Multiproduct Resin Reuse for Clinical and Commercial Manufacturing-Methodology and Acceptance Criteria.

Chromatography resins used for purifying biopharmaceuticals are generally dedicated to a single product. In good manufacturing practice (GMP) facilities that manufacture a limited amount of any particular product, this practice can result in the resin being used for a fraction of its useful life. A methodology for extending resin reuse to multiple products is described. With this methodology, resin and column performance, product carryover, and cleaning effectiveness are continually monitored to ensure that product quality is not affected by multiproduct resin reuse (MRR). Resin and column performance is evaluated in terms of (a) system suitability parameters, such as peak-shape and transition, and height equivalent theoretical plate (HETP) data; (b) key operating parameters, such as flow rate, inlet pressure, and pressure drop across the column; and (c) process performance parameters, such as impurity profiles, product quality, and yield. Historical data are used to establish process capability limits (PCLs) for these parameters. Operation within the PCLs provides assurance that column integrity and binding capacity of the resin are not affected by MRR.Product carryover defined as the carryover of the previously processed product (A) into a dose of the subsequently processed product (B) (COA→B), should be acceptable from a predictive patient safety standpoint. A methodology for determining COA→B from first principles and setting acceptance limits for cleaning validation is described.Cleaning effectiveness is evaluated by performing a blank elution run after inter-campaign cleaning and prior to product changeover. The acceptance limits for product carryover (COA→B) are more stringent for MRR than for single-product resin reuse. Thus, the inter-campaign cleaning process should be robust enough to consistently meet the more stringent acceptance limits for MRR. Additionally, the analytical methods should be sensitive enough to adequately quantify the concentration of the previously processed product (A) and its degradants in the eluent.General considerations for designing small-scale chromatographic studies for process development are also described. These studies typically include process-cycling runs with multiple products followed by viral clearance studies with a panel of model viruses. Small-scale studies can be used to optimize cleaning parameters, predict resin performance and product quality, and estimate the number of multiproduct purification cycles that can be run without affecting product quality. The proposed methodology is intended to be broadly applicable; however, it is acknowledged that alternative approaches may be more appropriate for specific scenarios.LAY ABSTRACT: Chromatography resins used for purifying biopharmaceuticals are generally dedicated to a single product. In good manufacturing practice (GMP) facilities that make a limited amount of any particular product, this practice can result in the resin being used for a fraction of its useful life. A methodology for extending resin reuse to multiple products is described. With this methodology, resin and column performance, product carryover, and cleaning effectiveness are continually monitored to ensure that product quality is not affected by multiproduct resin reuse.General considerations for designing small-scale chromatographic studies for process development are described. These studies typically include process-cycling runs with multiple products followed by viral clearance studies with a panel of model viruses. Small-scale studies can be used to optimize cleaning parameters, predict resin performance and product quality, and estimate the number of multiproduct purification cycles that can be run without impacting product quality.The proposed methodology is intended to be broadly applicable; however, it is acknowledged that alternative approaches may be more appropriate for specific scenarios.

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile.

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract.

The role of more than 40 years of improvement in protein A chromatography in the growth of the therapeutic antibody industry.

Protein A chromatography has been used as the mAb capture step in the majority of FDA submissions. In this study, the performance of protein A chromatography, as indicated by capacity, operational flow rate, and productivity (rate of mAb production per liter of resin) was examined over its full history to gain insights into the reasons for its consistent use. Protein A productivity and capacity have increased 4.3 and 5.5% a year, respectively, since 1978. In contrast, protein A operational flow rate increased between 1978 and 2001 and then remained constant or declined as further improvements provided only marginal benefits. The productivity of protein A resin and also the mAb bioreactor titer (14% growth) rapidly improved starting in about 1990 to economically provide material for clinical trials. Technology improvement is typically driven by product sales. The sales of protein A resin, as indicated by sales of protein A ligand (21% growth), have closely paralleled the sales of mAbs (20% growth). Both increased rapidly in 2000 after the first major mAb therapeutics were approved and the markets were developed. It is likely that alternatives to protein A chromatography have not been implemented because of the order of magnitude improvement in protein A performance. Protein A membrane adsorbers and monoliths have higher productivity than packed columns due to their short bed heights and high operational flow rates. These devices are not currently practical for large-scale manufacturing but may represent a format for future improvements in protein A productivity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1193-1202, 2016.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.

Top-Down Versus Bottom-Up Nanoengineering Routes to Design Advanced Oropharmacological Products.

Energy intensive and chemical routes predominately govern modern dental material fabrication involving complex physicochemical approaches. Current interest in dental material design is shifting towards biomineralization method and green chemistry synthesis to support oral tissue biocompatibility and oropharmacology. This review article describes the context of biophysical approaches based on development in nanoengineering to design advance nanomaterials for clinical dentistry. We particularly focus on approaches governing surface texture and hierarchical assembly emphasis based on micro-nanoscale tooth anatomy. Further, this article provides an overview about the merit of micro-nanoscale material design techniques exchanging the traditional dental material. In this context, top-down and bottom-up approaches involving biomimetic nanoengineering route, opportunities and challenges are discussed.

Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface.

Bacteriolytic enzymes often possess a C-terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real-world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age-restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyß, an enzyme with improved cell wall-binding ability and age-independent bactericidal activity. Although PlyPH and Plyß have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyß by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad-based protection from bacterial pathogens.

Biomineralized anisotropic gold microplate-macrophage interactions reveal frustrated phagocytosis-like phenomenon: a novel paclitaxel drug delivery vehicle.

This study reports a facile biomineralization route for gold microplates (GMPs) synthesis using bovine serum albumin (BSA) as a reductant and stabilizing agent. Adding BSA to HAuCl4 solution yields spontaneous versatile anisotropic and partially hollow GMPs upon aging. We hypothesize that the instantaneous protein denaturation at low pH enabled access to serine and threonine hydroxyl, and sulfhydryl groups of BSA, which act as a reductant and stabilizer, respectively. This reaction could be hastened by increasing the temperature well beyond 65 °C. Transmission electron microscopy/X-ray diffraction studies revealed highly crystalline and anisotropic structures (triangle, pentagon, and rectangle). Atomic force microscopy/scanning electron microscopy analyses demonstrated unique morphology of microplates with a partially void core and BSA mineralized edge structure. RAW 264.7 mice peritoneal macrophage-microplate interaction studies using live cell confocal imaging reveal that cells are capable of selectively internalizing smaller GMPs. Large GMPs are preferentially picked with sharp vertices but cannot be internalized and exhibit frustrated phagocytosis-like phenomenon. We explored particle phagocytosis as an actin mediated process that recruits phagosome-like acidic organelles, shown by a lysosensor probe technique. The biocompatible GMPs exhibited ∼70% paclitaxel (PCL) loading and sustained release of PCL, showing antitumor activity with the MCF-7 cell line, and could be a novel drug carrier for breast cancer therapy.

Growth inhibition of Mycobacterium smegmatis by mycobacteriophage-derived enzymes.

We report the ability of mycobacteriophage-derived endolysins to inhibit the growth of Mycobacterium smegmatis. We expressed and purified LysB from mycobacteriophage Bxz2 and compared its activity with that of a previously reported LysB from mycobacteriophage Ms6. The esterase activity of Bxz2 LysB with pNP esters was 10-fold higher than that of the previously reported LysB but its lipolytic activity was significantly lower. The presence of surfactant - Tween 80 or Triton X-100 - significantly increased the activity of LysB. Characterization of LysB-treated M. smegmatis cells and LysB-treated purified cell wall by mass spectroscopy confirmed the hydrolytic activity of the enzyme. Both enzymes were equally effective in inhibiting the growth of M. smegmatis, demonstrating their potential as bacteriostatic agents.

Carbon nanotube-induced loss of multicellular chirality on micropatterned substrate is mediated by oxidative stress.

Carbon nanotubes (CNTs) are receiving much attention in medicine, electronics, consumer products, and next-generation nanocomposites because of their unique nanoscale properties. However, little is known about the toxicity and oxidative stress related anomalies of CNTs on complex multicellular behavior. This includes cell chirality, a newly discovered cellular property important for embryonic morphogenesis and demonstrated by directional migration and biased alignment on micropatterned surfaces. In this study, we report the influence of single-walled carbon nanotubes (SWCNTs) on multicellular chirality. The incubation of human umbilical vein endothelial cells (hUVECs) and mouse myoblasts (C2C12) with CNTs at different doses and time points stimulates reactive oxygen species (ROS) production and intra- and extracellular oxidative stress (OS). The OS-mediated noxious microenvironment influences vital subcellular organelles (e.g., mitochondria and centrosomes), cytoskeletal elements (microtubules), and vinculin rich focal adhesions. The disorientated nuclear-centrosome (NC) axis and centriole disintegration lead to a decreased migration rate and loss of directional alignment on micropatterned surfaces. These findings suggest that CNT-mediated OS leads to loss of multicellular chirality. Furthermore, the in vitro microscale system presented here to measure cell chirality can be extended as a prototype for testing toxicity of other nanomaterials.

Enzyme-driven Bacillus spore coat degradation leading to spore killing.

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable.

Profile of dengue infection in Jamnagar city and district, west India.

Background and Methods India is one of the countries in the World Health Organization South-East Asia Region that regularly reports outbreaks of dengue fever (DF)/dengue hemorrhagic fever (DHF). As effective control and preventive programmes depend upon improved surveillance data, this study was carried out to report the seroprevalence of dengue virus infection in an area around Jamnagar city, Western India. [1] Methods The laboratory records of clinically suspected dengue patients from July 2008 to June 2011 were analysed retrospectively for the results of immunoglobulin M (IgM) anti-dengue antibodies, tested by dengue monoclonal antibody (IgM) capture enzyme-linked immunosorbent assay (MAC ELISA). Variations in disease incidence by sex, age group and season were assessed. Results A total of 903 serum samples were tested, of which 253 were positive. The majority were males (72%) and in the age group of 16-30 years. The incidence of dengue peaked in October and slowly tapered by December. Conclusion Dengue cases were higher during September to December, in the post-monsoon season. This observation is useful for planning special preventive strategies. The study draws attention to the susceptibility of the male, young adult age group.

Seropositivity of hepatitis B, hepatitis C, syphilis, and HIV in antenatal women in India.

INTRODUCTION: The epidemiology of viral hepatitis during pregnancy is of paramount importance for health planners and program managers. Data on viral hepatitis during pregnancy are not readily available. This study was conducted to assess the extent of seropositivity of hepatitis B, hepatitis C, HIV, and syphilis in pregnant women and to re-evaluate the need for routine antenatal care screening. METHODOLOGY: All samples were tested to detect HBsAg by enzyme linked immunosorbent assay (ELISA). Samples were tested to detect anti-HCV by ELISA. Samples were also tested for antibodies to Treponema Pallidum by qualitative rapid plasma reagine (RPR); finally, samples were tested for antibodies to HIV by three different methods as per Strategy III of the National AIDS Control Organization by using different systems of testing to establish a diagnosis of HIV. RESULTS: Seropositivity of hepatitis B was 2.9%, hepatitis C was 0.19%, syphilis was 0.48%, and HIV was 0.38%. Out of the 1038 samples, no co-infection was found between hepatitis B, hepatitis C, syphilis, or HIV. CONCLUSION: The data from this study can help health professionals to treat antenatal patients more effectively. The data also reinforces the need for establishing effective prevention programs, which could lead to a reduction in the prevalence of HBV, HCV, syphilis, and HIV.

Profile of The Chikungunya Infection: A Neglected Vector Borne Disease which is Prevalent In The Rajkot District.

BACKGROUND: Chikungunya Virus has been responsible for significant human morbidity probably for several hundred years; yet in spite of its prevalence, the Chikungunya Virus epidemiology and the mechanisms of virulence and pathogenesis are still poorly understood and undetermined. AIMS: This study was done to show that the Chikungunya infection has shown a change in its pattern of occurrence with respect to the clinical features, the gender and the age group which are predominant and the season of the outbreak. The present study was conducted to evaluate the features of the Chikugunya infection in patients with acute febrile illness from various geographical regions of Rajkot district, Gujarat, India. TYPE OF STUDY: A cross-sectional study, multi centric study. STATISTICAL METHOD: The Chi-square test for the goodness of the fit and independence. METHODS: One hundred ninty three serum samples of suspected cases of patients who attended the outdoor and indoor patients departments at a tertiary care hospital, Rajkot and the primary health centres, the community health centre and the urban health centres that were covered in the Rajkot district, which were collected during the period of one year from 1(st) January 2011 to 25(th) December 2011, were studied. The sera were processed and tested for the detection of the Chikungunya IgM antibody by using a solid phase, capture micro well ELISA technology. RESULTS: Out of the total 193 cases, 84 were positive for the Chikungunya IgM antibody. Out of the total 84 positive cases, 32 were males (38.09%) and 52 were females (61.9%). Female patients showed more prevalence of this disease. A majority of the patients presented with fever, headache and joint pain: 44(52.38%). The highest prevalence of Chikungunya was found in the 40-50 years age group, which occurred in 34 (40.47%) cases. In the months of November and December, the occurrence of Chikungunya was more. CONCLUSION: This study emphasizes the need for a continuous surveillance on the disease burden by using multiple diagnostic tests and it also warrants the need for appropriate molecular diagnostic techniques for an early detection of the Chikungunya virus.

Expression of low endotoxin 3-O-sulfotransferase in Bacillus subtilis and Bacillus megaterium.

A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10(4)-10(5)-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.

Characterization of AmiBA2446, a novel bacteriolytic enzyme active against Bacillus species.

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.

Enzyme-based listericidal nanocomposites.

Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion. These Ply500 formulations were effective in killing L. innocua (a reduced pathogenic surrogate) at challenges up to 10(5) CFU/ml both in non-growth sustaining PBS as well as under growth conditions on lettuce. This strategy represents a new route toward achieving highly selective and efficient pathogen decontamination and prevention in public infrastructure.

To Study the Prevalence of Various Enteric Parasitic Infections Among HIV Infected Individuals in the P.D.U. Medical College and Hospital, Rajkot, Gujarat, India.

BACKGROUND AND OBJECTIVES: Enteric parasites are a major cause of diarrhoea in HIV infected individuals. The present study was undertaken to detect the enteric parasites in HIV infected patients with diarrhoea, who were at different levels of immunity. METHODS: This study was carried out in the P.D.U Medical College and Civil Hospital, Rajkot, India. during the period from June 2009 to June 2010. A total of 100 stool samples from HIV seropositive patients were examined for opportunistic, gastrointestinal parasitic infections. The samples were classified according to the age groups, sex, and occupation, a history of diarrhoea and different categories of the CD4 cell count. The stool samples were collected and examined for enteric parasites by microscopy and by special staining methods. The CD4 cell counts were estimated by using the FACS count system. RESULTS: The intestinal parasitic pathogens were detected in 28% patients. Among all, Isospora appeared to have the highest prevalence (18%), followed by Giardia lamblia (5%), Strongyloides stercoralies (3%) and Cryptosporidium parvum (2%). In the HIV infected patients with a CD4 count of < 200 cells/µl, Isospora was the most commonly observed (56%) pathogen. The proportion of the opportunistic pathogens in the patients with CD4 counts of <200 cells/µl was significantly higher as compared to those in the other two groups of patients with CD4 counts of >200 respectively. INTERPRETATION AND CONCLUSIONS: Parasitic infections were detected in 28% of the HIV infected patients and a low CD4 count was significantly associated with an opportunistic infection. The detection of the aetiologic pathogens might help the clinicians in deciding the appropriate management strategies.