Methods for harvesting biomolecules
The disclosure provides methods of purifying native and recombinant biomolecules, e.g., proteins, from mammalian cells using purification protocols incorporating harvest recovery operations involving decanter centrifugation of at least one target biomolecule from at least one particulate component of cell culture fluid. The unexpected capacity of decanter centrifuge separation of biological materials of similar densities found in mammalian cell culture fluid has been found to yield high quantities of functional protein in efficient, low-cost harvest recovery steps of biomolecule purification protocols.
1 . A method for separating protein produced in a mammalian cell from at least one other mammalian cell component comprising:
(a) introducing a continuous stream of a fluid comprising mammalian cells from a bioreactor into at least one decanter centrifuge;
(b) operating the decanter centrifuge, thereby separating fluid components into a low density composition and a high density composition;
(c) recycling the low density composition through the bioreactor;
(d) collecting the low density composition or recycling the low density composition through the decanter centrifuge; and
(e) recycling the high density composition through the decanter centrifuge,
wherein the fluid components comprise mammalian cells and components of mammalian cells, further wherein the low density composition comprises the protein.
2 . The method of claim 1 wherein the fluid comprising mammalian cells is a culture harvest from a bioreactor comprising cultured mammalian cells.
3 . The method of claim 1 wherein the protein is an antibody or an antigen-binding fragment thereof.
4 . The method of claim 3 wherein the antigen-binding fragment is a peptibody, an antibody derivative, an antibody analog, a fusion protein, a mutein, a bispecific protein or a multispecific protein.
5 . The method of claim 3 wherein the antibody is a whole antibody, a single-chain variable fragment, a Fv, a Fab, a Fab′, a F(ab′)2, a diabody, a triabody, a tetrabody, a BiTE, a Fd, a dAb, a minibody, or a maxibody.
6 . The method of claim 4 wherein the fusion protein comprises an antibody chain or an antigen-binding fragment thereof comprising light chain complementarity determining regions 1, 2 and 3 or heavy chain complementarity determining regions 1, 2 and 3.
7 . The method of claim 4 wherein the fusion protein is a Chimeric Antigen Receptor (CAR).
8 . The method of claim 1 wherein the protein is a granulocyte colony-stimulating factor, an erythropoiesis stimulating agent, a HER receptor, a cell adhesion molecule, a growth factor, an osteoinductive factor, insulin, a coagulation protein, a colony stimulating factor, a blood group antigen; a growth hormone, a growth hormone receptor, a T-cell receptor; a neurotrophic factor, a neurotrophin, a relaxin, an interferon, an interleukin, a viral antigen, a lipoprotein, an integrin, a rheumatoid factor, an immunotoxin, a surface-membrane protein, a transport protein, a homing receptor, an addressin, a regulatory protein, or an immunoadhesin.
9 . The method of claim 1 further comprising subjecting the low density composition to a second centrifugation step performed in a disk-stack centrifuge.
10 . The method of claim 1 further comprising subjecting the low density composition to a depth filtration step or a tangential flow filtration step.
11 . The method of claim 1 further comprising subjecting the low density composition to any one or more of a second centrifugation step, a filtration step, an acoustic wave separation step, a flocculation step or a precipitation step.
12 . A method for purifying a protein from a mammalian cell culture comprising:
(a) introducing a continuous stream of a cell culture fluid comprising mammalian cells into at least one decanter centrifuge;
(b) operating the decanter centrifuge, thereby separating fluid components into a low density fluid composition and a high density fluid composition;
(c) subjecting the low density fluid composition to depth filtration to yield a filtered low density fluid composition;
(d) recycling the filtered low density composition through the bioreactor to obtain a recycled low density composition;
(e) recycling the high density composition through the decanter centrifuge;
(f) contacting the recycled low density fluid composition with at least one chromatography medium; and
(g) collecting fluid containing the protein from the chromatography medium, thereby purifying the protein.
13 . The method of claim 12 further comprising subjecting the low density composition to any one or more of a second centrifugation step, a second filtration step, an acoustic wave separation step, a flocculation step or a precipitation step.
14 . The method of claim 12 wherein the protein is an antibody, an antigen-binding fragment thereof, an antibody derivative, an antibody analog, a fusion protein, a peptibody, a mutein, a chimera, a bispecific protein or a multispecific protein.
15 . The method of claim 14 wherein the antibody or antigen-binding fragment thereof is a single-chain variable fragment, a Fv, a Fab, a Fab′, a F(ab′)2, a diabody, a triabody, a tetrabody, a BiTE, a Fd, a dAb, a minibody, or a maxibody.
16 . The method of claim 14 wherein the chimera comprises an antibody or antigen-binding fragment thereof.
17 . The method of claim 14 wherein the chimera is a Chimeric Antigen Receptor.