FIELD OF THE INVENTION
The present invention relates to a method of reducing endotoxins from an antibody composition by introducing a filtration step before every downstream processing step.
BACKGROUND OF THE INVENTION
Bioburden is the degree of microbial contamination or the number of viable microorganisms contaminating an object, a medical product, device or raw material. Any therapeutic product or component requires a strict control on the bioburden level, since bacterial bioburden release endotoxins or lipopolysaccharides, a major component of the outer membrane of gram-negative bacteria. The endotoxins released by them are major contaminants in commercially available biologically active substances or proteins and often complicate the study of the biological effects of the main ingredient. The presence of even small amounts of endotoxin in recombinant protein preparations can cause major pathophysiological effects such as endotoxin shock, tissue injury, and in rare cases, death. Due to the high toxicity of endotoxins, their presence in therapeutic preparations is a potential health risk, and their removal from the final product is a regulatory requirement.
Endotoxins induce pyrogenic reactions and shock in mammals upon intravenous injection at concentrations as low as a few ng/mL (Fiske J M et al. J Chrom B, 753:269-278, 2001). Therefore to regulate the levels of endotoxins in pharmaceutical products, the United States Federal Register, has proposed guidelines for determining endotoxins with the Limulus Amebocyte Lysate (LAL) Test on January 18, 1980. The United States Pharmacopeia recognizes the LAL Test to determine acceptable levels of endotoxins.
The K/M formula was presented in the FDA LAL-Test guideline as a way to calculate the endotoxin limit as shown below:
Endotoxin Limit (EL) = K (tolerance limit) / M (maximum dose/kg/hour)

The EL represents the maximum safe amount of endotoxin that is allowed in a dose of a specific drug. The tolerance limit varies with the type of parenteral product, its prescribed dosage and route of their administration. M is the dosage of a parenteral drug where the dosage is expressed in mL per kg per hour. K is typically 5 EU/kg of body weight for parenterals (except intrathecals) pertaining to human or veterinary drugs and biologies.
Therefore reducing the level of endotoxins to the tolerance limit (threshold value) is governed by strict regulatory guidelines and creates a significant challenge in the development of methods for the purification of therapeutic proteins.
A number of approaches are typically utilized to reduce endotoxin contamination in pharmaceutical therapeutics, including ion-exchange chromatography, affinity chromatography, ultrafiltration etc. The prior art discloses various methods for purification of proteins by removal of endotoxins.
Sweadner KJ, Forte M and Nelsen LL, Appl Environ Microbiol 34(4):382-385. 1977, have described ultrafiltration to remove aggregated Escherichia coli lipopolysaccharides from aqueous solutions.
Ultrafiltration although effective in removing endotoxins from water, might be inefficient in the presence of proteins because they can be damaged by physical forces. Anion exchangers, which take advantage of the negative net charge of endotoxins, have been described for their use in endotoxin adsorption by Pyo SH et al, Protein Expr. Purif 23:38-44. 2001.
Shibatani T, Kakimoto T, Chibata I, Thromb Haemost 28:49(2):91-5. 1983, described use of ion exchange chromatography for removing endotoxin contaminants.
Issekutz AC, J. Immunol. Methods 61:275-81. 1983, describes a process of removal of gram-negative endotoxin from solutions by affinity chromatography.

US 2004198957 teaches a method of removing endotoxin contaminants from protein solutions using hydrophobic charge induction chromatography sorbents.
US 6942802 and US 6365147 describes a process for the purification of polypeptides and the removal of endotoxin via immobilized metal affinity chromatography.
US 7919589 describes a method for removing endotoxin bound to a protein comprising binding the protein to a cation exchange resin and eluting the endotoxin using a low-ionic strength solution.
Though prior-art teaches the use of membrane or column based chromatography, these chromatographies have several inherent disadvantages like limited binding capacity, specific flow-rate requirements, larger buffer volume consumption etc. In techniques such as ion-exchange chromatography that utilize electrostatic or charge based interactions as the main driving force in the removal process, endotoxins are shown to form complexes with proteins of different isoelectric points, making the removal process more difficult. Thus a simple and powerful technique which would alleviate the difficulties in prior-art processes and that will reduce endotoxins to or below the acceptable limit has been a central requirement in the development of downstream process for any therapeutic product.
The objective of the present invention is to provide an efficient and cost effective method for removal of bioburden by using absolute filtration throughout the downstream process which in turn reduces the endotoxin level below the threshold limit. In addition, incorporation of the filtration step in the process reduces the pressure build up on the chromatographic columns used in the downstream process.

SUMMARY OF THE INVENTION
The present invention discloses a method of purification of antibody containing sample by incorporating absolute filters prior to every downstream processing step. In particular this invention employs the use of absolute filters of membrane pore size of 0.22 urn prior to every downstream processing step. The said filters are employed to remove bio-burden, thereby reducing the endotoxin level significantly lower than the threshold limit of the said antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Flow diagrams in Panel A, Panel B and Panel C refer to exemplary process comprising an absolute filtration step prior to every downstream unit operation. "F" represents the absolute filtration step.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for removal of bioburden comprising an absolute filtration step prior to every downstream unit operation which in turn reduces the endotoxin levels below the threshold limit.
In an embodiment, the invention provides a method of reducing endotoxins from an antibody composition comprising an absolute filtration step prior to any downstream unit operation wherein the membrane pore size of the absolute filter is 0.22 urn
In yet another embodiment, the invention provides a method of reducing endotoxins from an antibody composition comprising an absolute filtration step prior to any downstream unit operation wherein the membrane pore size of the absolute filter is 0.22 urn and wherein the endotoxin levels are reduced below the threshold levels required for a therapeutic product.
In yet another embodiment, the invention provides a method of reducing endotoxins by removal of bioburden from an antibody composition comprising an absolute filtration step prior to any downstream unit operation wherein the

membrane pore size of the absolute filter is 0.22 um and wherein the endotoxin levels are reduced below the threshold levels required for a therapeutic product.
The therapeutic product mentioned in the embodiments in an anti-VEGF antibody.
The term "threshold levels" or "tolerance limit" or "endotoxin acceptable levels" used interchangeably herein represents the maximum amount of endotoxin that is allowed in a dose of a therapeutic product that is considered safe for therapeutic administration in human or veterinary drug products.
An absolute filter is a filter capable of cutting off 100% by weight of particles greater than a stated micron size, "absolute filtration" is a filtration step performed using an absolute filter of a specific pore size.
The term "downstream processing" or "downstream process" or "downstream unit operation" used interchangeably herein refers to recovery and purification of antibody which include one or more of the following steps: removal of insolubles from the product, product isolation, product purification and product polishing.
Removal of insolubles or clarification involves the capture of the product as a solute in a particulate-free liquid, for example the separation of cells, cell debris or other particulate matter from fermentation broth containing the product. Typical operations to achieve this may include filtration using nominal filters; centrifugation; sedimentation; precipitation; flocculation; electro-precipitation; gravity settling; grinding; homogenization; or leaching.
Product isolation may involve the removal of those components whose properties vary markedly from that of the desired product and may include but not limited to the steps of: solvent extraction, adsorption, ultrafiltration or precipitation.
Product purification is generally done to separate the product from the contaminants and exemplary purification procedures may include but not limited to fractionation, fractional precipitation, precipitation, immunoaffinity,

chromatography such as affinity, ion-exchange, adsorption, size exclusion, mixed-mode / multi-mode, hydrophobic interaction, size exclusion, or reversed phase etc.,
Product polishing as described herein is the final processing step and may include concentration, crystallization, dilution, sterilization, virus filtration, depyrogenation, desiccation, lyophilization or spray drying etc.,
The invention is more fully understood by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.
Example 1: Expression and harvest of anti-VEGF antibody
Anti-VEGF antibody was cloned and expressed in a CHO cell line as described in U.S. Patent No. 7,060,269, which is incorporated herein by reference. The cell culture broth containing the expressed antibody was harvested
Example 2: Filter membranes
The filters used in the current invention are Millipak® (0.22 um membrane filters), Durapore® (Opticap XL and XLT capsule filters, 0.22 um) all having a hydrophilic polyvinylidene fluoride (PVDF) membrane from Millipore, though the invention may be carried out with filters/membranes such as but not limited to polyethersulfone, polypropylene, nylon, cellulose acetate, hydrophilic or hydrophobic polytetrafluoroethylene filter membranes.
Example 3: Experimental Setup
The culture harvest from example 1 was passed through an absolute filter with a microfiltration membrane of 0.22 urn pore size to obtain clarified cell culture broth (CCCB). CCCB was again passed through a microfiltration membrane of 0.22 um. The filtrate was then loaded onto an affinity chromatography resin, followed by one or more ion-exchange resins. The eluate or flow-through from the ion-exchange chromatography column was passed through a prefilter

(membrane pore size of ~ 0.2 um) followed by nanofiltration. The filtrate was then subject to an absolute filter with a microfiltration membrane pore size of 0.22 um. The filtrate obtained was subjected to a tangential flow filtration followed by another absolute filter of 0.22 um pore size before drug substance preparation.
Table 1: Shows consistent reduction of endotoxin levels of Anti-VEGF antibody across four batches of purification comprising absolute filtration step prior to every downstream unit operation

WE CLAIM:
1. A method of reducing endotoxins from an antibody composition comprising an absolute filtration step prior to any downstream unit operation wherein the membrane pore size of the absolute filter is 0.22 urn
2. A method of reducing endotoxins from an antibody composition comprising an absolute filtration step prior to any downstream unit operation wherein the membrane pore size of the absolute filter is 0.22 um and wherein the endotoxin levels are reduced below the threshold levels required for a therapeutic product.
3. A method of reducing endotoxins by removal of bioburden from an antibody composition comprising an absolute filtration step prior to any downstream unit operation wherein the membrane pore size of the absolute filter is 0.22 um and wherein the endotoxin levels are reduced below the threshold levels required for a therapeutic product.
4. A method according to claims 1, 2 and 3, wherein the therapeutic product is an anti-VEGF antibody.