Analysis of glycosylation heterogeneity in antibody production by Pichia pastoris

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

The aims of this M.Sc. thesis are the production of an antibody fragment in Pichia pastoris and to assess O-glycosylation heterogeneity theoretically and experimentally. All possible glycoforms (glycans that are attached to proteins) attached to this antibody were calculated and a simple mathematical model was developed in MATLAB to predict glycoforms heterogeneity. The production of Anti-(ED-B) scFv by Pichia pastoris was made in a 50 L fedbatch fermenter. The maximum product concentration obtained at approximately 100 hours of operation was 8,4mg/L. The Anti-(ED-B) scFv has no sequence of N-linked glycosylation and for the sequence of O-linked glycosylation there are 56 possible spots. Two mathematical models were developed in MATLAB: a deterministic and a stochastic model. Both models predict a given glycoforms distribution under the premise that the Endoplasmatic Reticulum enzymes and Golgi enzymes (O-Mannosyltranferase, α-1,2- Mannosyltransferase and β-1,2-Mannosyltransferase) are active. The models predict when the ratio of protein concentration per initial mannose concentration is low the prevalent glycoforms are protein with two mannoses. While when the ratio of protein concentration per initial mannose concentration is very low, the prevalent glycoforms are protein with five mannoses. For high number of mannose molecules at the beginning of the glycosylation process there is a convergence of results predicted by the deterministic and stochastic models. However, both models diverge when the number of mannose molecules is very low. In this situation, the stochastic predictions are more consistent with the true nature of the system than the deterministic ones. Despite of a theoretical O-linked glycoforms distribution, experimental test by Glycoprotein Detection Kit showed that the anti-(ED-B) scFv produced by Pichia pastoris is not glycosylated at the tested culture conditions. The posttranslational modifications that lead to the formation of monomers or dimers are not related to glycosylation but probably to the folding process. In future studies it would be interesting to study if the occurrence of Oglycosylation and its heterogeneity can be controlled by reactor operation parameters such as Temperature, pH and methanol feeding rate. It is known that such operational parameters have an important impact on the conformation of the scFv.