SELECTION WORK WITH STOCK OF TRANSGENIC GOATS IN THE REPUBLIC OF BELARUS

Genealogical structure of the livestock of goats producing the bio-analogue of human lactoferrin has been studied for the fi rst time in the Republic of Belarus. Successors of lines and families have been determined, and a comparative assessment of using various selection profi les for obtaining goats and dams of the stock of genetically engineered animals has been carried out.

The concentration of recombinant human lactoferrin in the milk of transgenic goats of the third and fourth year of lactation

The concentration of recombinant human lactoferrin (rhLF) in the milk of 22 transgenic goats of different ages was studied: the average concentration of rhLF in the milk of goats-producers of the 4th year of lactation was 13.7% higher than that of goats of the 3rd year of lactation (6.07 and 5.24 g/l, respectively). It was found that the maximum level of recombinant glycoprotein was produced by the mammary gland of animals in the 2nd and 4th quarters of the studied year period. Protein’s concentration in milk measured in the 2nd and 4th quarters was 6.4 g/l and 6.33 g/l for producers of the third year of lactation and 8.14 g/l and 7.02 g/l for goats of the fourth year of lactation, respectively. The correlation between the season of the year and the content of the target protein in milk was detected (p <0.01), it was found that the most active rhLF production in goat milk occurred in late spring and winter periods. Variability in the content of the protein of interest in milk between individuals was noted. During the study a relatively low level of rhLF was established in animal №0747 - 0.11–2.52 g/l, while its maximum value was detected in the producer № 0403 - 4.52–9.06 g/l (more than 4 times difference). The range of individual variability in terms of rhLF content in the milk of goats of the third and the fourth lactation was 0.11–9.06 g/l. Milk of genetically modified goats contains 3 times more lactoferrin than human breast milk. Therefore, rhLF concentration testing in goat milk and tracking the dynamics of this indicator allows us to obtain data that can be used later for breeding pro-grams formation, milk production planning and also to improve the gene pool of transgenic animals.

Transgenic goats producing an improved version of cetuximab in milk

ABSTRACTTherapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

DETERMINATION OF BIOLOGICALLY ACTIVE RECOMBINANT HUMAN LACTOFERRIN IN MILK OF TRANSGENIC GOATS AND PROTEIN FRACTIONS ISOLATED FROM MILK

Specific immunoreagents for the immunoassay technologies of recombinant human lactoferrin in food and biotechnological products have been obtained and characterized. This work was financially supported by the State program of scientific research «Chemical technologies and materials» for 2016 – 2020 years.

Physicochemical properties of lactoferrin under oxidative/halogenative stress

Lactoferrin (Lf) was discovered in the thirties of the twentieth century. Since that time a number of useful properties of Lf (antibacterial, antiviral, pro- and anti-inflammatory, etc.) have been found. That’s why Lf became a promising candidate for pharmaceuticals use. The concentration of Lf strikingly increases in inflammatory focuses due to neutrophil degranulation. At the same time, activated neutrophils starts to generate reactive oxygen and halogen species (ROS and RHS), which leads to the development of oxidative/halogenative stress. In this work, using the fluorescence analysis we found the change of the Lf structure and properties in the inflammation conditions (under oxidatives/halogenative stress). We use two forms of Lf – human Lf, excreted from human milk, and recombinant Lf, excreted from milk of transgenic goats. It was established that the amino acids of Lf (decreasing the number of tryptophanils and primary amines) and protein restructuring undergo modification under the HOCl action, while H2O2 has no influence. These changes in the molecule under the HOCl treatment result in decreasing the iron-binding capacity of Lf.