Characterization of Co-Formulated High-Concentration Broadly Neutralizing Anti-HIV-1 Monoclonal Antibodies for Subcutaneous Administration.

The discovery of numerous potent and broad neutralizing antibodies (bNAbs) against Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein has invigorated the potential of using them as an effective preventative and therapeutic agent. The majority of the anti-HIV-1 antibodies, currently under clinical investigation, are formulated singly for intra-venous (IV) infusion. However, due to the high degree of genetic variability in the case of HIV-1, a single broad neutralizing antibody will likely not be sufficient to protect against the broad range of viral isolates. To that end, delivery of two or more co-formulated bnAbs against HIV-1 in a single subcutaneous (SC) injection is highly desired. We, therefore, co-formulated two anti-HIV bnAbs, 3BNC117-LS and 10-1074-LS, to a total concentration of 150 mg/mL for SC administration and analyzed them using a panel of analytical techniques. Chromatographic based methods, such as RP-HPLC, CEX-HPLC, SEC-HPLC, were developed to ensure separation and detection of each antibody in the co-formulated sample. In addition, we used a panel of diverse pseudoviruses to detect the functionality of individual antibodies in the co-formulation. We also used these methods to test the stability of the co-formulated antibodies and believe that such an approach can support future efforts towards the formulation and characterization of multiple high-concentration antibodies for SC delivery.

Biopsy Proven Fibrosis in Non-Alcoholic Fatty Liver Disease: An Analysis of Risk Factors.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is emerging as an important cause of liver disease in India. NAFLD is characterized by hepatic steatosis in absence of a significant alcohol use or other known liver disease. Non-alcoholic steatohepatitis (NASH) is a progressive form of NAFLD which deserves particular attention because it is more prone for development of fibrosis. Liver biopsy is the gold standard for diagnosis of NASH by evaluating necroinflammatory activity and stages of fibrosis. The aim of the study was to analyze liver biopsy specimens and identify risk factors associated with fibrosis in patients of NAFLD in eastern coastal India. METHODS: A total of 216 subjects with fatty liver in ultrasonography (USG) were selected for needle biopsy. Those NAFLD cases showing fibrosis in biopsy were analyzed for risk factors association. RESULTS: Definite NASH was diagnosed in 50 (23.14%), borderline NASH in 66 (30.55%) and not NASH in 100 (46.39%) of cases. Those patients with fibrosis (22%) were taken as cases and those without fibrosis (78%) were taken as controls for risk factor analysis. Age > 40 [odds ratio (OR) 2.01 (1.09-4.04)], female gender [OR 2.74 (1.24-6.05)], body mass index (BMI) > 23 [OR 15.36 (4.59-51.37)] and moderate fatty change in USG [OR 1.89 (1.01-3.62)] were observed as risk factors for progression to fibrosis in NAFLD cases. CONCLUSION: Older age, females, obesity and moderate fatty liver on USG are risk factors for development of fibrosis in patients with NAFLD. Patients with these risk factors should be selected for liver biopsy and to be kept for close follow-up.

Use of Cyclodextrin as a Novel Agent in the SEC-HPLC Mobile Phase to Mitigate the Interactions of Proteins or Peptide or their Impurities with the Residual Silanols of Commercial SEC-HPLC Columns with Improved Separation and Resolution.

PURPOSE: Accurate quantification of the intact proteins, antibodies or peptides and their impurities without interaction to silanols of HPLC column. METHODS: Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Different commercial SEC-HPLC columns and biologics with a molecular weight ranging from 5.8 kDa to 150kDa were assessed with and without cyclodextrin. RESULTS: Addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate and reduced peak width with increasing concentration of cyclodextrin studied. DISCUSSION: High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate and ion pairing agents in the mobile phase used for separation of peptides, proteins and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biologic, but are sometimes, not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation but prevents the nonspecific binding or interaction of protein itself and thereby allowing for improved resolution, and accurate quantification of aggregates in antibodies, and peptides. The data on the separation in presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products. CONCLUSION: Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved SEC-HPLC resolution, and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.

Biochemical Profile of Nonalcoholic Fatty Liver Disease Patients in Eastern India with Histopathological Correlation.

Aminotransferase assay is often used as a screening test as well as an endpoint for resolution of disease in nonalcoholic fatty liver disease (NAFLD). Aim of the study was to evaluate the relationship of transaminase level with metabolic variables and histology in NAFLD. Single center observational study was conducted in a gastroenterology clinic at Cuttack in coastal Odisha. Subjects were consecutive patients presenting with functional bowel disease and undergoing abdominal sonography. All participants were evaluated for the presence of metabolic syndrome (MS), insulin resistance, liver function test and lipid profile. Various parameters were compared between NAFLD subjects and controls. 53.5 % of NAFLD had normal serum transaminases, whereas 20.8 % of healthy controls had transaminitis. NAFLD patients had significantly higher BMI, fasting plasma glucose, serum transaminases, serum triglycerides, serum insulin and homeostatic model assessment (HOMA) IR than controls. NAFLD patients who had transaminitis had significantly higher incidence of MS and higher mean HOMA IR than those without. There was no significant difference in histopathological features between NAFLD with and without transaminitis. To conclude, over half of NAFLD subjects do not have transaminitis while transaminitis is present in a fifth of healthy people without fatty liver. Hence serum transaminase should not be used as screening test for NAFLD. NAFLD patients with transaminitis had a higher incidence of MS and insulin resistance than those without. However, there was no significant difference in histopathological features between these two groups.

Histological Evaluation of Non-alcoholic Fatty Liver Disease and Its Correlation with Different Noninvasive Scoring Systems with Special Reference to Fibrosis: A Single Center Experience.

BACKGROUND: Although liver biopsy remains the gold standard for the diagnosis of non-alcoholic fatty liver disease [NAFLD], many non-invasive markers of liver fibrosis have recently been proposed and assessed as surrogates of liver biopsy. AIMS AND OBJECTIVE: To evaluate the degree of liver fibrosis by different non-invasive fibrosis scoring systems and to compare each non-invasive fibrosis scoring system with histological fibrosis stage. MATERIALS AND METHODS: The study population consists of consecutive patients with biopsy proven NAFLD. Complete medical history was taken and physical examination was done in all patients along with appropriate biochemical evaluations. NAFLD fibrosis score, BARD score, BAAT score and APRI score were calculated and each score was compared with histological fibrosis staging. RESULTS: The study population consisted of 60 patients having mean age 39.73 years (SD 9.62, range 17-63 years) including 51 (85%) males and 9 (15%) females. On histology fibrosis was present in 68.3% (41/60) patients. Out of 60 patients 41 had fibrosis and among them 17, 22, 2 patients had grade 1, 2, 3 fibrosis respectively and no one had grade 4 fibrosis. 61.67% (37/60) had definite NASH. Comparing the fibrosis of histology with the noninvasive scoring systems, the sensitivity and specificity of NAFLD fibrosis score were 5.56% and 100% respectively. BARD score had 45.83% sensitivity and 80.55% specificity. The sensitivities of BAAT score and APRI score were 0% and 29.16% respectively and the specificities were 100% and 97.22% respectively. CONCLUSION: The noninvasive scoring systems like NFS, BARD, BAAT, and APRI are not sensitive enough to detect fibrosis but highly specific to include fibrosis if scores are more than cut-off values in our cohort, however they cannot replace liver biopsy. Newer more efficient non-invasive scoring systems have to be devised for the Indian NAFLD population.

Acute-on-Chronic Liver Failure (ACLF) in Coastal Eastern India: "A Single-Center Experience".

BACKGROUND AND OBJECTIVES: Acute-On-Chronic liver failure (ACLF) is an emerging entity. The present study was undertaken to analyze the clinical profile and natural course of ACLF patients. PATIENTS AND METHODS: ACLF was defined as per Asia Pacific Association for the Study of Liver consensus criteria 2009. Patients fulfilling these criteria with some deviations were included and prospectively evaluated for clinical profile, etiologies of acute decompensation (AD) and underlying chronic liver disease, and short-term natural course [3 months]. RESULTS: Out of 123 patients with ACLF (mean age: 45.83 ± 12.05 years; male:female 109:14), 45.53% cases had prior history of AD, and 54.47% presented for the first time as ACLF. Etiologies of cirrhosis were alcohol, cryptogenic, and chronic hepatitis B virus infection in 65.04%, 23.57%, and 11.38% cases, respectively. Recent history of alcohol intake (within 4 weeks) [42.27%] followed by bacterial infections [36.58%] were the common etiologic precipitants for AD. Only 87 (70.73%) out of 123 cases could be followed up for a duration of 3 months; 62 (71.26%) cases died by 3 months. Most deaths occurred in the alcoholics compared to nonalcoholics [(43/53) 81.13% vs. (19/34) 55.88%; P = 0.01]. No significant difference in mortality rate was observed between ACLF cases with history of prior AD compared to newly diagnosed ACLF cases [30/40 (75%) vs. 32/47 (68.09%); P = 0.477]. The prognostic markers [MELD, MELD-Na, CTP] were not significantly different between survivors and nonsurvivors. CONCLUSION: ACLF patients in our population had high short-term mortality rates with majority of deaths in alcoholics. Alcohol intake and bacterial infections were mainly responsible for AD in our study.

Risk Factors Associated With Non-Alcoholic Fatty Liver Disease in Indians: A Case-Control Study.

BACKGROUND/AIMS: NAFLD has today emerged as the leading cause of liver disorder. There is scanty data on risk factors associated with NAFLD emanating from India. The present study was conducted to identify the risk factors associated with NAFLD. METHODS: 464 consecutive NAFLD patients and 181 control patients were subjected to detailed questionnaire regarding their lifestyle and dietary risk factors. Anthropometric measurements were obtained and biochemical assays were done. Comparison of different variables was made between NAFLD patients and controls using principal component analysis (PCA). RESULTS: NAFLD patients had higher BMI [26.25 ± 3.80 vs 21.46 ± 3.08 kg/m(2), P = 0.000], waist-hip ratio [0.96 ± 0.12 vs 0.90 ± 0.08, P = 0.000] and waist-height ratio [0.57 ± 0.09 vs 0.50 ± 0.06, P = 0.000] compared to controls. Fasting blood sugar [101.88 ± 31.57 vs 90.87 ± 10.74 mg/dl] and triglyceride levels [196.16 ± 102.66 vs 133.20 ± 58.37 mg/dl] were significantly higher in NAFLD group. HOMA-IR was also higher in NAFLD group [2.53 ± 2.57 vs 1.16 ± 0.58, P = 0.000]. Majority (90.2%) of NAFLD patients were sedentary. Family history of metabolic syndrome (MS) was positively correlated with NAFLD. Dietary risk factors associated with NAFLD were non-vegetarian diet [35% vs 23%, P = 0.002], fried food [35% vs 9%, P = 0.000], spicy foods [51% vs 15%, P = 0.001] and tea [55% vs 39%, P = 0.001]. Diabetes, hypertension, snoring and sleep apnoea syndrome were common factors in NAFLD. On multivariate PCA, waist/height ratio and BMI were significantly higher in the NAFLD patients. CONCLUSION: The risk factors associated with NAFLD are sedentary lifestyle, obesity family history of MS, consumption of meat/fish, spicy foods, fried foods and tea. Other risk factors associated with NAFLD included snoring and MS.

Nonalcoholic fatty liver disease (NAFLD) without insulin resistance: Is it different?

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of insulin resistance [IR]. However, a significant proportion of NAFLD patients are devoid of IR. Is NAFLD sans IR a different entity? The aim of the study was to compare the anthropometric, metabolic, biochemical, ultrasonography, and histological profile of NAFLD patients with and without IR. METHODS: Retrospective analyses of 336 NAFLD patients diagnosed during the last two years was done. Patients without IR were compared with those with IR. RESULTS: Out of 336 patients, 153 [45.53%] were without IR. Although age, gender, BMI and transaminase levels were comparable, significantly higher proportion of patients in non-IR group were non-obese [43.14% vs. 25/14%; P=0.0005], and had mild fatty change on ultrasonography; [78.43% vs. 67.21%; P=0.022]. Higher proportion of them had elevated transaminases; [67.97% vs. 56.83%; P=0.036]. Serum triglyceride [178.52±78.78 vs. 204.86±94.72 mg/dl; P=0.02], FBG [85.39±13.80 vs. 98.93±31.56 mg/dl; P=0.00], PGBG [123.76±36.77 vs. 148.07±64.67m g/dl; P=0.00], and serum insulin [6.33±2.18 vs. 15.39±12.56 µIU/ml; P=0.00] were significantly lower in patients without IR. Although there was no difference in histology, interestingly fibrosis was seen in one third of patients despite absence of IR. CONCLUSION: Nearly half of our NAFLD population was without IR; one third of them had significant fibrosis. NAFLD is probably a heterogeneous disease and IR is not the sole factor responsible for NAFLD; further studies are needed to find out other possible etiological factors.

Profile of patients with incidentally detected nonalcoholic fatty liver disease (IDNAFLD) in coastal eastern India.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) has emerged as the commonest hepatic problem in both developed and developing countries. This study was conducted to delineate the clinical profile of incidentally detected NAFLD patients. METHODS: A retrospective analysis of clinical, biochemical and histological parameters was performed for 632 patients in whom fatty liver was diagnosed by ultrasonography during 2006-2010. Every patient's clinical presentation, history, liver function tests, blood sugar, lipid profile, ultrasonogram, histology and other relevant investigations were analyzed. Patients referred for transaminitis or overt chronic liver disease were excluded. RESULTS: Out of 632 consecutive NAFLD patients, 484 (76.6%) were males and 148 (23.4%) were females. Mean age of our patients was 42.44 +/- 10.18 years. Their mean body mass index (BMI) was 26.14 +/- 3.38 kg/m2. Obesity (BMI > 25), diabetes mellitus, hypertension, hyperlipidemia, metabolic syndrome and insulin resistance were present in 61.7%, 15.2%, 19.8%, 62.0%, 40.0%, and 54.4% patients, respectively. SGOT and SGPT were elevated in 38.29% and 55.1% patients, respectively. The common primary diseases for which these patients sought consultation were non-ulcer dyspepsia (NUD) (54.5%) and irritable bowel syndrome (IBS) (29.4%). Of the 64 patients who underwent liver biopsy, one-fourth had definite NASH while one-third patients had fibrosis. CONCLUSION: In India, incidentally detected NAFLD (IDNAFLD) patients are predominantly middle aged males, most of whom are not lean. Most of these patients seek consultation for functional bowel disease.

Insulin resistance and beta cell function as prognostic indicator in multi-organ dysfunction syndrome.

Insulin resistance (IR) is a major factor in the pathogenesis of sepsis. Critically ill patients with multi-organ dysfunction syndrome (MODS), diagnosed as per modified Acute Physiology and Chronic Health Evaluation II (modified APACHE II) score criteria and admitted to the medical ward in our hospital, were assessed for IR and beta cell function by using the homeostasis model assessment A (HOMA-A) and HOMA-B models, respectively. Of 80 consecutive patients, 60 were followed up to day 7; 16 patients died and 4 did not agree to follow up. The mean value of IR in all the 80 patients studied on the day 1 of hospitalization was 6.67 +/- 10.65. The initial high values of serum insulin and IR were significantly reduced (p < 0.05) as these patients recovered from their critical illness. Of those who died, the first day mean insulin levels were high (13.80 +/- 14.72 micro/ml as well as IR 5.14 +/- 6.76 values), but they had statistically low beta cell function (46.45 +/- 433.64%) as compared to those who recovered (227.60 +/- 430.36%; p < 0.05). This suggests that, beta cell overexhaustion occurs in critically ill patients, because it was required to overcome the prevailing state of IR and has more bearing in patients having less than 4 organ failures. beta cell failure ensued from the onset in those who were more moribund and had more than four organs failing or those who died. IR and beta cell function are reliable indicators of the state of severity of critical illness, and they corroborated with mortality in patients with MODS.