Quality by Design approaches to assessing the robustness of tangential flow filtration for MAb.

Quality by Design (QbD) is a modern approach for quality assurance in pharmaceutical production. This article illustrates a case study of TFF robustness performed for a process characterization of a monoclonal antibody under QbD principles by exploring functional relationships that link the process parameters to quality/process attributes with prior process knowledge, risk assessment, and multivariate experiments. In every case of quality or process attributes, all measured values were in alignment with the allowable specification range, and the developed models were non-significant and had no lack of fit, thus confirming the robustness of the TFF process within the tested ranges of process parameters.

Epigenetic regulation of the potential tumor suppressor gene, hLHX6.1, in human cervical cancer.

It is well known that the Homo sapiens LIM homeobox domain 6 gene (hLHX6), a putative transcription regulator, controls the differentiation and development of neural and lymphoid cells, particularly in the central nervous system. In this study, we investigated hLHX6.1 (an isoform of hLHX6), which functions as a tumor suppressor gene in the cervix. Firstly, the methylation levels of the hLHX6 and hLHX6.1 promoters were investigated in 8 cervical cancer cell lines and human tissue samples with a distinctive degree of malignant transformation. In spite of the presence of multiple cytosine guanine dinucleotides (CpG islands) in 2 proximal promoters of the hLHX6 and hLHX6.1 genes, only the hLHX6.1 promoters were found to be mostly hypermethylated and associated with transcriptional silencing by promoter methylation, whereas the hLHX6 promoters were not. Methylation levels in the hLHX6.1 promoter were also found to be strongly related to cervical cancer development. The level of hLHX6.1 gene expression was found to be relatively high in normal cells, in which the hLHX6.1 promoter was mostly unmethylated. However, the hLHX6.1 gene expression was down-regulated or undetectable in cervical cancer cell lines and cancer tissues, in which the hLHX6.1 promoter was hypermethylated. This epigenetic alteration in the hLHX6.1 promoter begins at a relatively early stage, suggesting its potential as a biomarker for the early diagnosis and prevention of cervical cancer. Moreover, the overexpression of the hLHX6.1 gene in cervical cancer cells suppressed the tumorigenic phenotype, as shown by soft agar colony formation and migration assays, suggesting that hLHX6.1 could be a new tumor suppressor gene in the cervix.

The role of hLHX6-HMR as a methylation biomarker for early diagnosis of cervical cancer.

The homo sapiens LIM homeobox domain LHX6 gene, hLHX6, is a putative transcription regulator with homeo-domain. Multiple cytosine guanine dinucleotides (CpG island) are found in the genomic sequences between exon 4a and exon 5 of the gene encoding hLHX6s (alternative short iso-form of hLHX6 gene). This specific CpG island, hLHX6-HMR, is found frequently hypermethylated in 7 cervical cancer cell lines as shown in MSP, BSP, and COBRA assays. Methylation densities were also investigated with human tissue samples with a distinctive degree of malignant transformation. Our data showed that the hLHX6-HMR was rarely or partly methylated in the normal and CIN I cells, respectively. In contrast, it was frequently hypermethylated in CIN II, CIN III, and invasive carcinoma cells. In summary, this methylation study led to two conclusions. First, hLHX6-HMR hypermethylation is exclusively associated with cervical carcinogenesis. Second, the epigenetic change in hLHX6-HMR seems to start at CIN I, relatively early stage of cervical cancer development. Therefore, hLHX6-HMR can be used as an effective and sensitive methylation biomarker for early diagnosis of cervical cancer.