Methods for manufacturing a polyclonal protein
A protein, a technology for preparing drugs, used in immunoglobulins, chemical instruments and methods, allergic diseases, etc.
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[0060] different preparation options
[0061] Expression of recombinants in culture vessels
[0062] Cultured mammalian cells have become the primary system for the production of recombinant proteins for clinical use due to their ability to correctly fold, assemble and post-translationally modify. Two main formats have been employed for the production of recombinant proteins in mammalian cells: cultures of adherent cells and suspension cultures. The latter is by far the most common.
[0063] In simple batch or fed-batch processes, scale-up to very large volumes can be achieved by diluting the contents of the bioreactor into 5-20 volumes of fresh medium kept prewarmed in the larger reactor occur. The process from thawing stored cells to actual large-scale production consists of three separate phases: seed culture, inoculum culture, and production phase. Seeding is usually done in smaller cell culture vessels, starting from small volumes of a few mL of culture immediately af...
Embodiment 1
[0200] Example 1: Production of split cell expansion, mixing in shake flasks
[0201] overview
[0202] Six different cell populations were counted and mixed in equal cell ratios (1:1:1:1:1:1) and used to inoculate bioreactors where each cell population produced a different antibody. Cells were cultured for 7 days before ion exchange chromatography (IEX) was used to quantify the amount of each of the six antibodies. The experiment was repeated twice and the batch-to-batch reproducibility of the obtained polyclonal antibody composition was investigated.
[0203] Materials and methods
[0204] cell line
[0205] The parental producer cell line used was a derivative of the DHFR negative CHO cell line DG44 obtained from Lawrence Chasin (Columbia University). DG44 cells were transfected with the cDNA of the adenovirus type 5 transactivator E1A in the vector pcDNA3.1+ (Invitrogen). Transfectants were selected with Geneticin (Invitrogen) at a concentration of 500 μg / ml. After s...
Embodiment 2
[0234] Example 2: Separate Upstream Cultivation in a Fed-Batch Bioreactor, Mixing, followed by Purification
[0235] overview
[0236] Five different ECHO cell lines expressing five different IgG antibodies were cultured individually in a bioreactor for 13-14 days. After incubation, the antibody-containing fractions (2:1:1:1:1) were mixed during purification to obtain the antibody composition in a bulk drug substance (BDS).
[0237] Expression plasmids, cells, etc. were as in Example 1, except that only five antibodies were expressed (see Table 4).
[0238] Table 4. Antibodies and ECHO clones
[0239] Antibody
ECHO clone name
Sym002-186
37-11
Sym002-482
39-3
Sym002-303
26-9
Sym002-286
28-6
Sym002-235
38-8
[0240] melt and expand
[0241] In ProCHO4 heated to 37 °C TM Each of the five different cell lines was thawed in culture medium. Centrifuge the cell suspension at 800 rpm (160G) for 4 minut...
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