Field of the Invention

The present invention relates to a process of increasing protein recovery and quality of the protein of interest by pre-treatment of the depth filter. More specifically the present invention relates to a process to increase the recovery of Etanercept by washing the depth filter with a high concentration salt solution, prior to the clarification process.

Background of the Invention

Large scale production of proteins for biopharmaceutical applications is a complex process that comprises of multiple steps, such as, filtration, centrifugation, and various chromatographic steps etc. Inundated by the complexity of each step, as well complexity of protein chemistry of the protein of interest, each step could be critical in improving end product recovery in both qualitative as well as quantitative terms.

Clarification of cell culture harvests and high-solid feedstocks often face the challenge associated with large harvest volumes from the upstream production batches, furthermore these batches often contain cells in high densities. Hence, often requiring a primary, as well as secondary clarification steps prior to the subsequent downstream operations.

High titers of protein of interest during biopharmaceutical processes may facilitate increased cell culture productivity. However, as a drawback high density cell culture also produces cell culture harvests with greater amounts of biomass and cell debris. Cell culture harvest media, which act as the feed stream for further filtration processes, containing such larger amounts of biomass and cell debris can lead to high turbidity after centrifugation. This poses significant challenge for the Secondary depth filters to handle the primary clarification of high-solid feed-streams. As a consequence, this often leads to reduced throughput for the secondary clarification, when using a depth filter. Furthermore, certain proteins are pre-disposed to sticking to the depth filter matrix, which may pose a major concern for effective utilization of depth filter, as well as protein yield in quality and quantity at the end of the manufacturing process. Hence, there is a need to improve the clarification process by increasing the life and efficiency of secondary depth filter in removing large amounts of cell culture biomass containing of impurities such as cell mass, cell debris, particle suspensions, host cell protein (HCP) and host cell DNA (HCDNA) along with the protein of interest at relatively low cost. The removals of such impurities are advantageous as it has potential to impact product characteristics, product stability, product safety and product efficacy. Summary of the Invention

In one embodiment the present invention relates to a process of increasing the recovery of protein by using clarification through depth filtration process, wherein the depth filter is washed or conditioned with a high concentration salt solution prior to the clarification step. In another embodiment the present invention relates to a process of increasing recovery of a protein by using one or more depth filters, the process comprising the steps of:

a) Pre-treating the depth filter by contacting a high concentration salt solution with the depth filter

b) clarifying the cell culture harvest comprising the protein of interest by using depth filters;

c) recovering the protein of interest by washing with equilibration buffer. In another embodiment the present invention relates to a process of increasing the recovery of Etanercept by using one or more depth filters, the process comprising the steps of: a) Pre-treating the depth filter by contacting a high concentration salt solution with the depth filter

b) clarifying the cell culture harvest comprising the protein of interest by using depth filters;

c) recovering the protein of interest by washing with equilibration buffer. In certain embodiments the step (a) could be optionally preceded by additional steps of contacting the secondary filter with low conductivity aqueous solution comprising pre- treating agents, wherein the conductivity is less than 100, or 50 or 20 or 10 or 5 mS/cm. In certain embodiments the protein recovery is improved by at least about 1.1 times compared to when the process is performed in absence of the high salt concentration pre- treatment.

In an embodiment the depth filters of the invention comprises of primary depth filter and secondary depth filter.

In yet another embodiment the present invention relates to a process to recycle the secondary depth filter by washing the depth filter with high concentration of salt solution prior to performing the clarification steps.

Detailed description of the Invention

Definition;

The terms“contaminant,”“impurity,” and“debris,” as used interchangeably herein, refer to any foreign or objectionable material, including a biological macromolecule such as a DNA, RNA, one or more host cell proteins (HCPs or CHOPs), endotoxins, viruses, lipids and one or more additives which may be present in a sample containing a protein or polypeptide of interest (e.g., an antibody or fusion protein) being separated from one or more of the foreign or objectionable molecules using a stimulus responsive polymer according to the present invention. In some embodiments, a stimulus responsive polymer described herein binds and precipitates a protein or polypeptide of interest from a sample containing the protein or polypeptide of interest and one or more impurities. In other embodiments, a stimulus responsive polymer described herein binds and precipitates one or more impurities, thereby to separate the polypeptide or protein of interest from one or more impurities.

As used herein the term washing or conditioning has been used synonymously with the term pre-treatment.

As used herein the term“about” has been defined to ±15% variability. As used herein the term“depth filter” (e.g., gradient-density depth filter) achieves filtration within the depth of the filter material. A common class of such filters are those that comprise a random matrix of fibers bonded (or otherwise fixed), to form a complex, tortuous maze of flow channels. Particle separation in these filters generally results from entrapment by or adsorption to, the fiber matrix. The most frequently used depth filter media for bioprocessing of cell culture broths and other feed stocks consists of cellulose fibers and a positively charged resin binder. Depth filter media, unlike absolute filters, retain particles throughout the porous media allowing for retention of particles both larger and smaller than the pore size. Particle retention is thought to involve both size exclusion and adsorption through hydrophobic, ionic and other interactions. The fouling mechanism may include pore blockage, cake formation and/or pore constriction. Depth filters are advantageous because they remove contaminants and also come in disposable formats thereby eliminating the validation issues.

In some embodiments, a depth filter, as described herein, is used for clarification, following which the clarified cell culture can continuously flow onto the next step in the purification process.

The present invention provides an increased recovery of Etanercept from cell harvest through depth filtration process by washing the depth filter with high concentration of salt solution prior to performing the clarification. In this process conditioning the secondary depth filter with high concentration of salt solution results in improved yields.

The depth filter consists of primary and secondary filters. Primary removes cells and cellular debris and secondary filter removes process and product related impurities.

In another embodiment the present invention relates to a process of increasing recovery of a protein by using one or more depth filters, the process comprising the steps of:

a) Pre-treating the depth filter by contacting a high concentration salt solution with the depth filter

b) clarifying the cell culture harvest comprising the protein of interest by using depth filters;

c) recovering the protein of interest by washing with equilibration buffer. In another embodiment the present invention relates to a process to increase the recovery of Etanercept by using one or more depth filters, the process comprising the steps of: a) Pre-treating the depth filter by contacting a high concentration salt solution with the depth filter

b) clarifying the cell culture harvest comprising the protein of interest by using depth filters;

c) recovering the protein of interest by washing with equilibration buffer. In certain embodiments the step (a) could be optionally preceded by additional steps of contacting the secondary filter with low conductivity aqueous solution pre-treating agents other, wherein the conductivity is less than 100, or 50 or 20 or 10 or 5 mS/cm.

In certain embodiment the low conductivity aqueous is WFI (Water for Injection).

In certain embodiment the protein recovery is greater than about 69%.

In certain embodiments the protein recovery in improved by at least about 1.1 times compared to when the process is performed in absence of the high salt concentration pre- treatment.

In another embodiment the primary depth filter pore size is at least from about 9µ to about 0.1 µ. In more another embodiment the primary depth filter pore size is at least from about 2 µ to about 0.5 µ.

In another embodiment the primary depth filter is selected from D0HC, C0HC, B1HC, A1HC, X0HC and F0HC

The primary depth filtration devices are able to filter cell culture harvest comprising a high solids feeds containing particles having a particle size at flow rate selected from about 10 to about 600 LMH(liters/square meter/hour)

The filtrate of primary filter comprises turbidity about from 2 to 40 NTU, 30NTU, high molecular weight impurities about from 10 to 30%, from 12 to 25% and low molecular weight impurities from about 3 to 30 %. In another embodiment the secondary depth filter pore size is lower or equal to that of the primary filter. In certain embodiment the secondary depth filter pore size ranges from about 0.4 µ to less than 0.1µ. In another embodiment the secondary depth filter pore size is less than about 0.1µ. In certain embodiment the secondary depth filter pore size ranges from about 2 µ to less than 0.1µ.

In certain embodiment the secondary depth filtration are able to filter filtrate of primary filter, as described, at a flow rate from about 10 to about 600 LMH (liters/square meter/hour)

In certain embodiment the primary and secondary depth filters are washed with water for injection (WFI). Flux of WFI is 20 LMH for not less than 60 L/ m2. The flux of WFI could be 70 LMH for not less than 90 L/ m2.

In certain embodiment primary and secondary filter are conditioned with high salt buffer at flux of 70 LMH for not less than 20 L/ m2.

In yet another embodiment at least the secondary filter is conditioned with high salt buffer at flux of 70 LMH for not less than 20 L/ m2.

In another embodiment the primary and secondary filters are treated with suitable buffer at flux of 70 LMH for not less than 50 L/ m2. In another embodiment the primary and secondary filters are treated with suitable buffer at flux of 70 LMH for not less than 30 L/ m2 using peristaltic pump. In another embodiment the high salt buffer comprises Tris and sodium chloride at pH 8 to pH10 with conductivity at least by 100mS/cm, wherein Tris is present at a concentration range of about 5 mM to 30 mM, and sodium chloride is present at a concentration range of about 1 mM to 5 mM.

In certain embodiments the high salt buffer comprises phosphate buffer and sodium chloride at about pH 6 to pH8 with a conductivity of at least 100mS/cm. In certain embodiment the high salt buffer concentration is suitable enough to saturate the filter. In certain embodiment the high salt buffer concentration is selected from 10mM, 50mM, 100mM, 150 mM, 200mM, 250mM, 300mM, 2M, and 2.5M.

In another embodiment the high salt buffer comprises 20 mM Tris and 2 M sodium chloride. In another embodiment the high salt buffer comprises 50 mM Tris and 2 M sodium chloride. In such embodiment the pH is selected in the range of about pH 7 to pH 10 and conductivity is selected in the range of about 45 to 180 mS/cm. In another embodiment the pH is selected in the range of about pH 9 to pH 10 and conductivity is selected in the range of about 100 to 180 mS/cm.

A suitable buffer comprises of disodium hydrogen phosphate dihydrate, Sodium chloride, EDTA. The disodium hydrogen phosphate dihydrate present in concentration between 10mM to 30mM, Sodium chloride present in concentration between 100mM to 150 mM and EDTA present in concentration between 5mM to 20 mM at pH selected from pH 7 to pH 7.5 and conductivity selected from 15 to 20 mS/cm.

In another embodiment the suitable buffer comprises 20 mM disodium hydrogen phosphate dihydrate, 150mM Sodium chloride, 5-10mM EDTA at pH pH 7.2 and conductivity at 18 mS/cm.

In certain embodiment conditioning the depth filter by performing a washing step with high concentration of salt solution prior to the clarification increased the product recovery at least by about 5 % without impacting the quality of clarified harvest. In another embodiment the product recovery is increased at least by about 15 %.

In yet another embodiment the present invention relates to a process to recycle the secondary depth filter by washing the depth filter with high concentration of salt solution prior to performing the clarification step. The secondary depth filter may be recycled for up to 5 cycles, in a preferred mode up to 3 cycles.

Tris, MOPs, Histidine, Glycine, and Sodium Chloride are non-limiting examples of various buffers that could be used. Numerous salts are known in the art, certain non-limiting P

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Two experimental conditions were performed to demonstrate head to head effect of pre- treatment or conditioning of the depth filter with a high salt solution prior to the clarification step. In condition A the secondary depth filter was not pre-treated with high concentration of salt solution and in condition B the filter was pre-treated with high concentration of salt solution.

Example 1 - Condition A– No Pre-Treatment:

The primary and/or secondary filter was wetted with WFI followed by equilibrating the membrane with equilibration buffer to make the membrane in same condition as that of the cell culture fluid (CCF). Later, clarification performed by passing CCF on to primary and secondary filter in series with each other. After completion of filtration post buffer flush given to recover the product. The collected product was then analyzed for process and product related impurities.

Example 2 - Condition B– Pre-Treatment with High Salt Concentration Solution:

The primary and/or secondary filter was wetted with WFI. This was followed with the high concentration salt solution flush for condition of membranes for better recovery. In case of secondary filter the same operation shall be done for reusing the membrane (only secondary). After pretreatment the same membrane is equilibrated with equilibration buffer to make the membrane in same condition as that of the cell culture fluid (CCF). Later, clarification performed by passing CCF on to primary and secondary filter in series with each other .After completion of filtration post buffer flush given to recover the product. The collected product was then analyzed for process and product related impurities. Results of experiment A and experiment B are mentioned in below Table 1;