Method Development and Qualification of pH-Based CEX UPLC Method for Monoclonal Antibodies.

Post-translational modifications ("PTMs") in monoclonal antibodies (mAbs) contribute to charge variant distribution, which will affect biological efficacy and safety. For the characterization of mAbs, charge variants are used as a critical quality attributes for product quality, stability consistency and effectiveness. Charge variants in mAbs are characterized by a time-consuming and a multistep process starting from cation/anion exchange chromatography, acidic/basic fractions collection and subsequent reverse phase (RP) liquid chromatography, coupled with mass spectrometry (MS) analysis. Hence, an alternative characterization approach that would be highly selective for ion exchange chromatography-based charge variant analysis, which is compatible with on-line MS detection, is needed in the biopharma industry. Against this backdrop, multiple studies are being conducted to develop a simple straight on-line charge variant analysis method. In this regard, we apply the current study, which aims to develop a charge variant analytical method, based on volatile buffers with low ionic strength that can be used for on-line MS detection of charge variants of mAbs. This would enable the detection on "PTMs" using low ionic strength mobile phase compatible with MS. Hence, fruitful data can be obtained with a single chromatography run without any test sample preparation, eliminating the need for multiple steps of analysis, time-consuming process and multiple sample preparation steps. Thus, Charge Variant Analysis-MS technique will allow the characterization of charge-related PTMs on the intact protein stage. In this regard, this study is about development of a method having combination of chromatography and volatile mobile phase for mass spectrometry detection of mAbs being analyzed in native form. The method is qualified considering pharmacopeia guidelines because the ultimate aim is to transfer this method for Quality Control (QC) release testing of a monoclonal antibody, which is critical for batch release and the regulatory point of view. Acidic and basic variants have been separated with high resolution peak profile. Furthermore, there was no matrix interference and good separation selectivity in terms of specificity was obtained using this method. The experimental data suggested for the linearity of the method are 2.4 mg/mL to 3.6 mg/mL with % RSD below 2.0%. Additionally, Limit of Quantitation is found to be 0.15 mg/mL, which is 5% of loading amount. Consistently, the data show that the method is precise under the same operating conditions with a short time interval. Overall a simple, accurate, robust and precise pH gradient cation exchange chromatography method was developed and qualified for the characterization of a therapeutic native mAb. Additionally, this method can be used to claim a biosimilar product profile of an in-house product compare to an innovator.

p53 polymorphism and association of human papillomavirus in oral submucous fibrosis and oral squamous cell carcinoma: A case-control study.

INTRODUCTION: The tumor-suppressor p53 protein is inactivated by the human papillomavirus (HPV) E6 oncoprotein, causing polymorphism of the p53 at codon 72 of exon either proline (Pro) or arginine (Arg). Specific allele predisposition has been reported in the literature. The association between the p53 allele and HPV types has been reported. We analyzed the association between p53 polymorphism at codon 72 and HPV 16 and 18 genotypes in control, oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Of the total 90 cases, biopsy tissues of all groups (30 cases of OSF, OSCC and control each) were collected to extract DNA. Polymerase chain reaction was used to detect HPV 16 and 18 and alleles of codon 72 in p53 were evaluated in all the samples. RESULTS: In control, OSF and OSCC samples showed the presence HPV 63.3%, 33.3% and 60%, respectively. In OSF, HPV 16 and 18 was detected in four and four cases, respectively, whereas in OSCC, HPV 16 and 18 was detected in ten and nine cases, respectively. In all three groups, predominantly, Arg/Arg protein was present followed by Pro/Pro and Arg/Pro. Among the control, Arg/Arg type protein was frequently seen followed by Arg/Pro, Pro/Pro in the presence of HPV. OSF and OSCC were associated homologous genes in the presence of HPV. CONCLUSION: The definite association between p53 codon 72, polymorphism and HPV 16 and 18 was seen in OSCC with low frequency in OSF. Frequency of homozygous genotype is at high risk in the presence of HPV 16 and 18 in developing OSCC.

Genomic DNA of MCF-7 breast cancer cells not an ideal choice as positive control for PCR amplification based detection of Mouse Mammary Tumor Virus-Like Sequences.

The identification of the etiology of breast cancer is a crucial research issue for the development of an effective preventive and treatment strategies. Researchers are exploring the possible involvement of Mouse Mammary Tumor Virus (MMTV) in causing human breast cancer. Hence, it becomes very important to use a consistent positive control agent in PCR amplification based detection of MMTV-Like Sequence (MMTV-LS) in human breast cancer for accurate and reproducible results. This study was done to investigate the feasibility of using genomic DNA of MCF-7 breast cancer cells to detect MMTV-LS using PCR amplification based detection. MMTV env and SAG gene located at the 3' long terminal repeat (LTR) sequences were targeted for the PCR based detection. No amplification was observed in case of the genomic DNA of MCF-7 breast cancer cells. However, the 2.7 kb DNA fragment comprising MMTV env and SAG LTR sequences yielded the products of desired size. From these results it can be concluded that Genomic DNA of MCF-7 cell is not a suitable choice as positive control for PCR or RT-PCR based detection of MMTV-LS. It is also suggested that plasmids containing the cloned genes or sequences of MMTV be used as positive control for detection of MMTV-LS.

Molecular basis of ß-thalassemia in Karnataka, India.

In ß-thalassemia, point mutations in the ß-globin gene are largely responsible for either decreased or no ß-globin synthesis. The ß-globin gene has three exons and two introns. The molecular characterization of ß-thalassemia is absolutely necessary for carrier screening, for genetic counseling, and to offer prenatal diagnosis. The objective of the present study was to identify the rare mutations in ß-globin gene of ß-thalassemia patients. We have sequenced the entire ß-globin gene in 36 clinically identified thalassemia patients from the Karnataka region using polymerase chain reaction and sequencing. Our analysis revealed 11 ß-thalassemia variants. The most common being IVSII-16 G>C, IVSI-5G>C, IVSII-74 T>G, codon 3 (T>C), and Poly A site (T>C). In addition, we have also documented a novel deletion at codon 6 (-CT) (HBB:c.16delCT). These data are useful in future molecular screening of the population for implementing a thalassemia prevention and control program. Further it is found that family studies and comprehensive hematological analyses would provide useful insights for accurate molecular diagnosis of thalassemia phenotype and offers an interesting subject for further investigations in the Indian populations.

Prevalence and distribution of high risk human papillomavirus (HPV) Types 16 and 18 in Carcinoma of cervix, saliva of patients with oral squamous cell carcinoma and in the general population in Karnataka, India.

BACKGROUND: In view of conducting HPV vaccination in India it is most important to understand the prevalence of HPV genotypes in this population, not only in squamous cell carcinoma of cervix and oral cavity but also in the general population. In this study we explored the prevalence and distribution of high-risk HPV types 16 and 18 in carcinoma of cervix, saliva of patients with oral squamous cell carcinoma and in general population in Karnataka. METHODS: Cervical cancer specimens after punch biopsy (n=60) were obtained from women attending Karnataka Institute of Medical Sciences and Karnataka Cancer Therapy and Research Institute, Hubli (KCTRI). Saliva rinse of (n=34) OSCC patients from KCTRI and (n=396) normal individuals from different regions of North Karnataka, were collected and PCR based high-risk HPV genotyping was carried out. RESULTS: Using consensus PCR primers it was observed that 96.7% patients were infected with HPV irrespective of specific type in cervical cancer. Among them, HPV 16 was observed in 89.7%, HPV 18 in 86.2% and both HPV 16 and 18 in 79.3% patients. In OSCC, 70.6% were positive for HPV, among which HPV 16 prevalence was observed in 45.8%, HPV 18 in 54.2%, and HPV 16 and 18 multiple infection in 4.18%. In general population, HPV prevalence was observed in 84.4%. Among them, HPV 16 was observed in 2.75% and HPV 18 in 22.0% patients. In general population, multiple infection with HPV 16 and 18 was not observed but 75.3% were found to be infected by HPV genotypes other than HPV 16 and 18. CONCLUSIONS: Our study reveals that multiple infection of HPV 16 and 18 is quite high in cervical cancer and in case of OSCC, it was in conformity with the other studies. In general population HPV 18 prevalence was observed to be high. With this, we can conclude that both HPV 16 and 18 vaccinations will reduce the burden of cervical cancer and OSCC in Karnataka.