Identification of bottlenecks in antibody expression using targeted gene integration

Abstract

Antibody engineering allows proper design of improved properties in antibody products such as a high binding affinity, stable biophysical properties and low immunogenicity. Besides these obvious features improved by elegant design the primary amino acid sequence also has tremendous effects on the performance of the expression host itself influencing cell culture parameters including specific productivities and growth rates therefore having major impact on the finally obtained antibody expression titers [1]. By understanding the underlying mechanisms how certain primary sequence combinations influence culture performance in conjunction with biophysical features of the antibody protein, we might rationally improve the production process of these expensive but essential biotherapeutic products. Many different antibody variants were expressed in our group in different projects based on various isotypes. For several antibody variable regions we could observe a direct correlation of advantageous cell performance and a high degree of similarity to the closest human germline sequence of the respective mature antibody leading to higher specific productivities. However, the fundamental principles of these consistent observations and the correlation of cell behavior and biophysical product properties remain quite elusive. Therefore, in this project we aim to set the proper foundations to investigate the generalization of a concept to improve cell performance and antibody expression based on the primary sequence at a high germinality degree (Figure ​(Figure1).1). To have proper control of transgene integration locus, gene copy numbers and mRNA transcript level a suitable host for recombinase-mediated cassette exchange (RMCE) was developed to compare different antibody variants under isogenic conditions. Open in a separate window Figure 1 Observed correlation of specific productivity and sequence identity to the closest human germline sequence (germinality) (A). Antibody expression levels are influenced by several factors. Recombinase-mediated cassette exchange was applied to control for gene copy number and mRNA transcript level. Low expressing antibody variants (2F5, 4B3 and 2G12) were compared to its closest human germline sequence together with a therapeutic antibody (Ustekinumab) with low degree of germinality (B). Respective germline antibody variants were constructed by combination of germline V-, D- and J-segments.