VEGF (vascular endothelial growth factor) resistant protein composition and preparation method thereof
A harvested, chromatographic technology, applied in the field of anti-VEGF composition and its preparation, which can solve the problems of vision loss, submacular effusion and the like
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[0323]As used herein, the term "protein" or "protein of interest" may include any polymer of amino acids having covalently attached amide bonds. Examples of target proteins include, but are not limited to, Aflibercept and MiniTrap. Proteins comprise one or more polymer chains of amino acids and are commonly referred to in the art as "polypeptides". "Polypeptide" means a polypeptide consisting of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked by peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof composed of polymers. "Synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid phase peptide synthesis methods are known to those skilled in the art. A protein may contain ...
Embodiment 1
[0725] Example 1: Protein production using chemically defined media
[0726] 1.1 Cell source and harvest
[0727] Aflibercept-producing cell lines were used in this study. Aflibercept-producing cell lines were grown and harvested using chemically defined media (CDM).
[0728] 1.2 Proteolytic cleavage of aflibercept
[0729] With immobilized IdeS enzyme (obtained from Genovis (Cambridge, MA) ) column was used to generate MT1. Aflibercept (20 mg in 1.0 mL cleavage buffer) obtained from the cell culture harvest was added to the column and incubated at 18°C for 30 minutes. After 30 minutes, the column was washed with cleavage buffer (1.0 mL). Combine the digestion mixture with the wash solution. The mixture was loaded on an analytical protein A affinity column (Applied Biosystems TM , POROS TM 20 μΜ Protein A cartridge 2.1 x 30 mm, 0.1 mL (Catalog No. 2-1001-00) was applied to and eluted from. According to POROS TM 20 μM Protein A Cartridge 2.1 x 30 mm, 0.1 mL (Catalo...
Embodiment 2
[0730] Example 2. Anion Exchange Chromatography (AEX) for Minimizing Color
[0731] (A) Reduce color formation using AEX
[0732] AEX chromatography was performed to remove the staining obtained during the production of aflibercept expressed with CDM1.
[0733] 2.1 Design
[0734] As detailed in Table 2-1, five AEX separations were performed for this study using the AEX protocol as described in Table 2-2. A 15.7mL Q Sepharose Fast Flow column (19.5cm bed height, 1.0cm I.D.) and a 14.1mL POROS 50 HQ column (18.0cm bed height, 1.0cm I.D.) were integrated into an AKTA Avant benchtop LC controller.
[0735]The pH of the AEX loading solution was adjusted to target ±0.05 pH units using 2M Tris base or 2M acetic acid. The conductivity of the AEX loading solution was adjusted to target ±0.1 mS / cm using 5M NaCl or deionized water. All pooled samples were analyzed for high molecular weight (HMW), color and yield.
[0736] Table 2-1. Overview of study design for AEX color reduction ...
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