Biologicals (Biologicals)

Publications in this journal

• Article: Safety of the production process of SURFACEN(®) to inactivate and remove virus.

  Yordank Sánchez, Enrique Noa, Wilma Alfonso, Marta Dubed, Giselle Alvarez, Leonor Navea, Nivian Montes de Oca, Leonor Lobaina, Elaine Díaz
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  ABSTRACT: SURFACEN(®) is a biological product produced from pig lungs. Since these animals can be potential sources of microbial pathogens such as viruses, the manufacturing process of this product should guarantee safety from health hazards. The SURFACEN(®) production procedure is capable of effective viral clearance (inactivation/removal) by involving two stages of organic solvent extraction followed by acetone precipitation and heat treatment. In this study, we evaluated the clearance capacity of these four stages for a wide range of viruses by performing spiking experiments. Residual contamination was assessed using a Tissue Culture Infectious Dose assay (log10 TCID50). The validation study demonstrated that, for all viruses tested, the TCID50 titers were reduced by more than 2 log10 in each stage. Total log reduction values achieved were between ≥17.82 log10 and ≥27.93 log10, depending on the virus physical properties, titer, and the number of processing stages applied. Results indicated that the production procedure of SURFACEN(®) can inactivate or remove contaminant viruses from the raw material.
  Biologicals 05/2013;
• Article: Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines from China.

  Matthew C Cook, Sabrina Gibeault, Vasilisa Filippenko, Qiang Ye, Junzhi Wang, Jeremy P Kunkel
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  ABSTRACT: The active components of most meningococcal vaccines are four antigenic serogroup capsular polysaccharides (A, C, Y, W135). The vaccines, monovalent or multivalent mixtures of either free polysaccharides or polysaccharides conjugated to antigenic carrier proteins, may be in liquid or lyophilised formulations, with or without excipients. Acid hydrolysis and chromatographic methods for serogroup quantitation, which were previously optimised and qualified using polysaccharide-based standards and a narrow range of real vaccines, are here challenged with multiple lots of a broad assortment of additional multivalent polysaccharide-based meningococcal vaccine products. Centrifugal filtration successfully removed all interfering lactose excipient without loss of polysaccharides to allow for the determination of Y and W135 serogroups. Replicate operations by three different analysts indicated high method reproducibility. Results indicated some lot-to-lot and product-to-product variations. However, all vaccines were within general specifications for each serogroup polysaccharide, with the exception of all lots of one polysaccharide vaccine - which by these methods were found to be deficient in the serogroup A component only. These robust techniques are very useful for the evaluation of antigen content and consistency of manufacture. The deformulation, hydrolysis and chromatographic methods may be adaptable for the evaluation of other types of polysaccharide-based vaccines.
  Biologicals 05/2013;
• Article: Rapid clearance of intranasally administered DNA from rat tissues.

  David E Tabor, Sachin Mani, Xuan Shen, Xiaomu Chen, Charles Engbers, Scott Jacobson, Rosemary Broome, Jonathan Liu, Dominic Justewicz, Mark S Galinski
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  ABSTRACT: The cold-adapted (ca) live attenuated influenza vaccine (LAIV) strains are manufactured in embryonated hens' eggs. Recently, a clonal isolate from Madin Darby Canine Kidney (MDCK) cells was derived and characterized to assess its utility as a potential cell substrate for the manufacturing of LAIV [1]. Since MDCK cells are a transformed continuous cell line [2], and low levels of residual cellular components (DNA and protein) are found in the intermediates and final filled vaccine, we sought to characterize the uptake and clearance of MDCK DNA from tissues in order to assess theoretical risks associated with manufacturing LAIV in MDCK cell culture. In order to address this concern, MDCK DNA uptake and clearance studies were performed in Sprague Dawley rats. DNA extracted from MDCK Master Cell Bank (MCB) cells was administered via an intranasal (IN) or intramuscular (IM) route. Tissue distribution and clearance of MDCK DNA were then examined in fourteen selected tissue types at selected time points post-administration using a quantitative PCR assay specific for canine (SINE) DNA. Results from these studies demonstrate that the uptake and clearance of MDCK DNA from tissues vary depending on the route of administration. When DNA was administered intranasally, as compared to intramuscularly, detectable DNA levels were lower at all time points. Thus, the intranasal route of vaccine administration appears to reduce potential risk associated with residual host cell DNA that may be present in cell culture produced final vaccine products.
  Biologicals 05/2013;
• Article: Phage display antibodies for diagnostic applications.

  Nur Hidayah Hairul Bahara, Gee Jun Tye, Yee Siew Choong, Eugene Boon Beng Ong, Asma Ismail, Theam Soon Lim
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  ABSTRACT: With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.
  Biologicals 05/2013;
• Article: The hidden threat of unidentified agents of disease in human and veterinary biologicals.

  Charles Chiu
  Biologicals 04/2013;
• Article: Reverse-vaccinology strategy for designing T-cell epitope candidates for Staphylococcus aureus endocarditis vaccine.

  Mihaela Oprea, Felicia Antohe
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  ABSTRACT: Staphylococcus aureus is an opportunistic pathogen causing various inflammatory diseases from skin and tissue local infections, to serious life threatening infections including endocarditis. Experimental models for endocarditis demonstrated that virulence factors of S. aureus, that are very important in infection of heart vegetations, are surface proteins which promote bacterial adherence. Until now, efforts to develop effective vaccines against S. aureus were unsuccessful, partly due to the fact that different vaccine formulations have targeted mainly B-cell immunity. Reverse vaccinology is applied here, in order to identify potential vaccine epitope candidates. The basic epitopes prediction strategy relied on detection of a common antigenic 9-mer epitope meant to be able to stimulate both the B-cell and T-cell mediated immunity. Ten surface exposed proteins were chosen for antigenicity testing. Using a web-based system, five T-cell epitopes corresponding to fibronectin binding protein A (FDFTLSNNV and YVDGYIETI), collagen adhesin (FSINYKTKI), serine-rich adhesin for platelets (LTFDSTNNT) and elastin binding protein (FAMDKSHPE) were selected as potential vaccine candidates. Epitopes sequences were found to be conserved among the different S. aureus genomes screened from NCBI GenBank. In vitro and in vivo immunological tests will be performed in order to validate the suitability of the epitopes for vaccine development.
  Biologicals 04/2013;
• Article: Neutralization of five subgenotypes of Enterovirus 71 by Taiwanese human plasma and Taiwanese plasma derived intravenous immunoglobulin.

  Chi-Yu Wu, Hsiu-Chi Wang, Kun-Teng Wang, Shu-Ching Weng, Wei-Hao Chang, Daniel Yang-Chih Shih, Chi-Fang Lo, Der-Yuan Wang
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  ABSTRACT: Enterovirus 71 (EV71) commonly occurs in children, causing hand, foot and mouth disease (HFMD) in about 29% of patients. Studies have suggested that patients develop meningitis and encephalopathy with a mortality rate of 4-26%. EV71 subgenotypes including B4, B5, C2, C4 and C5 have caused HFMD epidemics in Taiwan. In terms of therapeutical strategy, intravenous immunoglobulin (IVIG) has been shown to improve patient conditions. In this study, the EV71 neutralizing titer was evaluated in 75 human plasmas and 8 lots of Taiwanese plasma derived IVIG. Results showed that human plasmas and IVIG significantly neutralized B4 and C2 subgenotypes. Four percent of human plasma contained neutralizing antibody titer of 1:128 against B4 and C2. Most IVIG lots possessed a median effective dose of over 100 against B4 and C2. IVIG lots had an average neutralizing capacity of 5.60, 0.90, 4.30, 1.12 and 0.77 log10 CCID50/ml against B4, B5, C2, C4 and C5, respectively. In conclusion, effective neutralization of B4 and C2 could be due to their earlier appearance in the EV71 epidemiology timeline of Taiwan. IVIG derived from Taiwanese plasma may be desirable for treatment of patients infected with EV71 of specific subgenotypes.
  Biologicals 03/2013;
• Article: An in vitro method for evaluating endotoxic activity using prostaglandin E2 induction in bovine peripheral blood.

  Masaru Usui, Hidetaka Nagai, Yutaka Tamura
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  ABSTRACT: Severe side effects of veterinary vaccines, in particular Histophilus somni-containing vaccines for cows, have frequently been reported in Japan. These side effects are probably caused by endotoxins. Contamination levels of endotoxins could be monitored using the Limulus amebocyte lysate (LAL) test; however, the LAL test is not completely adequate for evaluation of in vivo endotoxic activities. In this study, we established a method for evaluating endotoxic activities using prostaglandin E2 (PGE2) induction in bovine peripheral blood. Blood and standard endotoxin, derived from Escherichia coli, were mixed and incubated. The concentration of induced PGE2 in the culture supernatant reached a maximum after 24-h incubation. A linear dose-response curve was observed for PGE2 concentration and the logarithmic transformed standard endotoxin concentration (5-5000 ng/ml). The endotoxic activity of H. somni in cows was the highest among those of several tested endotoxins. However, the LAL activities of H. somni were not as high as those of the other tested endotoxins. These results may provide a reason for the many report of side effects of H. somni-containing vaccines. The PGE2 detection assay described here could be a valuable method for evaluating the endotoxic activities of vaccines in cows.
  Biologicals 03/2013;
• Article: The codon-optimization of cfaE gene and evaluating its high expression capacity and conserved immunogenicity in Escherichia coli.

  Maysam Mansouri, Seyed Jafar Mousavy, Zahra Ehsaei, Shahram Nazarian, Mohammad Reza Zali, Seyed Mohammad Moazzeni
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  ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) is the most common cause of children diarrhea in the world. Adhesion of ETEC to small intestine is an important virulence trait. One of the most prevalent colonization factors (CFs) in human is CFA/I fimbriae and CfaE which is the required binding factor for adhesion of ETEC to intestinal mucosa. We optimized cfaE gene codons according to codon bias of E. coli to achieve a high level of recombinant protein expression. The optimized gene was expressed in E. coli and rCFaE protein was used for mice immunization. Blocking activity of the obtained antibody was examined by microplate agglutination inhibition test. SDS-PAGE analysis indicated that the optimized sequence of cfaE produces a suitable amount of rCFaE in comparison with native gene sequence. This optimized rCFaE protein could induces strong humoral response in mice and the antibody obtained against rCFaE inhibited the adhesion of ETEC to human group A erythrocytes. It is concluded that codon optimization is a useful approach for obtaining large quantities of recombinant rCFaE protein. With regard to the results of hemagglutination inhibition test, codon optimization and increased production of recombinant protein expressed in E. coli did not affect the immunogenicity potential of CFaE.
  Biologicals 02/2013;
• Article: Evaluation of recombinant outer membrane protein based vaccine against Salmonella Typhimurium in birds.

  Prejit, Rajesh Kumar Agarwal, Kannan Porteen, Zunjar B Dubal, Karthikeyan Asha, Singh Shweta, Biswas Ripan
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  ABSTRACT: Food-borne diseases caused by Salmonella enterica from poultry sources represent an important public health problem and no reliable control by vaccination has proved effective despite research. The aim of the present study was to evaluate the use of recombinant OmpC protein for immunization of birds to elucidate its protection against virulent Salmonella Typhimurium. The recombinant OmpC protein was prepared after cloning and expressing ompC gene and was characterized by SDS-PAGE and Western blot analyses. The protein preparations were tested as vaccine candidate in layer birds by comparing the immune response, protection and organ clearance against crude lysate and control. The biologically functional recombinant 43 kDa truncated OmpC protein proved to be a good immunogen which induced a significantly high humoral immune response than control. At the same time, it primed a stable cell-mediated immune response. A protective index (based on faecal shedding of organism) of rOmpC based preparations ranged between 50 and 75% as observed for 3 weeks after challenge. Therefore, the protein preparations conferred satisfactory protection against challenge infections with virulent strains of S. Typhimurium as evidenced by limited faecal shedding and minimal detection of Salmonella from edible tissues and eggs. These findings suggest the possibility to explore the use of S. enterica OMP protein for the production of novel vaccine.
  Biologicals 02/2013;
• Article: Protein sieving characteristics of sub-20-nm pore size filters at varying ionic strength during nanofiltration of Coagulation Factor IX.

  Clint J Winkler, Nuria Jorba, Kenneth T Shitanishi, Steven W Herring
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  ABSTRACT: Nanofiltration assures that protein therapeutics are free of adventitious agents such as viruses. Nanofilter pores must allow passage of protein drugs but be small enough to retain viruses. Five nanofilters have been evaluated to identify those that can be used interchangeably to yield a high purity Coagulation Factor IX product. When product preparations prior to nanofiltration were analyzed using electrophoresis, Western blot, liquid chromatography - tandem mass spectrometry and size exclusion HPLC, factor IX, inter - α - trypsin inhibitor and C4b binding protein (C4BP) were observed. C4BP was removed from product by all five nanofilters when nanofiltration was performed at physiological ionic strength. However, at high ionic strength, C4BP was removed by only two nanofilters. HPLC indicated that the Stokes radius of C4BP was larger at low ionic strength than at high ionic strength. The results suggest that C4BP exists in an open conformation at physiological ionic strength and is removed by nanofiltration whereas, at high ionic strength, the protein collapses to an extent that allows passage through some nanofilters. Manufacturers should be aware that protein contaminants in other nanofiltered protein drugs could behave similarly and conditions of nanofiltration must be evaluated to ensure consistent product purity.
  Biologicals 02/2013;
• Article: Oligodeoxyribonucleotides derived from salmon sperm DNA: An alternative to defibrotide.

  Chang-Ye Hui, Yan Guo, Xi Zhang, Jian-Hua Shao, Xue-Qin Yang, Wen Zhang
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  ABSTRACT: Defibrotide is a single-stranded nucleic acid polymer originally derived from porcine mucosa. Cheap salmon sperm DNA is commercially available and widely used in drug production. In this study, oligodeoxyribonucleotides were successfully obtained from the controlled depolymerization of salmon sperm DNA. The obtained product shared similar chemical and biological properties with defibrotide produced by Gentium SpA, Italy. It was also found that oligodeoxyribonucleotides derived from non-mammalian origins could also directly stimulate tissue plasminogen activator (t-PA) release from cultured human endothelial cells, and enhance fibrinolytic activity in the rabbit.
  Biologicals 01/2013;
• Article: Antibody responses of Macaca fascicularis against a new inactivated polio vaccine derived from Sabin strains (sIPV) in DTaP-sIPV vaccine.

  Y Sato, K Shiosaki, Y Goto, K Sonoda, Y Kino
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  ABSTRACT: Antibody responses of Macaca fascicularis against a new tetravalent vaccine composed of diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, and inactivated poliovirus derived from Sabin strains (sIPV) was investigated to predict an optimal dose of sIPV in a new tetravalent vaccine (DTaP-sIPV) prior to conducting a dose-defined clinical study. Monkeys were inoculated with DTaP-sIPVs containing three different antigen units of sIPVs: Vaccine A (types 1:2:3 = 3:100:100 DU), Vaccine B (types 1:2:3 = 1.5:50:50 DU), and Vaccine C (types 1:2:3 = 0.75:25:25 DU). There was no difference in the average titers of neutralizing antibody against the attenuated or virulent polioviruses between Vaccines A and B. The average neutralizing antibody titers of Vaccine C tended to be lower than those of Vaccines A and B. The sIPV antigens did not affect the anti-diphtheria or anti-tetanus antibody titers of DTaP-sIPV. Furthermore, the average neutralizing antibody titers of Vaccine A against the attenuated and virulent polioviruses were comparable between M. fascicularis and humans. These results suggest that M. fascicularis may be a useful animal model for predicting the antibody responses to sIPVs in humans, and that it may be likely to reduce the amount of sIPVs contained in DTaP-sIPVs, even for humans.
  Biologicals 01/2013;
• Article: Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis.

  Xuan Shen, Xiaomu Chen, David E Tabor, Yi Liu, Methal Albarghouthi, Yi-Fan Zhang, Mark S Galinski
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  ABSTRACT: Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.
  Biologicals 01/2013;
• Article: Potential use of inflammation and early immunological event biomarkers in assessing vaccine safety.

  Béatris Mastelic, David J M Lewis, Hana Golding, Ian Gust, Rebecca Sheets, Paul-Henri Lambert
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  ABSTRACT: Highly effective vaccines have traditionally been designed in a rather empirical way, often with incomplete understanding of their mode of action. Full assessment of efficacy and reactogenicity takes time and, as a result, vaccine introduction to the market is usually slow and expensive. In addition, in rare cases, unacceptable reactogenicity may only become apparent after years of development or even widespread use. However, recent advances in cell biology and immunology offer a range of new technologies and systems for identifying biological responses or "biomarkers" that could possibly be used to evaluate and predict efficacy and safety during vaccine development and post-marketing surveillance. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the potential use of biomarkers to assess vaccine safety which was held in Baltimore, Maryland, USA, from 10 to 11 May 2012 and organized by the International Association for Biologicals (IABS). The conference focused particularly on determining which biomarkers might relate to vaccine efficacy and reactogenicity and whether our knowledge base was sufficiently robust at this time for the data to be used for decision-making. More information on the conference output can be found on the IABS website, http://www.iabs.org/.
  Biologicals 11/2012;
• Article: Assessment of snake antivenom purity by comparing physicochemical and immunochemical methods.

  Alvaro Segura, María Herrera, Mauren Villalta, Mariángela Vargas, José María Gutiérrez, Guillermo León
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  ABSTRACT: Purity is a characteristic that, together with effectiveness and safety, must be tested to determine the quality of biopharmaceutical products. In therapeutic immunoglobulins, such as human intravenous immunoglobulin (IVIG), purity is evaluated on the basis of physicochemical properties, and is usually assessed by chromatography and electrophoresis. However, in the case of antivenoms these methods fail to discriminate between antibodies towards venom antigens, which constitute the active substance, and antibodies towards non-venom antigens, which are the major impurities in most of the current formulations. The assessment of this aspect of purity requires the use of the immunochemical methods. In this study, it was demonstrated that antivenoms showing physicochemical purity higher than 90% might present immunochemical purity lower than 40%. It is proposed that a comprehensive analysis of antivenom purity should combine physicochemical and immunochemical parameters. In addition, these results are crucial to decide the more appropriate strategies to improve antivenom purity. Taking into account that the current methods of antivenom purification remove most non-antibodies proteins, we propose that efforts must be primarily directed to the improvement of immunization protocols to enhance the antibody response towards venom components in hyperimmunized animals, and secondarily, in the realm of immunoglobulin purification technology.
  Biologicals 11/2012;
• Article: The pharmacology study of a new recombinant TNF receptor-hyFc fusion protein.

  Jung-Hwan Lee, Jong Ho Cho, Jiwoo Yeo, Sung Hee Lee, Se Hwan Yang, Young Chul Sung, Ju-Hee Kang, Chang-Shin Park
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  ABSTRACT: TNF-α-blocking agents such as infliximab, adalimumab and etanercept are widely used for the treatment of severe inflammatory diseases including rheumatoid arthritis and psoriasis. The currently used TNF-α blockers have Fc regions of the human IgG1 subtype, which is advantageous in terms of in vivo half-life but also raise the potential for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein by fusing the TNF receptor (TNFR) with a hybrid Fc (hyFc) consisting of the CH2 and CH3 regions of IgG4 and the highly flexible hinge regions of IgD which would not have ADCC and CDC activity. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO and pharmacological characteristics were evaluated in comparison with the structurally similar etanercept. TNFR-hyFc effectively neutralized TNF-α in L929 bioassay and showed a 1.5-fold higher neutralizing activity compared to etanercept. In a pharmacokinetic study in cynomolgus monkeys, TNFR-hyFc showed plasma half-life and AUC comparable to etanercept. In a mouse collagen induced arthritis model, TNFR-hyFc showed significant amelioration of arthritis compared to etanercept or vehicle control. In an LPS-induced septic shock model, TNFR-hyFc showed a similar level of protection against mortality as etanercept. These results confirm the feasibility of the TNFR-hyFc as an effective TNF-α blocker for the treatment of inflammatory diseases.
  Biologicals 11/2012;