Animal-derived Raw Materials Research Articles

A comprehensive evaluation of lipid profiles and nutritional quality in different animal source muscle tissues

Lipid composition is crucial for assessing the nutrition and flavor of animal-derived raw materials, a key concern for consumers. Based on high-resolution mass spectrometry and chemical pattern recognition, the intermuscular lipids fingerprint of seven types of livestock, poultry, and fish muscle were analyzed. In total, 704 lipids were detected, containing fourteen lipid molecules, with significant content differences among samples, notably triglycerides, phosphatidylcholines, phosphatidylethanolamines, diglycerides, and fatty acids. Livestock and poultry muscle had a low content of lipids with an unsaturation degree above 4 (around 10% and 25%). These unsaturated lipid molecules in fish muscle ranged from approximately 60%–75%. Rainbow trout had higher contents of triglycerides and phosphatidylcholines with chain lengths exceeding 13. Mutton had more of these lipids within the 13–18 chain length, exceeding 0.3 g/(g·protein), which may be crucial for their unique texture and flavor. The results will help the deep processing and nutritional assessment of muscle foods.

Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model

Probiotics are living microorganisms that give beneficial health effects while consumed, and each strain possesses diverse and unique properties and also different technological characteristics that affect its ability to be produced at large scale. Limosilactobacillus fermentum is a widely studied member of probiotics, however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications.In the present study a novel L. fermentum strain was isolated from buffalo milk and used as test example for biotechnological process development. The strain was able to produce up to 109 CFU/mL on a (glucose based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media rich of casein and beef extract. The study of strain behavior in batch experiments indicated that the highest concentration of viable cells was reached after only 8 h of growth, greatly shortening the process. Moreover, initial concentrations of glucose in the medium above 30 g/L, if not supported by higher nitrogen concentrations, reduced the yield of biomass and increased production of heterolactic fermentation by-products. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover about 5.7 × 1010CFU/gpowder of viable cells, indicating strain resistance to harsh processing conditions.Overall, these data demonstrate the possibility to obtain and maintain adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. Moreover, since often exopolysaccharides produced by lactobacilli contribute to the strain's functionality, a partial characterization of the EPS produced by the newly identified L. fermentum strain was carried out.Finally, the effect of L. fermentum versus H. pylori in a gastric epithelial cell model was evaluated demonstrating its ability to stimulate the response of the immune system and displace the infective agent.

Modular microwave unit for heat treatment of raw materials at factory-farm enterprises

The objective of the work is to develop a microwave unit with removable modules for continuous heat treatment and disinfection of animal-derived raw materials of different structures with resonators, providing electromagnetic safety without shielding and high electric field intensity with reduced operating costs while preserving the feeding value of the product. The study includes 3D modeling of resonators for each module using CST Microwave Studio software and multi-criteria evaluation of regression models of process parameters, allowing one to estimate the feeding value of the product. The first module is designed for heat treatment of secondary animal-derived raw materials, including frozen blocks of meat waste. There are modules can be installed simultaneously or separately in the microwave unit, depending on demand and available raw materials. All modules are removable and easy to clean after completion of heat treatment, and the source block is closed and is subject only to maintenance inspection. Studies of electromagnetic safety for the perforated cylindrical resonator were performed. The operating modes of the second module were found, the duration of defrosting and heating of the animal colostrum was 10–15 min with the generator power of 4.8 kW and energy costs of 0.18–0.20 kW·h/kg.

Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms.

ABSTRACTListeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry.IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen’s transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Switching Kogenate FS® to Kovaltry® for the Treatment of Severe Haemophilia A Patients: A Successful Single Center Experience

Background: Haemophilia A is an X-linked hereditary disorder caused by deficient levels of the coagulation protein factor VIII (FVIII). Standard therapy for haemophilia A comprises replacement of FVIII with plasma-derived or recombinant FVIII concentrate, which can be administered as on-demand or prophylaxis regimen. The most challenging complication of replacement therapy in hemophilia A is the occurrence of alloantibodies against infused factor FVIII, thus predisposing the patients to an increased morbidity and disability. Historically, first FVIII was isolated from cryoprecipitated plasma concentrates. Thirty years ago, after the cloning of F8 gene, therapy for hemophilia A patients underwent a new exciting era that developed technological strategies aimed to improve haemostatic efficacy and safety of the replacement factor. In this line, a new recombinant FVIII, BAY 81-8973 (octocog alfa; Kovaltry®, Bayer) has recently been introduced onto pharmaceutical market for treatment of these patients. Kovaltry® is an unmodified, full-length, recombinant FVIII concentrate with the same amino acid sequence as Kogenate FS® (Bayer), but produced with innovative manufacturing technologies that include production without addition of human- or animal-derived raw materials from purification or formulation processes, inclusion of a higher degree of sialic acid capping of N-terminal glycans on the molecular surface, coexpression of the human chaperone protein heat shock protein 70, and a 20-nm nanofiltration step to remove small non-enveloped viruses and potential protein aggregates. On this basis we considered beneficial for our patients changing Kogenate FS® therapy for Kovaltry®.Objective: We aimed to demonstrate that switching from Kogenate FS® to Kovaltry® could be easily performed without increasing the risk of bleeding or FVIII consumption in severe haemophilia A patients.Methods: Sixty two adult and pediatric patients with severe haemophilia A, without inhibitors, treated with Kogenate FS® either on on-demand treatment (n=13) or on prophylaxis regimen (n=49), were recruited. On November 2016, we began to switch Kogenate FS® treatment to Kovaltry® in these patients.The trend of prophylaxis with Kovaltry® was evaluated by determining mean injection frequency (per week), mean dose of each injection (IU/kg), mean total dose injected/week (IU), baseline and post-infusion levels of FVIII and annual bleeding rate (ABR). Same data for Kogenate FS® were collected retrospectively from clinical histories of patients.For determining baseline and post-infusion levels of Kovaltry®, whole blood was collected in 3.2% sodium citrate tubes (BD Vacutainer, Becton Dickinson, San José, CA, USA) at pre-dosing and 30 minutes after Kovaltry® infusion. Plasma FVIII:C was measured by chromogenic assay.Results: Switching from Kogenate FS® to Kovaltry® caused no changes, except in two patients, either in mean injection frequency or mean dose of each injection or mean total dose injected/week or baseline and post-infusion levels of FVIII or annual bleeding rate. Increment observed in mean total dose injected/week in two of the pediatric patients was due to an increased physical activity (patient 2) and to weight gain (patient 8). For sake of example, Table 1 shows these variables for 10 pediatric patients under prophylactic treatment.None of the patients treated with Kovaltry® developed inhibitors.Conclusion: Changing severe haemophilia A treatment from Kogenate FS® to Kovaltry® can be performed easily and did not pose any risk to the patient since Kovaltry® proved to be equally effective than Kogenate FS® to prevent bleeding episodes and did not induce the occurrence of inhibitors. [Display omitted] DisclosuresÁlvarez-Roman:Novo Nordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

Feasibility of defined, animal component free, protein free, salt base freezing solution composed of 5% DMSO for cryopreservation of HMSC toward clinical applications

Background & Aim hMSC are a critical raw material for cell-based therapies and usually a large number of cells are required for clinical applications. Cryopreservation is currently the only method to preserve cells with maintained functional properties and genetic stability for long term storage. To date, a common practice is use of homebrew cryo-medium that is composed of animal-derived raw materials and low Mw Cryoprotective agents (CPAs), usually 10-20% DMSO. Alternatively, animal component free (ACF) freezing solutions were developed for clinical applications, but they are still based on culture medium and are composed of 10% DMSO (e.g. NutriFreez™ D10). In addition, a few products were developed with a reduced concentration of DMSO or even without DMSO. However, these products appear not to be optimal for hMSC. Moreover, most of the commercially available DMSO-free products are actually composed of other potentially toxic permeable CPAs (e.g. Ethylene Glycol, EG). Exposure of cells to these materials can impact the quality, safety and efficacy of cell-based therapeutic product. Facing strict regulatory requirements, the development of defined, ACF, salt base freezing solution with reduced concentration of DMSO is essential. Methods, Results & Conclusion The current study presents the feasibility of a novel defined, ACF, protein free, salt base freezing solution composed of 5% DMSO for cryopreservation of hMSC from various sources. Results show that the novel cryopreservation solution efficiently maintains high cell yield and viability, and supports the recovery of hMSC while maintaining the normal hMSC features: typical fibroblast-like cell morphology, phenotypic surface marker profile, differentiation capability as well as self-renewal potential. Clinically accepted, cryopreservation solution for hMSC holds a unique opportunity to facilitate the translation of these cells to cellular therapy applications.

Defining a sample preparation workflow for advanced virus detection and understanding sensitivity by next-generation sequencing.

The application of next-generation sequencing (also known as deep sequencing or massively parallel sequencing) for adventitious agent detection is an evolving field that is steadily gaining acceptance in the biopharmaceutical industry. In order for this technology to be successfully applied, a robust method that can isolate viral nucleic acids from a variety of biological samples (such as host cell substrates, cell-free culture fluids, viral vaccine harvests, and animal-derived raw materials) must be established by demonstrating recovery of model virus spikes. In this report, we implement the sample preparation workflow developed by Feng et. al. and assess the sensitivity of virus detection in a next-generation sequencing readout using the Illumina MiSeq platform. We describe a theoretical model to estimate the detection of a target virus in a cell lysate or viral vaccine harvest sample. We show that nuclease treatment can be used for samples that contain a high background of non-relevant nucleic acids (e.g., host cell DNA) in order to effectively increase the sensitivity of sequencing target viruses and reduce the complexity of data analysis. Finally, we demonstrate that at defined spike levels, nucleic acids from a panel of model viruses spiked into representative cell lysate and viral vaccine harvest samples can be confidently recovered by next-generation sequencing.

Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

Meeting Report: 2013 PDA Virus & TSE Safety Forum.

The report provides a summary of the presentations and discussions at the Virus & TSE Safety Forum 2013 organized by the Parenteral Drug Association (PDA) and held in Berlin, Germany, from June 4 to 6, 2013. The conference was accompanied by a workshop, "Virus Spike Preparations and Virus Removal by Filtration: New Trends and Developments". The presentations and the discussion at the workshop are summarized in a separate report that will be published in this issue of the journal as well. As with previous conferences of this series, the PDA Virus & TSE Safety Forum 2013 provided again an excellent opportunity to exchange information and opinions between the industry, research organizations, and regulatory bodies. Updates on regulatory considerations related to virus and transmissible spongiform encephalopathy (TSE) safety of biopharmaceuticals were provided by agencies of the European Union (EU), the United States (US), and Singapore. The epidemiology and detection methods of new emerging pathogens like hepatitis E virus and parvovirus (PARV 4) were exemplified, and the risk of contamination of animal-derived raw materials like trypsin was considered in particular. The benefit of using new sequence-based virus detection methods was discussed. Events of bioreactor contaminations in the past drew the attention to root cause investigations and preventive actions, which were illustrated by several examples. Virus clearance data of specific unit operations were provided; the discussion focused on the mechanism of virus clearance and on the strategic concept of viral clearance integration. As in previous years, the virus safety section was followed by a TSE section that covered recent scientific findings that may influence the risk assessment of blood and cell substrates. These included the realization that interspecies transmission of TSE by blood components in sheep is greater than predicted by assays in transgenic mice. Also, the pathogenesis and possibility of productive TSE infection of cell substrates were considered, and cell-based assays that may be suitable for use in TSE clearance studies were discussed. The current report provides an overview about the outcomes of the 2013 PDA Virus & TSE Safety Forum, a unique event in this field.

Investigations of porcine circovirus type 1 (PCV1) in vaccine-related and other cell lines

Porcine circovirus type 1 (PCV1) is highly prevalent in swine and was recently reported in some rotavirus vaccines. Since animal-derived raw materials, such as cells, trypsin, and serum, can be a major source of introducing virus contamination in biological products, we have investigated PCV1 in several cell lines obtained from ATCC that have broad use in research, diagnostics, or vaccine development. It is expected that these cell lines have been exposed to bovine and porcine viruses during their establishment and passage history due to the use of serum and trypsin that was not qualified according to current testing guidances or processed using new virus-inactivation methods. This study showed that Vero, MRC-5, and CEFs, which represent cell substrates used in some U.S. licensed vaccines, and other cell lines used in investigational vaccines, such as MDCK, HEK-293, HeLa, and A549, were negative for PCV1 using a nested PCR assay; some were also confirmed negative by infectivity analysis. However, MDBK cells, which are used for some animal vaccines, contained PCV1 sequences, although no virus was isolated. Although the results showed that PCV infection may not have occurred under previous culture conditions, the recent cases of vaccine contamination emphasizes the need for continued efforts to reduce the likelihood of introducing viruses from animal-derived materials used in product manufacture.

Safety and Preliminary Efficacy of Recombinant Activated FVII Analog (NN1731) In the Treatment of Joint Bleeds In Congenital Hemophilia Patients with Inhibitors

Safety and Preliminary Efficacy of Recombinant Activated FVII Analog (NN1731) In the Treatment of Joint Bleeds In Congenital Hemophilia Patients with Inhibitors

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.

Influence of Carboxymethyl Dextran and Ferric Citrate on the Adhesion of CHO Cells on Microcarriers

Due to the inherent risks of animal-derived raw materials, the biopharmaceutical industry has an increasing demand for serum-free and protein-free media for industrial cell culture bioprocesses. The absence of serum often changes the characteristics of mammalian cells, especially growth, productivity, and adherence properties. This study is mainly focused on the influence of media additives on cell adherence characteristics. An array of different carboxymethyl dextrans and different ferric citrate concentrations was tested with a number of CHO clones, using standard cell culture Roux-flasks and Cytoline 1 macroporous microcarriers. A prototype mixing system with controlled shear force input was developed as a screening system for adherence characteristics. The results of this evaluation revealed a negatively correlated dose-dependent influence on adhesion for ferric citrate. It was also found that certain carboxymethyl dextrans are capable of increasing the adherence on Roux-flasks and microcarriers.

Evaluation of Vaccines, Interferons and Cell Substrates for Pestivirus Contamination

Pestiviruses are potential contaminants of biological products produced in bovine or porcine cells or manufactured via processes using animal-derived raw materials such as bovine serum. In order to investigate possible contamination of products including those manufactured and/or licensed in the US, 38 lots of viral vaccines and five lots of interferon alpha (IFNα) were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of bovine viral diarrhoea virus (BVDV). All vaccines and interferons were negative for contaminating BVDV RNA when tested by RT-PCR, with the exception of an experimental live viral vaccine that had been produced in BVDV contaminated rabbit kidney cells. Cell lines commonly used to produce biological products and vaccines were experimentally infected with the NADL strain of BVDV to determine if they were permissive for virus replication. MRC-5 and Wl-38 cells were not infected. In contrast, Vero, CHO and CEF cells showed evidence of pestivirus infection. Taken together these data suggested that currently licensed viral vaccines were unlikely to be contaminated with pestiviruses. However, cell banks derived from non-human primate, hamster or rabbit kidney cell lines, or cultures of primary chick embryo fibroblasts, may be infected with BVDV if exposed to pestivirus contaminated raw materials during manufacture.

Collective experiences of adventitious viruses of animal-derived raw materials and what can be done about them.

Contamination of animal-derived raw materials with viruses, mycoplasmas, bacteria and fungi is common. These contaminants can interfere with the diagnosis of viral infection, and vaccines produced using infected cell cultures could lead to seroconversion or disease in the vaccinated animal. The purity, safety and efficacy of viral vaccines requires testing of the ingredients, cell substrates and final product. Methods for detection of viruses, especially bovine viral diarrhea virus, in nutrient serum, cell cultures, seed viruses and viral vaccines, and the frequency of their detection at the Center for Veterinary Biologics are discussed.

Large-scale management systems and parasite populations: Ectoparasites

An increased intensification of animal production is needed so that worldwide hunger can be fought successfully and the ever-increasing human population can be supplied with animal proteins and animal-derived raw materials. The author considers parasitism to be an arrangement of species — different populations basing on somatoxenia, that is, a body to body contact-relation. Arthropods have an exceptional position within the animal kingdom because of: 1. (1) the richness in species — out of the 1.26 million living species more than 1 million belong to arthropods, and nearly every 5th of these is a parasite! 2. (2) their ability to live in all 3 elementary biotopes: on the continental, in the fluvial and in the oceanal; 3. (3) their exceptionally high adaptability, and parasitic arthropods share this adaptability.