DESC:The following specification particularly describes the invention and the manner in which it is to be performed:

ANALYTICAL ION-EXCHANGE METHOD

INTRODUCTION
The present invention relates to analytical methods for therapeutic proteins. In particular, the invention discloses an ion exchange method for improved separation and quantitation of the glycosylated isoforms of the therapeutic protein.
BACK GROUND
Erythropoietin and darbepoetin-alfa are two major erythropoiesis stimulating agents (ESA’s) known to stimulate production of red blood cells in mammals. Darbepoetin-alfa is a second generation ESA, derived from erythropoietin alfa (EPO). Darbepoetin-alfa comprises of two additional N-glycosylation sites as opposed to erythropoietin, and thus comprises a total of five N-glycosylation sites. The N-linked glycans can be bi-, tri-and tetra-antennary glycans. On each of these N-glycosylation sites, the attached glycan moiety is capped at the end with a sialic acid molecule. Also, there is a single O-glycosylation site present on darbepoetin-alfa. Hence, darbepoetin-alfa can contain up to 22 sialic acid molecules (up to four sialic acids on each of the five N-linked glycosylation site and up to two sialic acids on one O-linked glycosylation site).
The sialic acid content present on darbepoetin-alfa is known to impact in-vivo half-life and therapeutic activity. The glycosylated isoforms of darbepoetin-alfa contain 18-22 of sialic acids. Ensuring desirable percentage of darbepoetin-alfa sialylated isoforms is important for ensuring adequate therapeutic efficacy. Thus, analytical methods need to be designed to ensure resolution of the sialylated isoforms in the darbepoetin composition.
Further, the analytical method so designed and employed should be less labor intensive, and involve minimal sample processing, so as to be feasible for analysis of samples at various steps of the purification scheme. Also, preferably the method should be semi-quantitative to evaluate the percentage recovery of desired isoforms after each step of the purification scheme.
SUMMARY OF THE INVENTION
The present invention discloses an analytical anion exchange chromatography method for improved resolution of sialylated isoforms of darbepoetin alpha composition. The aforementioned anion exchange method comprises use of buffer system comprising an organic solvent, more particularly comprising acetonitrile. The analytical anion exchange method, of the present invention, is a quantitative method for calculating the % recovery of desired isoforms from each step of the purification scheme designed for purification of darbepoetin alpha composition.
In addition, buffer conditions and flow rate are optimized as a time dependent factor during the course of the development of chromatographic technique for analysis of various possible isoforms of darbepoetin alpha with improved resolution.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Overlay of chromatograms indicating the separation of undesired and desired isoforms. The profile to the left of red vertical line indicates the undersialylated (undesired) isoforms.
Figure 2. Separation of desired and undesired isoforms on SAX column. Panel A shows the overlay of CCCB and Chrom-I with the DS; panel B shows overlay of Chrom-II and TFF-I retentate with the DS; panel C shows overlay of TFF-II retentate and Chrom-III with the DS.
DETAILED DESCRIPTION
The present invention discloses an analytical anion exchange method for analyzing sialylated isoforms in darbepoetin-alfa composition, wherein the buffer used in the said anion exchange method comprises an organic solvent.
Often sialic acids occupy the terminal positions of the oligosaccharides on the glycosylation. Sialylation contributes extra negative charges for the glycoprotein and therefore, a strong anion exchange is an ideal chromatography medium to separate the sialylated isoforms.
Salt and pH gradients are the most common for ion exchange chromatography. In the present invention, the salt gradient method has been employed in combination with the organic modifier (acetonitrile) in the buffer system. The iso-electric point of darbepoetin alpha molecule is around 3.0-3.9 (range is for differently sialylated isoforms) and the pH of the mobile phases was maintained at 3.5.
It is shown that the selectivity of ion-exchange chromatography can be significantly improved by adding organic solvents to the aqueous mobile phase. By increasing the amount of solvents the ions undergo maximum retention on the media. The phenomena is attributed to the competing effects of enhanced salvation of the buffer ions and reduced solute ionization.
In an embodiment of the invention, the darbepoetin-alfa composition can be an in-process sample or the drug substance.
In yet another embodiment of the invention, the buffer used in the said anion exchange method comprise acetonitrile solvent at 10% v/v of the total buffer introduced onto the column from the time when first isoform of darbepoetin starts to elute from column and till the time the last isoform is eluted out.
In a further embodiment of the invention, the organic solvent used is acetonitrile.
In an embodiment of the invention, the method can be used for quantifying the % of desired isoforms in the darbepoetin alpha samples at different stages of purification.
Definitions:
The term “Isoforms” refers to the protein product variants, which differs in post-translational modifications. Isoforms may be generated due to differential glycosylation, deamidation, oxidation etc. In particular, for the purpose of the present invention the term “isoforms” referred to are the product variants which differs in the no. of sialic acids present on terminal end of the glycan chain. More particularly, in the present invention the term “desired isoforms” are the isoforms having 18-22 number of sialic acids and the “undesired isoforms” refer to isoforms containing lower number of sialic acids. The “undesired isoforms” according to the methods of present invention elute before the “desired isoforms” in the anion exchange chromatogram.
“In-process samples” refer to the fractions obtained from the purification/chromatographic steps (chrom 1 to chrom 3) containing the partially purified darbepoetin-alfa. The in-process samples are further introduced onto the other purification/chromatographic steps to obtain darbepoetin purified to a desirable level which is then termed as drug substance (DS).
The term “resolution” refers to a quantitative measure of the peak spacing in a liquid chromatography (LC) separation. The most common formula for measuring resolution (Rs) is
Rs = 2 (t2 - t1) / (w1 - w2)
Where, t1 and t2 are the retention times of the two peaks of interest, and w1 and w2 are the peak widths measured at the baseline between tangents drawn to the peak sides.
Examples
Example 1: Expression and harvest of darbepoetin-alfa
Expression of darbepoetin-alfa in mammalian cells was accomplished as described in EP2456871 (B1), which is incorporated herein as reference. Harvested cell culture broth (CCB) was clarified by centrifugation to obtain clarified cell culture broth (CCCB). The CCCB was subjected to different chromatographic separation stages (anion, cation and bind/elute) to finally obtain the purified drug substance (DS). The present method employed Thermo Scientific Dionex ProPac© SAX-10; 250 x 4 mmID column. The mobile phases used for the strong anion exchange method were; A: 25 mM Sodium acetate (pH 3.5); B: 25 mM Sodium acetate + 250 mM sodium chloride (pH 3.5); and C: 100% acetonitrile (flowing at 10% into the column). The use of step gradient was found to improve the separation and resolution of the sialylated isoforms. The gradient program of the present invention is given below –

Time (mins) % of Mobile Phase-A % of Mobile Phase-B % of Mobile Phase-C
0-12.2 100 0 0
12.2-14 91.5 8.5 10
14-14.2 91.5 8.5 10
14.2-16 89 11 10
16-16.2 89 11 10
16.2-18 87 13 10
18-18.2 87 13 10
18.2-20 84.5 15.5 10
20-20.2 84.5 15.5 10
20.2-22 81 19 10
22-22.2 81 19 10
22.2-24 77.5 22.5 10
24-30 77.5 22.5 10
30-32 70 30 0
32-34 0 100 0
34-40 100 0 0

Table 1: Chromatographic gradient employed
A fluorescence-based detection system is attached to the chromatographic system, for analyzing the isoforms in samples at different purification stages.
Example 2: Quantitation of percentage isoforms
The CCCB was purified to obtain an eluate, which was further purified by a number of purification/chromatographic steps to yield a drug substance (DS). In particular, the said eluate was introduced onto the different ion exchange chromatography steps, along with two tangential flow filtration steps TFF-1 and TFF-II. The samples obtained during and after each such step are termed CCCB, Chrom 1, Chrom 2, TFF-I, TFF II, Chrom3, Chrom4 and DS. They were evaluated for recovery of the desired isoforms at each step. For each sample, percentage of the desired and undesired isoform (all pre-peaks eluted before the desired isoforms), is represented in Table-7.
Sample name % Undesired Isoforms % Desired Isoforms
CCCB 79.59 20.42
Chrom I 49.2 50.8
Chrom 2 44.6 55.4
TFF-I retentate 47.44 52.55
TFF-II retentate 41.38 58.61
Chrom 3 6.61 93.38
DS 4.27 95.74

Table-2: Percentage of desired and undesired isoforms in samples at different purification stages
,CLAIMS:WE CLAIM:

1. An anion-exchange method for quantifying sialylated isoforms in a darbepoetin-alfa composition, wherein the method comprises a salt gradient program in combination with use of an organic modifier in the buffer system.
2. A method according to claim 1, wherein the organic modifier is acetonitrile.
3. A method according to claim 1, wherein the pH of the mobile phase is maintained at 3.5.
4. A method according to claim 1, wherein the said buffer system comprises acetonitrile at 10% v/v of the total buffer introduced onto a chromatographic column.
5. A method according to claim 4, wherein said percentage of acetonitrile is maintained in the chromatographic column at least from the time when the first isoform of darbepoetin starts to elute from column and till the time the last isoform is eluted out.