Phage Display Library – Single-chain Fv Antibody Library Construction Service

A single-chain antibody consists of an antibody variable light (VL) and heavy (VH) fragment joined by a flexible peptide linker, which can be easily expressed in a functional form on the surface of phage, and is therefore commonly known as a single-chain variable fragment (scFv). scFv fragment is the smallest unit of immunoglobulin molecule (except nanobodies) with function in antigen-binding activities and allows protein engineering to improve the properties of scFv (single chain fragment variable) such as increase of affinity and alteration of specificity. In Alpha Lifetech Inc., scFv antibodies have been constructed mainly from hybridoma, spleen cells from immunized mice, and B lymphocytes from humans. Total RNA or mRNA is first isolated from hybridoma, spleen, lymph cells and bone morrow cells followed by reverse transcribed into cDNA to serve as a template for antibody genes amplification (PCR).

The diversity of a library is determined by the number of independent clones. To produce a large-sized scFv antibody library, our scientists will utilize our proprietary two-step strategy for library generation. In the first step of the procedure described, primary repertoires are prepared from PCR products encoding the V_H, V_κ, and V_λ domains, yielding typical medium-sized libraries (1 - 100 million clones). In the second step, VH (or V_L) fragments are isolated by digestion of plasmid DNA purified from the primary repertoires and cloned into the acceptor phagemid vector containing the V_L (or V_H) repertoires. This innovation increases the size of the libraries dramatically (10¹⁰-10¹² clones).

Engineering scFv for improved stability, yield and function

As we know, instability of the VH–VL interface due to exposed hydrophobic patches of the chains that favor aggregation, has been hypothesized to be the main cause of irreversible scFv inactivation. With years of experience from antibody affinity maturation to high-level expression of recombinant protein, Alpha Lifetech Inc. has a deep understanding of the engineering scFv for improved stability, yield and function, such as parameters including linker design, thermodynamically stable assay, ionic strength and pH.

Large-scale and high-throughput processing of scFvs

For large-scale preparation of scFvs, it is evident that the choice of expression host will have a significant impact on process design, downstream processing strategies and process cost. The choice of manufacturing route will be dependent on the relevance of post-translational modification and timeline for clinical development and economics, which are also essentially 'host system'-dependent. There is no universal expression system that ensures high product expression levels for a broad range of different antibodies or even proteins.