PROTEIN PURIFICATION METHODS TO REDUCE ACIDIC SPECIES
The instant invention relates to the field of protein production and purification, and in particular to compositions and processes for controlling the amount of charge variants, aggregates, and fragments of a protein of interest, as well as host cell proteins, present in purified preparations by applying particular chromatography conditions during such protein purification.
1 . A method of making a pharmaceutical composition, comprising mixing
(a) a composition comprising adalimumab, wherein the composition comprises less than 10% total acidic species of adalimumab, wherein the acidic species of adalimumab correspond to the peaks that elute earlier than the main peak in a WCX-10 HPLC chromatogram of adalimumab, wherein the WCX-10 HPLC chromatogram is generated using a first mobile phase of 10 mM Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and wherein the WCX-10 HPLC chromatogram is generated using detection at 280 nm; and
(b) a pharmaceutically acceptable carrier, thereby making a pharmaceutical composition.
2 . The method of claim 1 , wherein the acidic species of adalimumab comprise a first acidic region (AR1) and a second acidic region (AR2).
3 . The method of claim 1 , wherein the adalimumab is produced in a mammalian host cell grown in cell culture.
4 . The method of claim 3 , wherein the mammalian host cell is selected from the group consisting of a CHO cell, an NS0 cell, a COS cell, and an SP2 cell.
5 . The method of claim 1 , wherein the low acidic species composition comprises less than 3.8% total acidic species of adalimumab.
6 . The method of claim 2 , wherein the low acidic species composition comprises 0.8% AR1 and 3.0% AR2.
7 . The method of claim 1 , wherein the low acidic species composition comprises less than 2.4% total acidic species of adalimumab.
8 . The method of claim 2 , wherein the low acidic species composition comprises 0.2% AR1 and 2.2% AR2.
9 . The method of claim 1 , wherein the low acidic species composition comprises 4.7%-8.3% total acidic species of adalimumab.
10 . The method of claim 1 , wherein adalimumab is present in the pharmaceutical composition at a concentration of 0.1-250 mg/mL.
11 . The method of claim 1 , further comprising filling a syringe with the pharmaceutical composition.
12 . The method of claim 1 , wherein the pharmaceutically acceptable carrier comprises one or more excipient selected from the group consisting of a buffer, a surfactant and a polyalcohol, or a combination thereof.
13 .- 18 . (canceled)
19 . A method of making a pharmaceutical composition, comprising mixing
(a) a composition comprising adalimumab, wherein the composition comprises less than 10% total acidic species of adalimumab, wherein the acidic species of adalimumab are quantified based on the relative area percent of peaks that elute earlier than the main peak in a WCX-10 HPLC chromatogram of adalimumab wherein the WCX-10 HPLC chromatogram is generated using a first mobile phase of 10 mM Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10 mM Sodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5) and wherein the WCX-10 HPLC chromatogram is generated using detection at 280 nm; and
(b) a pharmaceutically acceptable carrier comprising a surfactant and a polyalcohol, thereby making a pharmaceutical composition; and
filling a syringe with the pharmaceutical composition.
20 . The method of claim 19 , wherein the acidic species of adalimumab comprise a first acidic region (AR1) and a second acidic region (AR2).
21 . The method of claim 19 , wherein the adalimumab is produced in a mammalian host cell grown in cell culture.
22 . (canceled)
23 . The method of claim 19 , wherein the low acidic species composition comprises less than 3.8% total acidic species of adalimumab.
24 . The method of claim 20 , wherein the low acidic species composition comprises 0.8% AR1 and 3.0% AR2.
25 . The method of claim 19 , wherein the low acidic species composition comprises less than 2.4% total acidic species of adalimumab.
26 . The method of claim 20 , wherein the low acidic species composition comprises 0.2% AR1 and 2.2% AR2.
27 . The method of claim 19 , wherein the low acidic species composition comprises 4.7%-8.3% total acidic species of adalimumab.
28 .- 30 . (canceled)