Sincere thanks may also be directed at Kathrin Ladetzki-Baehs and Tag Winderlich for advice about the preparation of figures and statistics. CDRs. Phage selection was performed against several antigens and exclusive antibodies with exceptional biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. Keywords: antibody engineering, human antibody library, phage display, Slonomics, CDR-H3 design, VH/VL pairing, Ylanthia Introduction Due to their high specificity and broad target space, monoclonal antibodies (mAbs) represent the most important class of biologics and have great potential for both diagnostic and therapeutic applications.1 A diverse set of in vivo and in vitro technologies are currently available for the generation of these versatile molecules. Fully human mAbs, for example, can be derived from human B cells by immortalization via viral transformation,2 through the generation of human hybridomas,3 or through direct cloning of Ig encoding gene transcripts.4-6 Target specific human B cell material, however, in some cases may not be Mycophenolate mofetil (CellCept) readily accessible. The hybridoma technology7 using immunoglobulin (Ig) transgenic mouse systems8 overcomes this limitation but still suffers from the restriction of the target space due to the need for immunogenic sequences and epitopes. On the other hand, the mentioned in Mycophenolate mofetil (CellCept) vivo methods share the advantage of delivering structurally diverse IgM, IgG and IgA antibodies with naturally paired and correctly folded variable Ig heavy and Ig light (VH/VL) chain frameworks. Among the in vitro technologies, the methods of choice for the generation of human mAbs are phage, ribosome and yeast display of recombinant antibody libraries.9-14 Various na?ve,15,16 immune,17 semi-synthetic18 and synthetic19-21 library formats have been described. They differ both in their design regarding framework composition and complementarity-determining region (CDR) diversification,22 as well as their quality, as defined by library size and correct sequences. Major advantages of phage display include the virtual independence of the selection process from target characteristics (e.g., immunogenicity or toxicity of the antigen), which allows application to a wide range of antigens, the possibility of accessing rodent cross reactive antibodies for animal models, the full control over the selection conditions and the technological robustness and relative velocity.23 Therapeutic antibodies must fulfill high standards with regard to their binding affinity, target and epitope specificity, and functional activity. An additional challenge of antibody discovery technologies is the generation of mAbs with appropriate biophysical properties. To reach the market, antibody therapeutics ideally should, for example, not aggregate or precipitate and resist degradation by proteases, as these factors contribute to optimal manufacturing and shelf life properties and ensure a long serum half-life. Furthermore, the increasing demands of regulatory authorities with respect to properties such as the homogeneity of the product drive these needs. Comparable stability requirements also apply for diagnostic and research mAb tools.24 Various approaches have been undertaken to combine the advantages of in vivo and in vitro technologies to generate recombinant libraries that include predominantly antibodies with superior properties. For example, the human combinatorial antibody library (HuCAL) Mycophenolate mofetil (CellCept) platform is based on a composition of selected VH and VL consensus grasp gene sequences that are diversified using modular CDR cassettes.19 HuCAL PLATINUM, the latest generation HuCAL Mycophenolate mofetil (CellCept) antibody library,20 implemented optimized antibody framework sequences with a reduction of unproductive sequences and a revised rational CDR design compared with the previous HuCAL scFv and HuCAL GOLD libraries.19,21 Another approach to combine structural CDR diversity with a biophysically optimal VH/VL combination is the n-CoDeR scFv antibody Tshr library, which uses a VH3C23/V1C47 pairing as scaffold.25 The Ylanthia antibody library introduces a new concept in which fixed VH/VL framework pairs are rationally selected based on biophysical characteristics that are relevant for antibody selection, development and manufacturing. Through this process, optimal biophysical properties can be built into the library using human germline antibody sequences, without the need to introduce stabilizing mutations. The work-flow leading to the final framework composition of the library comprised several actions. First, 20 VH, 12 V and 8 V Ig gene segments were chosen based on their prevalence in the human antibody repertoire and additional in silico analysis to cover all canonical CDR structures. The 400 VH/VL pairings resulting from the combination of those 40 variable domains were characterized in vitro to rank VH/VL framework pairs according to properties, such as relative protein expression level, thermal and serum.