approach to clinical testing.