The present invention relates to the field of recombinant protein manufacturing.
More particularly, the present invention provides a method for reducing host cell protein
5 content in a protein preparation comprising a protein of interest recombinantly produced
in a host cell in the manufacturing process of proteins intended for administration to a
patient, such as therapeutic or diagnostic proteins.
Host Cell Proteins (HCPs) are proteins of the host cells that are involved in cell
maintenance and growth, and protein synthesis and processing. However, in the realm of
10 therapeutic or diagnostic proteins, the presence ofHCPs threatens product quality and
patient safety by posing concerns such as aggregation, product fragmentation by catalytic
activity and/or immunogenicity. Hence, HCPs are identified as a critical quality attribute
(CQA) of protein formulations. The formation of undesired aggregates and product
fragmentation require additional purification steps to reduce/remove HCPs and these
15 additional purification steps often result in reduced yield of the desired protein and
increased overall manufacturing costs.
The challenges of eliminating HCPs from manufacturing processes, and attempts
to improve the processes to reduce HCPs have been disclosed, for example as set forth in
Gilgunn et al; Goey et al., Biotechnology Advances 36 (2018) 1223-1237; and Current
20 Opinion in Chemical Engineering 2018, 22:98-106. However, these processes to remove
HCPs have limitations. For example, in some instances, these disclosures demonstrate
one or more of, incomplete removal ofHCPs, inconsistency in processes in removal of
HCPs leading to aggregation, co-purification of the desired proteins and HCPs, impaired
product function, immunogenicity concerns in patients, and I or reduced pharmacokinetic
25 properties such as half-life. Furthermore, the processes developed to remove HCPs often
require for example, the need to work with increased volumes and additional purification
steps, often resulting in increased manufacturing costs and reduced yield. In some
instances, the applicability of the method is limited to a specific molecule and I or format.
As such, there remains a need for alternative methods of reducing HCPs in the
30 purification process of therapeutic or diagnostic proteins. Such alternative methods
reduce HCPs preferably without affecting product stability, yield, or cost to ultimately
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maintain product quality and is amenable to large scale manufacturing and ensuring
patient safety.
Accordingly, the present invention addresses one or more of the above problems
by providing alternative methods of reducing HCPs in the preparation of therapeutic or
5 diagnostic proteins. The methods of the present invention provide reproducible methods
that are highly effective in removing HCPs, whilst preserving protein stability, reducing
aggregation, maintaining product yield and has a potential to lower immunogenicity risk.
Such methods can effectively remove HCPs without requiring increased protein
preparation volume. Surprisingly, the methods of the present invention achieved HCP
10 counts well below the industry acceptable standards of< 100 ppm. Surprisingly, in
embodiments the methods of the present invention achieved HCP counts of< 50 ppm
whilst preserving protein stability, reducing aggregation, and maintaining product yield.
More surprisingly, other embodiments of the present invention achieved HCP counts of<
20 ppm whilst preserving protein stability, reducing aggregation, and maintaining product
15 yield. Furthermore, embodiments of the present invention provide methods ofHCP
removal that are applicable to a broad range of molecules. Other embodiments of the
present invention enable the elimination of additional purification steps, resulting in a
reduction in batch processing time, and decreased manufacturing costs.
Accordingly, particular embodiments, provide a method of reducing host cell
20 protein content in a protein preparation comprising a protein of interest recombinantly
produced in a host cell comprising, subjecting the protein preparation recombinantly
produced in a host cell to an affinity chromatography column, eluting the protein of
interest from the chromatography column with a combination of acids comprising of a
weak acid and a strong acid to obtain an eluate comprising the protein of interest, raising
25 the pH of the eluate to above about pH 6.0, subjecting the eluate to a depth filter, and
obtaining a filtered protein preparation. In some embodiments the ionic strength of the
eluate from the step of raising the pH to above about pH 6.0, is about 10 mM to about
45 mM. In some embodiments, the weak acid has no more than one pKa value less than
7.0, and the strong acid has no more than one pKa value less than 7.0. Preferably, the
30 host cell protein content in the filtered protein preparation is reduced. More preferably,
the host cell protein content in the filtered protein preparation is reduced to less than
about 100 ppm, to less than about 50 ppm, or to less than about 20 ppm.
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Accordingly, in particular embodiments, provided is a method of reducing host
cell protein content in a protein preparation comprising a protein of interest
recombinantly produced in a host cell comprising, subjecting the protein preparation
recombinantly produced in a host cell to an affinity chromatography column, eluting the
5 protein of interest from the chromatography column with a combination of acids
comprising of a weak acid and a strong acid to obtain an eluate comprising the protein of
interest, performing viral inactivation on the eluate, raising the pH of the eluate to above
about pH 6.0, subjecting the eluate to a depth filter, and obtaining a filtered protein
preparation. In some embodiments the ionic strength of the eluate from the step of raising
10 the pH to above about pH 6.0, is about 10 mM to about 45 mM. In some embodiments,
the weak acid has no more than one pKa value less than 7.0, and the strong acid has no
more than one pKa value less than 7.0. Preferably, the host cell protein content in the
filtered protein preparation is reduced. More preferably, the host cell protein content in
the filtered protein preparation is reduced to less than about 100 ppm, to less than about
15 50 ppm, or to less than about 20 ppm.
Accordingly, in particular embodiments, provided is a method of reducing host
cell protein content in a protein preparation comprising a protein of interest
recombinantly produced in a host cell comprising, subjecting the protein preparation
recombinantly produced in a host cell to an affinity chromatography column, eluting the
20 protein of interest from the chromatography column with a combination of acids
comprising of a weak acid and a strong acid to obtain an eluate comprising the protein of
interest, performing viral inactivation comprising adjusting the pH of the eluate from said
step of eluting the protein from the chromatography column, to below about pH 4.0, and
wherein the eluate is maintained at below about pH 4.0 for about 0 minutes to about 180
25 minutes, raising the pH of the eluate to above about pH 6.0, subjecting the eluate
comprising the protein to a depth filter, and obtaining a filtered protein preparation. In
some embodiments the ionic strength of the eluate from the step of raising the pH to
above about pH 6.0, is about 10 mM to about 45 mM. Preferably, the host cell protein
content in the filtered protein preparation is reduced. More preferably, the host cell
30 protein content in the filtered protein preparation is reduced to less than about 100 ppm,
to less than about 50 ppm, or to less than about 20 ppm.
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Accordingly, in particular embodiments, provided is a method of reducing host
cell protein content in a protein preparation comprising a protein of interest
recombinantly produced in a host cell comprising, subjecting the protein preparation
recombinantly produced in a host cell to an affinity chromatography column, eluting the
5 protein of interest from the chromatography column with a combination of acids
comprising of a weak acid and a strong acid to obtain an eluate comprising the protein of
interest, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or
lactic acid, adjusting the pH of the eluate comprising the protein from said step of eluting
the protein from the chromatography column, to below about pH 4.0, and wherein the
10 eluate is maintained at below about pH 4.0 for about 0 minutes to about 180 minutes,
raising the pH of the eluate to above about pH 6.0, subjecting the eluate comprising the
protein to a depth filter, and obtaining a filtered protein preparation. In some
embodiments the ionic strength of the eluate from the step of raising the pH to above
about pH 6.0, is about 10 mM to about 45 mM. Preferably, the host cell protein content
15 in the filtered protein preparation is reduced. More preferably, the host cell protein
content in the filtered protein preparation is reduced to less than about 100 ppm, to less
than about 50 ppm, or to less than about 20 ppm.
In some embodiments of the invention, the present disclosure provides a method
of reducing host cell protein content in a protein preparation comprising a protein of
20 interest recombinantly produced in a host cell comprising, subjecting the protein
preparation recombinantly produced in a host cell to an affinity chromatography column,
eluting the protein of interest from the chromatography column with a combination of
acids comprising of a weak acid and a strong acid to obtain an eluate comprising the
protein of interest, wherein the weak acid is acetic acid and the strong acid is phosphoric
25 acid, wherein the concentration of the acetic acid is about 20 mM, and wherein the
concentration of the phosphoric acid is about 5 mM to about 10 mM, adjusting the pH of
the eluate comprising the protein from said step of eluting the protein from the
chromatography column, to below about pH 4.0, and wherein the eluate is maintained at
below about pH 4.0 for about 0 minutes about 180 minutes, raising the pH of the eluate to
30 above about pH 6.0, subjecting the eluate comprising the protein to a depth filter, and
obtaining a filtered protein preparation. In some embodiments the ionic strength of the
eluate from the step of raising the pH to above about pH 6.0, is about 10 mM to about 45
5
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mM. Preferably, the host cell protein content in the filtered protein preparation is
reduced. More preferably, the host cell protein content in the filtered protein preparation
is reduced to less than about 100 ppm, to less than about 50 ppm, or to less than about 20
ppm.
In some embodiments of the invention, the present disclosure provides a method
of reducing host cell protein content in a protein preparation comprising a protein of
interest recombinantly produced in a host cell comprising, subjecting the protein
preparation recombinantly produced in a host cell to an affinity chromatography column,
eluting the protein of interest from the chromatography column with a combination of
10 acids comprising of a weak acid and a strong acid to obtain an eluate comprising the
protein of interest, wherein the weak acid is acetic acid and the strong acid is lactic acid,
wherein the concentration of the acetic acid is about 20 mM, and wherein the
concentration of the lactic acid is about 5 mM, adjusting the pH of the eluate comprising
the protein from said step of eluting the protein from the chromatography column, to
15 below about pH 4.0, and wherein the eluate is maintained at below about pH 4.0 for about
0 minutes to about 180 minutes, raising the pH of the eluate to above about pH 6.0,
subjecting the eluate comprising the protein to a depth filter, and obtaining a filtered
protein preparation. In some embodiments the ionic strength of the eluate from the step
of raising the pH to above about pH 6.0, is about 10 mM to about 45 mM. Preferably,
20 the host cell protein content in the filtered protein preparation is reduced. More
preferably, the host cell protein content in the filtered protein preparation is reduced to
less than about 100 ppm, to less than about 50 ppm, or to less than about 20 ppm.
In some embodiments, the present disclosure provides a method of reducing host
cell protein content in a protein preparation comprising a protein of interest
25 recombinantly produced in a host cell comprising, subjecting the protein preparation
recombinantly produced in a host cell to an affinity chromatography column, eluting the
protein of interest from the chromatography column with a combination of acids
comprising of a weak acid and a strong acid to obtain an eluate comprising the protein of
interest, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or
30 lactic acid, adjusting the pH of the eluate comprising the protein from said step of eluting
the protein from the chromatography column, wherein said step of adjusting the pH of the
eluate comprises adding about 20 mM HCl to the eluate, wherein the pH of the eluate is
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adjusted to about pH 3.3 to about pH 3.7, and wherein the eluate is maintained at about
pH 3.3 to about pH 3. 7 for about 0 minutes to about 180 minutes, raising the pH of the
eluate to above about pH 6.0, subjecting the eluate comprising the protein to a depth
filter, and obtaining a filtered protein preparation. In some embodiments the ionic
5 strength of the eluate from the step of raising the pH to above about pH 6.0, is about 10
mM to about 45 mM. Preferably, the host cell protein content in the filtered protein
preparation is reduced. More preferably, the host cell protein content in the filtered
protein preparation is reduced to less than about 100 ppm, to less than about 50 ppm, or to
less than about 20 ppm.
10 In some embodiments, the present disclosure provides a method of reducing host
cell protein content in a protein preparation comprising a protein of interest
recombinantly produced in a host cell comprising, subjecting the protein preparation
recombinantly produced in a host cell to an affinity chromatography column, eluting the
protein of interest from the chromatography column with a combination of acids
15 comprising of a weak acid and a strong acid to obtain an eluate comprising the protein of
interest, wherein the weak acid is acetic acid and the strong acid is phosphoric acid or
lactic acid, adjusting the pH of the eluate comprising the protein from said step of eluting
the protein from the chromatography column, wherein said step of adjusting the pH of the
eluate comprises adding about 20 mM HCl to the eluate, wherein the pH of the eluate is
20 adjusted to about pH 3.5, and wherein the eluate is maintained at about pH 3.5 for about 0
minutes to about 180 minutes, raising the pH of the eluate to above about pH 6.0,
subjecting the eluate comprising the protein to a depth filter, and obtaining a filtered
protein preparation. In some embodiments the ionic strength of the eluate from the step
of raising the pH to above about pH 6.0, is about 10 mM to about 45 mM. Preferably,
25 the host cell protein content in the filtered protein preparation is reduced. More
preferably, the host cell protein content in the filtered protein preparation is reduced to
less than about 100 ppm, to less than about 50 ppm, or to less than about 20 ppm.

CLAIMS
1. A method of reducing host cell protein content in a protein preparation comprising a
protein of interest recombinantly produced in a host cell, the method comprising the
steps of:
5 a. subjecting the protein preparation to an affinity chromatography column;
b. eluting the protein of interest from the chromatography column with a
combination of acids comprising of a weak acid and a strong acid to obtain an
eluate comprising the protein of interest;
c. raising pH of the eluate to above about pH 6.0; and
10 d. subjecting the eluate to a depth filter and obtaining a filtered protein preparation.
2. The method of Claim 1, wherein the chromatography column comprises a Protein A,
Protein G or Protein L affinity chromatography column.
3. The method of Claim 1, wherein the weak acid has no more than one pKa value less
than 7.0, and the strong acid has no more than one pKa value less than 7.0.
15 4. The method of Claim 1, wherein the weak acid is acetic acid and the strong acid is
phosphoric acid or lactic acid.
5. The method of claim 4, wherein the concentration of the acetic acid is about 20 mM,
and wherein the strong acid is phosphoric acid and wherein the concentration of the
phosphoric acid is about 5 mM to about 10 mM.
20 6. The method of Claim 4, wherein the concentration of the acetic acid is about 20 mM,
and wherein the strong acid is lactic acid and wherein the concentration of the lactic
acid is about 5 mM.
7. The method of Claim 1, further comprising a step of performing viral inactivation.
8. The method of Claim 1, further comprising a step of performing viral inactivation,
25 comprising adjusting the pH of the eluate from said step of eluting the protein from
the chromatography column, to below about pH 4.0, and wherein the eluate is
maintained at below about pH 4.0 for about 0 minutes to about 180 minutes.
9. The method of claim 8, wherein said step of adjusting the pH of the eluate comprises
adjusting the pH of the eluate to about pH 3.3 to about pH 3.7
30 10. The method of claim 9, wherein the pH of the eluate is adjusted to about pH 3.5.
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11. The method of any one of claim 8 to 10, wherein adjusting the pH of the eluate
comprises adding any one ofHCl, phosphoric acid, or a combination of acetic acid
and phosphoric acid.
12. The method of claim 1, wherein said step of raising the pH of the eluate comprises
5 raising the pH to about pH 6.5 to about pH 7.5.
13. The method of claim 12, wherein the pH of the eluate is raised to about pH 7.0.
14. The method of any one of claim 12 or 13, wherein the step of raising the pH of the
eluate comprises adding Tris.
15. The method of any one of claims 1 to 14, wherein the eluate at said step of raising the
10 pH to above about 6.0 has an ionic strength of about 10 mM to about 45 mM.
16. The method of any one of claims 1 to 15, further comprising a step of subjecting the
depth filtered protein preparation to ion exchange chromatography.
17. The method of any one of claims 1 to 16, wherein the host cell protein content in the
filtered protein preparation is reduced to less than 100 ppm.
15 18. The method of any one of claims 1 to 16, wherein the host cell protein content in the
filtered protein preparation comprises PLBL2, and wherein the PLBL2 is reduced to
less than 100 ppm.
19. The method of any one of claims 1 to 18, wherein the protein preparation comprises a
harvested cell culture fluid, a capture pool, or a recovered protein pool.
20 20. The method of any one of claims 1 to 19, wherein the protein is a therapeutic or
diagnostic protein.
21. The method of any one of claims 1 to 19, wherein the protein is an antibody, Fe
Fusion protein, peptide, an immunoadhesin, an enzyme, a growth factor, a receptor,
a hormone, a regulatory factor, a cytokine, an antigen, a peptide, or a binding agent.
25 22. The method of Claim 21, wherein the antibody is a monoclonal antibody, a
chimeric antibody, a humanized antibody, a human antibody, a bispecific
antibody, or an antibody fragment.
23. The method of Claim 22, wherein the antibody is an IgG1 antibody.
24. The method of any one of claims 1 to 23, wherein the protein is an anti-SARS-COV-2
30 antibody.
25. The method of claim 24, wherein the anti-SARS-COV-2 antibody is bamlanivimab.
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26. The method of claim 24, wherein the anti-SARS-COV-2 antibody comprises a VH of
SEQ ID NO: 1 and a VL of SEQ ID NO: 2.
27. The method of claim 24, wherein the anti-SARS-COV-2 antibody comprises a HC of
SEQ ID NO: 3 and a LC of SEQ ID NO: 4.
5 28. The method of claim 24, wherein the anti-SARS-COV-2 antibody is etesevimab.
29. The method of claim 24, wherein the anti-SARS-COV-2 antibody comprises a VH of
SEQ ID NO: 5 and a VL of SEQ ID NO: 6.
30. The method of claim 24, wherein the anti-SARS-COV-2 antibody comprises a HC of
SEQ ID NO: 7 and a LC of SEQ ID NO: 8.
10 31. The method of claim 24, wherein the anti-SARS-COV-2 antibody is bebtelovimab.
32. The method of claim 24, wherein the anti-SARS-COV-2 antibody comprises a VH of
SEQ ID NO: 9 and a VL of SEQ ID NO: 10.
33. The method of claim 24, wherein the anti-SARS-COV-2 antibody comprises a HC of
SEQ ID NO: 11 and a LC of SEQ ID NO: 12.
15 34. A method of reducing host cell protein content in an anti-SARS-COV-2 antibody
preparation recombinantly produced in a host cell comprising:
a. subjecting the anti-SARS-COV-2 antibody preparation recombinantly produced in
a host cell to a Protein A affinity chromatography column;
b. eluting the anti-SARS-COV-2 antibody with a combination of acids comprising of
20 acetic acid and phosphoric acid or a combination of acetic acid and lactic acid to
obtain an eluate comprising the anti-SARS-COV-2 antibody;
c. adjusting the pH of the eluate comprising the anti-SARS-COV-2 antibody by
addition of about 20 mM HCl, wherein the pH is lowered to about pH 3.3 to about
pH 3.7, and wherein the eluate is maintained at about pH 3.3 to about pH 3.7 for
25 about 0 minutes to about 180 minutes;
30
d. raising the pH of the eluate comprising the anti-SARS-COV-2 antibody by
addition of about 250 mM Tris Buffer, wherein the pH is raised to about pH 6.5 to
about pH 7.5; and
e. subjecting the eluate comprising the anti-SARS-COV-2 antibody to a depth filter,
and obtaining a filtered anti-SARS-COV-2 antibody preparation,
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wherein host cell protein content in the filtered anti-SARS-COV-2 antibody
preparation is reduced to about 0 ppm to about 20 ppm, and wherein the anti-SARSCOV-
2 antibody is an IgG1 antibody.
35. The method of claim 34, wherein the combination of acids of step b comprises 20 mM
5 acetic acid and 5 mM phosphoric acid, or 20 mM acetic acid and 5 mM phosphoric
acid, or 20 mM acetic acid and 5 mM lactic acid.
36. The method of claim 34, wherein step c of adjusting the pH of the eluate comprises
adjusting the pH of the eluate to about 3.5.
37. The method of any one of claims 34 to 36, wherein said step of adjusting the pH of
10 the eluate comprising the anti -SARS-COV -2 antibody by addition of about 20 mM
HCl achieves viral inactivation.
38. The method of claim 34, wherein said step of raising the pH of the eluate comprises
raising said pH to about pH 7.25.
39. The method of any one of claims 34 or 38, wherein the eluate after said step of raising
15 the pH has an ionic strength of about 10 mM to about 45 mM.
40. The method of any one of Claims 34 to 39, further comprising a step of subjecting the
depth filtered anti-SARS-COV-2 antibody preparation to ion exchange
chromatography.
41. The method of any one of claims 34 to 40, wherein the anti -SARS-COV -2 antibody is
20 bamlanivimab.
42. The method of any one of claims 34 to 40, wherein the anti-SARS-COV-2 antibody
comprises a VH of amino acid SEQ ID NO: 1 and a VL of amino acid SEQ ID NO: 2.
43. The method of any one of claims 34 to 40, wherein the anti -SARS-COV -2 antibody
comprises a HC of amino acid SEQ ID NO: 3 and a LC of amino acid SEQ ID NO: 4.
25 44. The method of any one of claims 34 to 40, wherein the anti-SARS-COV-2 antibody is
etesevimab.
45. The method of claim 34 to 40, wherein the anti-SARS-COV-2 antibody comprises a
VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 6.
46. The method of claim 34 to 40, wherein the anti-SARS-COV-2 antibody comprises a
30 HC of SEQ ID NO: 7 and a LC of SEQ ID NO: 8.
47. The method of any one of claims 34 to 40, wherein the anti-SARS-COV-2 antibody is
bebtelovimab.
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48. The method of any one of claims 34 to 40, wherein the anti-SARS-COV-2 antibody
comprises a VH of amino acid SEQ ID NO: 9 and a VL of amino acid SEQ ID NO:
10.
49. The method of any one of claims 34 to 40, wherein the anti-SARS-COV-2 antibody
5 comprises a HC of amino acid SEQ ID NO: 11 and a LC of amino acid SEQ ID NO:
12.
50. The method of any one of claims 1 to 49, wherein the depth filter comprises COSP,
XOSP, XOHC, Emphaze AEX Hybrid Purifier, or Zeta Plus (ZB Media).
51. The method of any one of Claims 1 to 50, wherein the host cell is a mammalian
10 cell.
15
20
25
30
52. The method of claim 51, wherein the mammalian cell is a CHO cell.
53. A composition produced by the method of any one of claims 1-52.
54. The composition of claim 53, wherein the host cell protein content in the
composition is less than about 100 ppm.