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Humanization workflow: 1) Fv modeling - Application of Schordinger to build up one Fv homology model and one Fv hetero model - Generate aggregation surface report - Generate PTM analysis report (including N-glycosylation, deamidation, oxidation and protease site analysis based on the displaying status of the amino acid on 3D structure surface) - Generate 3D PI prediction 2) Humanization design - Up to 5 humanized VH and 5 humanized VL versions will be designed respectively following INN new rule - Coden optimization (optional) 3.1) First round of leads selection and production: - DNA synthesis, cloning and transient transfection - Affinity (ELISA) assay with expression media - Aggregation potential and thermos-stability raw screening (ELISAs after heating) with expression media - Production of top 3 humanization leads and chimeric antibody (1mg each) 3.2) Production of all humanized versions plus chimeric Ab as the control - 2mg protein for each sample 4) Second round of final humanization lead selection a) Affinity: Biacore or cell based assay (such as FACS, if Biacore is not applicable) b) Aggregation potential: DLS c) Thermostability: DSC d) Purity: SEC or CE e) Non-specific binding (ELISA on Baculovirus) with in-house controls Price list, payment method and time line: Package A: Golden Design ($20,000) 1) + 2) Upfront payment: $12000 Design fee payment: $8000 Design starts after receiving the upfront payment, 2 weeks after receiving the payment, an antibody humanization report include: - Fv models - Schordinger aggregation surface prediction - Schordinger PTM analysis, - 3D based PI prediction - Human template information - Humanization percentage of each VH and VL version - Optimization interpretation (if any) will be sent to the client. If the client is satisfied with the report, please pay the remaining service fee ($8000) in two weeks. After receiving the final payment, sequences of all humanized VH and VL versions will be sent to the client via email in 2 days. If needed, the designer will provide clients free email consultation service within 2 months after the sequences are received by the client. Package B: Gold design + Preliminary screening ($40,000) 1) + 2) + 3.1) Upfront payment: $12,000 Preliminary screening payment: $15,000 Design fee payment: $8000 to $10,000 Golden design service starts after receiving $12,000 upfront payment. Design report (without sequence information) will be sent to customer in 2 weeks. Preliminary screening will be performed after clients approve the design and pay $15,000 service fee. Preliminary screening data will be sent to clients in 6 weeks. One week after the data is received by the client, $8000 remaining design fee and $5000 protein production fee need to be sent to Ab Studio. After receiving the final payment, Ab Studio will send to client humanized sequences information and 1mg/sample of purified chimeric antibody as well as that of 2-3 leads selected by the client in 6 weeks. Package C: Gold design + Protein production ($40,000) 1) + 2) + 3.2) Gold design service payment: $20,000 Protein production payment: $20,000 After Golden design service is done, if clients want to get purified proteins for characterization, $20,000 will be charged for producing 9 humanized versions and 1 chimeric antibody control at 2mg per sample in 8 weeks. Both designed humanized sequences and purified proteins will be delivered to the client after the full payment is received. Package D: Diamond full humanization service ($70,000) 1) + 2) + 3.1) + 4) Upfront payment: $12,000 Preliminary screening payment: $15,000 Design fee payment: $8000 Protein production payment: $15,000 Secondary screening payment: $20,000 Humanization design starts after receiving $12,000 upfront payment, design report without sequence information will be sent to client for approval in 2 weeks. After clients approve the report and pay $15,000 preliminary screening fee, preliminary screening will start and complete in 6 weeks. Screening data will be sent to clients for review and approval. One week after clients review and approve the report and select the leads for second round screening, $8000 design fee as well as $15,000 protein production fee (for producing 2 humanization leads and chimeric antibody control at 5mg/sample) need to be paid to Ab Studio. Protein production will complete in 6 weeks. After that, Ab Studio will start the secondary round screen service after receiving $20,000 service fee from clients. The secondary round screen service includes Biacore for affinity, DLS for aggregation, DSC for thermostability, SEC/CE for heterogenecity and Bac ELISA for non-specific tissue binding. All these data are required for analyzing the developability of a humanized antibody in CMC processing. These work will be done in 6 weeks. Both the remaining purified protein and the full humanization report containing all data as well as humanized sequence information will be sent to the client after full service fee is completed.