REUATED APPEIC ATION S

This application is related to Indian Provisional Application 201921009334 filed 11 ^th Mar, 2019 and is incorporated herein in its entirety.

FIEED OF THE INVENTION

The present invention provides a novel process for the purification of Adalimumab obtained from a fermentation harvest of CHO cell culture expressing said Adalimumab. The present invention further provides a novel purification process of Adalimumab by employing AEX-CEX tandem chromatography technique in a unique manner to obtain a highly purified preparation of Adalimumab while potentially preventing the formation as well as clearing of impurities such as aggregate species. The present invention also provides a highly scalable, reproducible and cost effective process for purification of Adalimumab.

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF) is a polypeptide cytokine involved in inflammatory and the acute phase responses. TNF-alpha is present in larger quantities in persons with rheumatoid arthritis or Crohn's disease. It is also involved in Juvenile Idiopathic Arthritis (JIA), Psoriatic Arthritis (PA), Ankylosing Spondylitis (AS), Ulcerative Colitis and Plaque Psoriasis. Direct inhibition of TNF-alpha by the biological agents has produced significant advances in the treatment of rheumatoid arthritis and other auto-immune disease and has validated the extra-cellular inhibition of this pro- inflammatory cytokine as an effective therapy. One such biological agent is Adalimumab.

Adalimumab (Anti-TNFa antibody), marketed as HUMIRA® by Abott Inc., is a recombinant human IgGl monoclonal antibody specific for human tumor necrosis factor (TNF). Adalimumab was created using phage display technology resulting in an antibody with human derived heavy and light chain variable regions and human IgGl constant regions. Adalimumab is produced by recombinant DNA technology in a mammalian cell expression system and is purified by a process that includes specific viral inactivation and removal steps. It consists of 1330 amino acids and has a molecular weight of approximately 148 kilodaltons.

US8946395 discloses method for purifying Adalimumab from a composition comprising the antibody and a contaminant having a reduced level of at least one impurity, comprising hydrophobic interaction chromatography (HIC) step.

US9109010 discloses method for producing host cell protein reduced antibody comprising low pH treatment, ion exchange chromatography and HIC chromatography, wherein sample mixture is not exposed to protein A.

US6417335 discloses method for purifying an antibody from a composition comprising the antibody and a contaminant, which method comprises: (a) loading the composition onto a cation exchange resin, wherein the amount of antibody loaded onto the cation exchange resin is from about 20 mg to about 35 mg of the antibody per mL of cation exchange resin; and (b) eluting the antibody from the cation exchange resin. US7863426 discloses method for producing a host cell protein-(HCP) reduced antibody preparation from a mixture comprising an antibody and at least one HCP, comprising an ion exchange separation step wherein the mixture is subjected to a first ion exchange material, such that the HCP-reduced antibody preparation is obtained.

In order to attain the appropriate degree of purity for the commercial use of therapeutic and diagnostic proteins, purification protocols often require use of two or more chromatographic purification steps. Challenges are presented by more complex purification schemes. For example, product pools processed by one purification mode must be made compatible for processing in a second purification mode. Also, partially processed product pools must be properly stored when downstream purification systems are not immediately available for use. Multiple systems necessitate multiple sampling and validation steps to ensure consistent and proper operation. Systems that integrate multiple purification modes would potentially reduce the complexity of current purification schemes, thereby enhancing the efficiency and reducing the cost of handling manufactured proteins.

Methods of purifying proteins using systems in which purification units; particularly chromatography are linked in tandem will results robust operation of purification units. Methods that maintain robustness over variations in operating parameters require less feedback control and are more cost effective, thereby, in practice, reducing the cost of goods of purified proteins. Integrating purification units eliminates the need to store partially processed product pools between runs. Product may be processed in single shifts, reducing purification time. Integration also reduces sampling, validation, and documentation requirements (e.g., bioburden sampling, batch record documentation, etc.). Variations in different chromatography parameters such as flow rates, back pressure, and mixing of titrant and eluate between columns may alter product binding and compromise product recovery and quality. Features of the present methods provide a high tolerance to such variations.

Protein aggregation is a common phenomenon that can be encountered during various stages of a commercial antibody manufacturing processes. For example, aggregates can form during the fermentation, purification, final formulation operations, or as a result of the storage of the drug substance or final drug product.

Downstream processing has been estimated to account for 50 to 80% of the total manufacturing costs of therapeutic antibodies. Numerous strategies have been developed to minimize antibody aggregation during the upstream and downstream unit operations required for antibody manufacturing including optimization of media components, culture feed conditions and operating parameters, genetic engineering of host cell/expression systems, protein engineering, formulation buffer screening and separation during downstream processing. However, it is widely recognized that it may not be possible, or commercially feasible, to optimize a particular manufacturing process to the point where protein aggregation is completely suppressed or prevented during manufacturing. Accordingly, separation of aggregates from drug substance using a downstream manufacturing step (unit operation) that is optimized for a particular protein of interest (POI) is a popular strategy which affords an opportunity to remove aggregates from the drug substance once they have been generated. Therefore, in order to effectively manage the issue of size heterogeneity during the manufacturing of biopharmaceuticals, including therapeutic antibodies, there is an unmet need for alternative chromatography processes which are capable of separating antibody from aggregates and complexes which may form as a result of process-driven modifications or manufacturing conditions. In light of the above, new purifying methods are needed which are effective in purifying Adalimumab with reduced formation of product related impurities such as aggregate species.

Accordingly, it would be desirable to provide a novel purification method for Adalimumab by decreasing formation of impurities such as aggregate species or charge variants to achieve higher final concentrations along with minimizing yield loss and impact on processing time. Also it would be preferred to have a purification method for Adalimumab by which holding at intermediate stage is avoided that potentially prevents formation of aggregate species, thereby providing better control on the quality of the product. Thus, by employing the novel purification method of Adalimumab, present invention eliminate the chances of increase in bioburden and bacterial endotoxin between the steps and thus eliminates additional steps during purification and helps in increasing the manufacturing ease and reducing processing time and cost.

OBJECTS OF THE INVENTION

The principal object of the present invention is to provide a novel process for purification of Adalimumab with decreased formation of impurities such as aggregates, through chromatographically contacting the sample with AEX-CEX tandem chromatography followed by subsequent purification steps.

Another objective of the present invention is to provide a novel process for purification of Adalimumab through AEX-CEX tandem chromatography followed by subsequent purification steps.

Another objective of the present invention is to provide a novel process for purification of Adalimumab through AEX-CEX tandem chromatography followed by subsequent purification steps wherein AEX-CEX tandem chromatography is preceded by protein A chromatography, low pH treatment and depth filtration step.

Another object of the present invention is to provide a novel process for purifying Adalimumab from a fermentation harvest of a Chinese Hamster Ovary (CHO) cell culture expressing said Adalimumab, said process comprising:

a) Binding Adalimumab from said fermentation harvest to a Protein A chromatography and collecting eluate;

b) Adjusting the output pH of Protein A chromatography to pH 7.0 after Low pH treatment;

c) Carrying out AEX - CEX tandem chromatography on an eluent obtained in step b); and

d) Optionally followed by Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

Another objective of the present invention is to provide cost effective novel process for purification Adalimumab, wherein said method comprising the steps of:

a) Protein A chromatography;

b) Low pH treatment;

c) AEX - CEX tandem Chromatography; and

d) Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

Another objective of the present invention is to provide cost effective novel process for purification Adalimumab, wherein said method comprising the steps of:

a) Protein A chromatography;

b) Low pH treatment;

c) Depth filtration;

d) AEX - CEX tandem Chromatography; and e) Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide a novel process for purification of Adalimumab with decreased formation of impurities such as aggregate species through chromatographically contacting the sample with AEX- CEX tandem chromatography followed by subsequent purification steps.

Another aspect of the present invention is to provide a novel process for purification of Adalimumab through AEX-CEX tandem chromatography followed by subsequent purification steps. Another aspect of the present invention is to provide a novel process for purification of Adalimumab through AEX-CEX tandem chromatography followed by subsequent purification steps wherein AEX-CEX tandem chromatography is preceded by protein A chromatography and low pH treatment step. Another aspect of the present invention is to provide a novel process for purifying Adalimumab from a fermentation harvest of a Chinese Hamster Ovary (CHO) cell culture expressing said Adalimumab, said process comprising:

a) Binding Adalimumab from said fermentation harvest to a Protein A chromatography and collecting eluate;

b) Adjusting the output pH of Protein A chromatography to pH 7.0 after Low pH treatment;

c) Carrying out AEX - CEX tandem chromatography on an eluent obtained in step b); and d) Optionally Followed by Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

Another aspect of the present invention is to provide cost effective novel process for purification Adalimumab, wherein said method comprising the steps of:

a) Protein A chromatography;

b) Low pH treatment;

c) AEX - CEX tandem Chromatography; and

d) Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

Another aspect of the present invention is to provide cost effective novel process for purification Adalimumab, wherein said method comprising the steps of:

a) Protein A chromatography;

b) Low pH treatment;

c) Depth filtration;

d) AEX - CEX tandem Chromatography; and

e) Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

BRIEF DESCRIPTION OF DRAWINGS

Figure-la: Conventional mode of chromatography (Non-tandem Mode)

Figure-lb: Tandem mode of chromatography

Figure-2: Preparative Chromatography Profile for non-tandem process:

Protein A chromatography

Figure-3: Preparative Chromatography Profile for non-tandem process:

Anion exchange chromatography

Figure-4: Preparative Chromatography Profile for non-tandem process:

Cation exchange chromatography

Figure-5: Preparative Chromatography Profile for tandem process: Protein A chromatography

Figure-6: Preparative Chromatography Profile for tandem process:

Anion-Cation Tandem mode chromatography

DETAILED DESCRIPTION OF THE INVENTION

Aggregates in therapeutic monoclonal antibody (mAh) drug products can be viewed as an evolving mixture of various types which can actively undergo transition under equilibrium states. At any given time, the heterogeneous molecular forms present in in such a mixture may reach a new equilibrium and transform into an even larger aggregate or complex structure. Once the higher order structure of a protein is disrupted, non-native interactions of exposed hydrophobic regions can promote intermolecular interactions leading to aggregation, or in some cases, precipitation.

Protein aggregates can be classified in several ways, including soluble/insoluble, covalent/non-covalent, reversible/non-reversible, and native/denatured. Soluble aggregates refer to those that are not visible as discrete particles and that may not be removed by a 0.22 pm filter. Conversely, insoluble aggregates may be removed by filtration and are often visible to the unaided eye. Covalent aggregates arise from the formation of a chemical bond between two or more monomers, or result from the chemical linking of partially unfolded molecules with each other. Disulfide bond formation resulting from previously unpaired free thiols is a common mechanism for covalent aggregation. Oxidation of tyrosines may also result in covalent aggregation through the formation of bityrosine. Reversible protein aggregation typically results from relatively weak noncovalent protein interactions. The reversibility is sometimes indicative of the presence of equilibrium between the monomer and higher order forms. This equilibrium may shift as a result of a change in solution conditions such as a decrease in protein concentration or a change in pH. As used herein, the term "aggregates" refers to protein aggregates. It encompasses multimers (such as dimers, tetramers or higher order aggregates) of the mAh to be purified and may result e.g. in high molecular weight aggregates.

As used herein the term "charge variants", refers to the full complement of product variants including, but not limited to acidic species and basic species (e.g., Lys variants). In certain embodiments, such variants can include product aggregates and/or product fragments, to the extent that such aggregation and/or fragmentation results in a product charge variation.

As used herein the term "acidic variant" refers to a variant or isoform of the antibody of interest which is more acidic and has a lower pi value than the antibody of interest.

The term "basic variant" as used herein refers to a variant or isoform of the antibody of interest which is more basic and has a higher pi value than the antibody of interest.

A “high-molecular- weight- species” or “HMW” comprises a preparation of Adalimumab having a molecular weight that is greater than the main species or intact Adalimumab.

A“low-molecular-weight-species” or“LMW” of Adalimumab comprises a fragment of Adalimumab that has a molecular weight less than that of main species or intact Adalimumab.

The terms " flow-through process," "flow-through mode," and "flow-through chromatography," as used interchangeably herein, to refer to a product separation technique in which at least one product (monomeric mAh) contained in a sample along with one or more contaminants is intended to flow through a chromatographic resin or media, while at least one potential contaminant or impurity binds to the chromatographic resin or media. The "flow- through mode" is generally an isocratic operation (i.e., a chromatography process during which the composition of the mobile phase is not changed).

The terms "bind and elute mode" and "bind and elute process," as used interchangeably herein, refer to a product separation technique in which at least one product contained in a sample (e.g., an Fc region containing protein) binds to a chromatographic resin or media and is subsequently eluted.

The term“ultrafiltration” refers to membrane filtration technique which employs controlled pore, semi permeable membranes to concentrate or fractionate dissolved molecules. Molecules much larger than the pores are retained in the feed solution and are concentrated in direct proportion to the volume of liquid that passes through the membrane. Molecules having a size which is close to the pore size of the membrane concentrate to a lesser extent with some of the molecules passing through the membrane in the permeate. The concentration of freely permeable molecules (salts) in the sample remains essentially unchanged. Membranes suitable for ultrafiltration (referred to as ultrafiltration or UF membranes) are defined by the molecular weight cut-off (MWCO) of the membrane used. Ultrafiltration can be applied in cross-flow or dead-end mode.

The term“diafiltration” refers to a technique that uses ultrafiltration membranes to completely remove, replace or lower the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. The process selectively utilizes permeable (porous) membrane filters to separate the components of solutions and suspensions based on their molecular size. An ultrafiltration membrane retains molecules that are larger than the pores of the membrane while smaller molecules such as salts, solvents and water, which are 100% permeable, freely pass through the membrane. During diafiltration, buffer is introduced into the recycle tank while filtrate is removed from the unit operation. In processes where the product is in the retentate, diafiltration washes components out of the product pool into the filtrate, thereby exchanging buffers and reducing the concentration of undesirable species. When the product is in the filtrate, diafiltration washes it through the membrane into a collection vessel.

The term“Tangential Flow Filtration” or“TFF” refers to a mode of filtration which is useful for clarifying, concentrating and purifying biological materials, e.g., proteins and vaccines. In Tangential Flow Filtration (TFF) mode of filtration, the fluid is pumped tangentially along the surface of the membrane. An applied pressure serves to force a portion of the sample through the membrane to the filtrate side (referred to as permeate). Biological materials and particulates that are too large to pass through the membrane pores are retained on the upstream side (referred to as retentate). However, in contrast to normal filtration mode, the retained materials do not build up at the surface of the membrane. Instead, they are swept along the face of the membrane by tangential flow of fluid. TFF may also be referred to as“cross-flow filtration.”

The platform process for purification of monoclonal antibodies involve capturing of the target protein secreted by CHO cells using a protein-A affinity chromatography column, a low pH treatment for inactivation of virus particles, one to three chromatographic steps with orthogonal working principles to separate the target molecule from different impurities such as size variants, charge variants, etc., followed by ultrafiltration and/or diafiltration, nanofiltration and sterile filtration, as required.

The principal embodiment of the present invention is to provide a novel process for purification of Adalimumab with decreased formation of impurities such as aggregate species through chromatographically contacting the sample with AEX- CEX tandem chromatography followed by subsequent purification steps.

Another embodiment of the present invention is to provide a novel process for purification of Adalimumab through AEX-CEX tandem chromatography followed by subsequent purification steps.

Another embodiment of the present invention is to provide a novel process for purification of Adalimumab through AEX-CEX tandem chromatography followed by subsequent purification steps wherein AEX-CEX tandem chromatography is preceded by protein A chromatography and low pH treatment step.

Another embodiment of the present invention is to provide a novel process for purifying Adalimumab from a fermentation harvest of a Chinese Hamster Ovary (CHO) cell culture expressing said Adalimumab, said process comprising:

a) Binding Adalimumab from said fermentation harvest to a Protein A chromatography and collecting eluate;

b) Adjusting the output pH of Protein A chromatography to pH 7.0 after Low pH treatment;

c) Carrying out AEX - CEX tandem chromatography on an eluent obtained in step b); and

d) Optionally followed by Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF). Another embodiment of the present invention is to provide cost effective novel process for purification Adalimumab, wherein said method comprising the steps of: a) Protein A chromatography;

b) Low pH treatment;

c) AEX - CEX tandem Chromatography; and

d) Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

Another embodiment of the present invention is to provide cost effective novel process for purification Adalimumab, wherein said method comprising the steps of: a) Protein A chromatography;

b) Low pH treatment;

c) Depth filtration;

d) AEX - CEX tandem Chromatography; and

e) Ultrafiltration - Diafiltration (UFDF)/Tangential flow filtration (TFF).

Yet in another embodiment of the present invention, one of the chromatographic steps used during the purification is anion exchange chromatography and cation exchange chromatography in tandem mode. Anion exchange chromatography is done in flow through mode where process-related impurities such as HCP, viral particles and product-related impurities such as size variants get adsorbed on to the column and the target protein, i.e., Adalimumab, will not get adsorbed when the loading of protein mixture is done at pH less than the isoelectric pH of Adalimumab (approximately 8.2). Cation exchange chromatography is done at bind-elute mode where binding happens at pH below the isoelectric pH of Adalimumab and elution is facilitated by increasing the salt concentration along with a change in pH. In the cation exchange chromatography described here, the elution conditions are chosen such that our protein of interest, Adalimumab, is selectively eluted, resulting in separation of product-related impurities such as size variants. The cation exchange output thus will have higher purity and lower levels of impurities. The target levels of impurities at this stage will be such that the product will match with the impurity profile of innovator (Brand name: Humira®) at the drug product stage.

Yet in another embodiment of the present invention, Clarified cell culture harvest is loaded on to the protein A chromatography resin with equilibration buffer (5-15 CVs) having 5-20mM tris-acetate, 100-130mM NaCl and l-5mM EDTA having pH of about 7.0-8.0 and conductivity of about 10-20mS/cm. Post load equilibration is carried out with 5-15 CV of same equilibration buffer followed by high salt wash with 2-lOCVs of high salt wash buffer (10-20mM tris-acetate, 1-2 M NaCl having pH of about 7.0-8.0, Conductivity of about 80-110 mS/cm). Following high salt wash, low pH wash with 7-15 CVs of low pH wash buffer (10-20 mM tris-acetate pH of about 6.0-7.0, Conductivity of about 0.2-1.0 mS/cm) is performed. Finally the elution is performed with elution buffer (200-300 mM tris-acetate pH of about 3.0- 4.0, Conductivity of about 0.2-1.0 mS/cm). Protein A chromatography output is subjected to low pH treatment at pH of about 3.0-4.0 at temperature: (25 ± 2) °C, for (60 ± 10) min followed by neutralization to pH 7.0 with 1M Tris. After low pH treatment depth filtration is performed. The stationary phase media used at anion exchange step is cross-linked agarose with the ligand quaternary amine, although other stationary phase media such as cross-linked cellulose or polymeric media with similar ligands can be used. The anion exchange column is first equilibrated with 5- 25 CVs of equilibration buffer 10-20 mM Tris-acetate (pH about 7.0-8.0; conductivity about 1-10 mS/cm) followed by loading of the protein mixture with binding capacity < 120 mg/ml of resin. The flow through from the anion exchange chromatography is collected and loaded to a cation exchange chromatographic column. The stationary phase media used at cation exchange step is polymeric media with the ligand containing sulphoisobutyl group, although other stationary phase media such as cross-linked agarose or cellulose with similar ligands can be used. The cation exchange column is first equilibrated with 5-25 CVs of equilibration buffer 20-40 mM Tris-acetate (pH about 7.0-8.0; conductivity about 1-10 mS/cm) followed by loading of the protein mixture with binding capacity < 120 mg/ml of resin. The loaded protein at cation exchange stage gets bound to the column and washed with 5 - 10 CVs of equilibration buffer 20-40 mM Tris-acetate (pH about 4.0-6.0; conductivity about 1-5 mS/cm). After washing, the protein of interest is eluted using 10-25CVs of 0.4-1 M Tris-acetate (pH about 5.0-6.0; conductivity about 1-5 mS/cm).

The major feature of current invention is, with the above chromatographic conditions, the flow through from anion exchange chromatographic column can be directly loaded on to the second column - the cation exchange chromatographic column - without any treatment such as dilution or pH adjustment. This enables the two chromatographic column procedures to be used in tandem mode, where the outlet of first chromatographic column is directly connected to the inlet of second chromatographic column. By employing this strategy, holding at intermediate stage is avoided that potentially prevents formation of impurities such as aggregate species, thereby providing a better control on the quality of the product.

Following flow chart illustrates process flow of non-tandem chromatographic process Vs tandem chromatographic process:

Moo-Tandem Chromatography Tandem Chromatography

Process Process

The novel process for purification of Adalimumab as described in the present invention has the following advantages:

a) By adopting a tandem mode for the two chromatographic steps will also eliminate the chances of increase in bioburden and bacterial endotoxin between the two steps.

b) By adopting a tandem mode there will be elimination of additional steps such as pre-column filtration that helps in increasing the manufacturing ease and reducing the processing time and cost.

c) By adopting a tandem mode for the two chromatographic steps either no sample conditioning is required for the second column or in-line sample conditioning can be done using the chromatographic system of second column. The invention will now be further described by the following examples, which are illustrative rather than limiting. The following examples illustrates the purification of Adalimumab described in the present invention through tandem chromatographic purification process. Further, it illustrates comparability, cost effectiveness and step reduction achieved by the tandem chromatographic purification process in compared to conventional non-tandem chromatographic purification process at manufacturing scale. EXAMPLES

Example 1: Purification of Adalimumab through non-tandem mode chromatographic process Adalimumab purification process starts with cell culture supernatant / clarified broth containing the product. The cell culture supernatant was obtained after clarification of cell culture harvest produced at bioreactor in upstream. The purification process was based on three chromatographic techniques: Protein A chromatography, anion exchange chromatography and cation exchange chromatography; and one ultrafiltration diafiltration (UFDF)/tangential flow filtration (TFF) step.

The Protein A chromatography was employed as the capture step in mAb purification process because of its specificity for the fragment-crystallisable (Fc) region of antibodies. IgG binds to Protein A ligand through affinity interactions. This step removes majority of the host related (HCPs, HCD, potential endotoxin like contaminants, viruses and virus like particles) and process related (media components and coloring matter) impurities with a very high yield (> 90%). Low pH treatment was performed to inactivate enveloped viruses at pH 3.5 for ~60 min. This step is performed immediately after the Protein A elution to inactivate enveloped viruses, if any. Neutralization was performed to adjust the pH and conductivity conditions for loading on anion exchange chromatography. After neutralization depth filtration is performed to control turbidity.

Following depth filtration, the depth filter output (DFOP) was loaded on Anion exchange column. Anion exchange chromatography was run in flow through mode where process-related impurities such as HCP, viral particles and product-related impurities such as size variants get adsorbed on to the column and the target protein, i.e., Adalimumab, will not get adsorbed when the loading of protein mixture is done at pH less than the isoelectric point of Adalimumab (approximately 8.4). After completion of Anion exchange chromatography step eluate can be stored till further chromatographic step. This holding at intermediate stage increases chances of increase in bioburden and bacterial endotoxin between the two steps. After this step anion exchange chromatography output was loaded onto cation exchange chromatography column. Cation exchange chromatography is operated in bind elute mode. The cation exchange chromatography output was then subjected to ultrafiltration diafiltration (UFDF)/Tangential flow filtration (TFF) to obtain the product in the final formulation buffer and target drug substance concentration. This step was followed by 0.2m filtration to obtain drug substance. This drug substance was then stored at -20°C.

Table: 1 and Table: 2 represents product quality in terms of size variants and product quality in terms of charge variants respectively, through non-tandem mode chromatographic process. Table: 1 Product quality in terms of size variants in non-tandem mode purification process

Table: 2 Product quality in terms of charge variants in non-tandem mode purification process

Example 2: Purification of Adalimumab through tandem mode chromatographic process Adalimumab purification process starts with cell culture supernatant /clarified broth containing the product. The cell culture supernatant is obtained after clarification of cell culture harvest produced at bioreactor in upstream. The purification process is based on three chromatographic techniques: Protein A chromatography, Anion exchange chromatography and Cation exchange chromatography (in tandem); and one ultrafiltration diafiltration (UFDF)/tangential flow filtration (TFF) step. Protein A chromatography & Low PH treatment step was same followed as mentioned in above Example 1.

The Depth filter output (DFOP) was then loaded onto AEX-CEX, which were connected in tandem. AEX was operated in flow through mode and CEX was operated in bind elute mode. The loaded protein flows through the AEX column and binds directly on to the CEX resin under the given flow, pH and conductivity conditions. The AEX step provides clearance of impurities such as HCD (Host cell DNA) and viruses that bind to the column, whereas the protein does not bind under the existing pH. Cation exchange chromatography on the other hand, provides a complementary chemistry, charge based binding and charge and salt based elution which allows most of the negatively charged impurities such as host cell proteins, residual DNA, residual leached Protein A and bacterial endotoxins to be removed in flow through and wash fractions, while the target product remains bound to the resin. CEX chromatography may also separate aggregates, low molecular weight/ degraded/ truncated impurities from the target product. Thus the combination of these two steps removes most of the product related and process related impurities and yields the product with purity more than 98 %.

The AEX-CEX output was then subjected to ultrafiltration diafiltration (UFDF)/Tangential flow filtration (TFF) to obtain the product in the final formulation buffer and target drug substance concentration. This step was followed by 0.2m filtration to obtain drug substance. This drug substance was then stored at - 20°C. Table: 3 and Table: 4 represents product quality in terms of size variants and product quality in terms of charge variants respectively, through tandem mode chromatographic process.

Table: 3 Product quality in terms of size variants in tandem mode purification process

Table: 4 Product quality in terms of charge variants in tandem mode purification process

Conclusion of Table 1-4:-

The control in HMW for both the cases is similar. However, these two processes can be compared in terms of manufacturing easiness, reduction of one separate chromatographic purification step thereby achieving reduction in processing time and cost with no significant impact on product quality between Tandem and Non tandem mode.