Formulations
A preparation and pharmaceutical preparation technology, applied in the field of bispecific protein and scFV pharmaceutical preparations, can solve the problems of TCR instability, low protein concentration, and maintaining protein stability are not easy
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Embodiment 1
[0073] Example 1 - Stable Aqueous Formulation of Soluble TCR-scFv Bispecific Protein
[0074] A)
[0075] A bispecific protein comprising a soluble TCR fused to an anti-CD3 scFv was prepared using known methods (alpha chain of SEQ ID No: 45 of WO2011 / 001152, wherein amino acids 1 to 109 were replaced with SEQ ID No. 8 of WO2011001152 , and the amino acid at position 1 is A, and based on the numbering of SEQ ID No: 45, the C-terminus of the α chain is truncated by 8 amino acids from F196 to S203 (inclusive); and the SEQ ID No of WO2011 / 001152: 36 β chain, wherein the 259th to 370th residues correspond to SEQ ID No.27 of WO2011 / 001152, and the amino acids at the 1st and 2nd positions are A and I respectively), and size exclusion chromatography (SEC ) for buffer exchange to formulation buffer. The protein was then diluted to a final concentration of 0.20 ± 0.02 mg / mL using formulation buffer and bottled in a laminate airflow (LAF) bench.
[0076] Formulation buffer contained 5...
Embodiment 2
[0094] Example 2 - formulation optimization
[0095] A- Detergent Protein Ratio
[0096] The weight ratio between detergent and protein was found to be a key factor in producing stable formulations.
[0097] Evidence for this was obtained from Light Obscuration (LO) measurements, which assessed the presence of invisible particles of 2 μm and larger in size after 5 freeze-thaw cycles. The formulation buffer contained the TCR-scFv bispecific protein of Example 1A, 50 mM phosphate-citrate, 5% trehalose, 1% mannitol, and Tween 20 at one of 5 different concentrations. The pH is 6.5. The protein concentration was kept constant at 0.2 mg / ml. The measurement is performed on a PAMAS SVSS-C or equivalent particle sizer. Samples were analyzed purely with a pre-run volume of 0.4 ml and a measurement volume of 0.1 ml in triplicate. Particle counts > 2 μm, > 10 μm and > 25 μm were determined.
[0098] image 3 It is shown that when the ratio of Tween 20 to protein is 1, the count of ...
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