Conjugation. The AFAIK2 product from the first engineering batch will be conjugated with 50 grams of high purity urease. The final product will be prepared in a stabilized buffer solution, the nature of the ingredients to be provided by Helix. The conjugated product will be available for use by Helix as well as be available to complete the qualification for the majority of the release tests, with others as FIO since the stability of the product prior to conjugation has not been established. The product will also be used for packaging trials of the bulk drug substance in the selected packaging container for shipment to the formulator. Site cGMP API Facility, Class 100K clean room Time 2 days Documentation Preliminary batch records and notebooks to prepare product against target specifications, draft QC test methods and procedures. Deliverables 50 grams L-DOS47 , Completed batch records, QC reports Development report, Draft Certificate of analysis 5.4 L-DOS-47 Engineering Batch #2
Conjugation. The AFAIK2 product from the second engineering batch will be conjugated with 50 grams of high purity urease. The final product will be prepared in a stabilized buffer solution, the nature of the ingredients to be provided by Helix. It is BioVectra’s understanding that the second engineering batch will be used as the toxicological batch. As such this batch will be used for the early stability study, as the batch prior to the clinical batch and a portion of this batch will allocated accordingly. The final product will be released against the same tests as proposed for the future clinical batch. Site cGMP API Facility, Class 100K clean room Time 2 days Documentation Batch records and notebooks to prepare product against target specifications, QC test methods and procedures. Deliverables 50 grams L-DOS47, Completed batch records, QC reports Development report, Certificate of analysis 5.5 L-DOS-47 Clinical Batch
Conjugation. During conjugate preparation, the single cysteine mutants will form Au-S bonds with AuNPs and the His tag mutant will strongly associate with the His-Tag. Each mutant was rehydrated with 1.42 mg/mL TCEP, to prevent disulfide bonds from forming,30 and were left to incubate for at least an hour. To make 5 nm conjugates, 400 L of 2.40 nM 5 nm AuNPs were added to 30 L of 50 mM free protein in a lo-bind Eppendorf tube and left to incubate overnight at 4°C to allow the conjugation to occur. The samples were centrifuged for 90 minutes at 13,200 rpm. The supernatant was removed, and the pellet was replenished with 0.005% Tween in 10 mM sodium phosphate buffer to wash the free protein from the pellet of DHFR-AuNP conjugates. After wash cycles, the pellet was stored in 0.005% Tween in 10 mM sodium phosphate buffer, and diluted accordingly. To make 15 nm conjugates, the same methods to make 5 nm conjugates were made except centrifuging speeds and times were changed to 8000 rpm and 35 minutes for 800 L of 7.88 nM 15 nm AuNP. To make 30 nm conjugates, the same methods to make 5 nm conjugates were made except centrifuging speeds and times were changed to 8000 rpm and 10 minutes for 400 L of 2.40 nM 15 nm AuNP.
