WO2000056768A2

WO2000056768A2 - Chromatographic albumin process

Info

Publication number: WO2000056768A2
Application number: PCT/US2000/006354
Authority: WO
Inventors: Rajesh Sharma; Susan Trukawinski; Hanns-Ingolf Paul; Richard Rose
Original assignee: Bayer AG; Bayer Corp
Current assignee: Bayer AG; Bayer Corp
Priority date: 1999-03-19
Filing date: 2000-03-10
Publication date: 2000-09-28
Anticipated expiration: 2001-09-19
Also published as: AU3624400A; WO2000056768A3

Abstract

The invention provides an improved method of manufacturing Albumin utilizing an addition caprylate for both separation and viral inactivation; and chromatography for additional purification. In the improved method the caprylate incubation is carried out at a pH of from about 5.5 to about 5.6 and at a temperature between 30 and 50 degrees centigrade. The required incubation time is decreased to one hour. No centrifugation is necessary, with additional purification to remove unwanted contaminants being accomplished with filtration steps including a chromatographic filtration through an anionic column. This method provides a significant decrease in manufacturing time with increased purity, without any measurable reduction in yield.

Description

CHROMATOGRAPHIC ALBUMIN PROCESS

BACKGROUND OF THE INVENTION

Field: This disclosure generally involves manufacturing plasma-derived therapeutic protein solutions, and more specifically, manufacturing selectively stabilized animal or human serum albumin (HSA).

Background: The Cohn fractionation method, which utilizes ethanol, temperature, pH, protein concentration, ionic strength, and time to insolubilize unwanted proteins during albumin manufacture, was originally published in 1946, and remains a primary method in the United States for processing plasma. T. Gerelough's subsequent use of 95% ethanol in the Cohn fractionation process greatly diminished process volumes required, and thereby reduced corresponding manufacturing costs. Gerelough's method is also a recognized standard in the United States for plasma fractionation. U.S. Pat. Nos. 2,710, 294;

2,710,293 (1955). In Europe, H. Nitschamam and P. Kistler describe a shorter method for processing albumin; however, the resulting product failed to satisfy regulatory guidelines imposed by United States agencies during that time period. Vox Sang., 5, 272 (1960).

Plasma fractionation methods in the United States have employed Cohn techniques since

Cohn et al's 1946 publication. Consequently, neither interest nor necessity encouraged manufacturers to identify alternative albumin fractionation techniques for more than twenty years. M. Steinbuch, Vox Sang., 23, 92 (1972). Furthermore, since conventional fractionation techniques produced albumin that could be successfully pasteurized (by heating for 10 hours at 60 degrees centigrade) to inactivate viruses, there was little motivation and much caution among albumin manufacturers in seeking alternative and improved fractionation methods.

Then in 1972 M. Steinbuch explored the ability of several reagents, other than ethanol, to separate plasma proteins via precipitation. Using Cohn Fraction in as a starting material.

Steinbuch studied the precipitation capacity of caprylic acid, which had previously been used to stabilize albumin (M. Steinbuch, Vox. Sang., 23:92-106, 1972, Yu L. Hao, U.S. Pat. No. 4,222,934, 1980), and subsequently to inactivate lipid-enveloped viruses, Seng et al., U.S. pat. No. 4,939,174, 1990. As a result of these studies, scientists later developed several techniques for purifying IgG, IgA, alpha- 1 acid glycoprotein and prealbumin, concurrently finding that the precipitation reaction was highly temperature and pH dependent.

During human immunoglobulin preparation caprylic acid is generally recognized as an effective precipitating agent for most plasma proteins at pH 4.8, so long as parameters such as temperature and ionic strength are optimized. Steinbuch et al., Preparative Biochemistry, 3(4), 363-373 (1973). Accordingly, Steinbuch et al. have described a method for isolating IgG from mammalian sera using caprylic acid, finding that extensive non-immunoglobulin precipitation is best obtained at slightly acidic pH, but not below pH 4.5. Steinbuch et al., Arch. Biochem. Biophys., 134, 279-294 (1969).

Habeeb et al. used caprylic acid precipitation to obtain plasma-derived IgG that was free of aggregates, plasmin and plasminogen; low in anticompliment activity; and stable during storage. Preparative Biochemistry, 14(1), 1-17 (1984). Also, IgA has been prepared as a routine fractionation by-product from Cohn fraction IH, based on IgA solubility with caprylic acid present at pH 4.8. Pejaudier et al., Vox Sang. 23, 165-175 (1972). Fraction HI additionally provides starting material for obtaining IgM-enriched plasma fractions.

Sodium caprylate has also been used to purify albumin. According to these methods, sodium caprylate is added to process plasma, and protects albumin when the process stream is exposed to high temperatures. Extreme temperatures not only denature process stream globulins, but often generate contaminant neo-antigens. Schneider et al., U.S. Pat.

No. 4,156,681 (1979); Institue Merieux, U.S. Pat. No. 3, 992,367; Bayer Corporation, U.S. Pat No. 5, 561,115.

Chromatographic methods of plasma purification utilizing caprylate separation are described in USSN 08/879,362 filed by June 20, 1997, pending, and owned by the same assignee as this application. Surprisingly, we have now found that albumin may be separated from impurities more efficiently than by previously disclosed methods such as US Patent No. 5,561,155 assigned to this manufacturer and incorporated herein by reference, by chromatography methods and by the inclusion of a caprylate addition under improved conditions.

SUMMARY OF THE INVENTION

The invention is an improved manufacturing process for preparing albumin from a solution of plasma proteins. The solution generally contains albumin, non-albumin proteins and contaminant manufacturing debris, including metal ion contaminants, ethanol and salts. Sodium caprylate is employed as a partitioning agent to separate albumin from the non-albumin proteins, and also provides inactivation of lipid enveloped viruses in the plasma protein solution. The albumin separation improvement comprises: a) incubating sodium caprylate with the plasma protein solution at a pH of about 5.5 or 5.6 at a temperature of between 30 and 50 degrees centigrade to separate the albumin from non-albumin proteins; b) separating the albumin from the non-albumin proteins; and c) diafiltering the separated albumin of step b) to remove caprylate, metal ion contaminants, ethanol and salts.

It is preferred to diafilter against sodium chloride or water for injection (WFI).

Preferably, the albumin solution obtained in step b) is processed through an anionic column to further purify the albumin product.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a flow diagram for the caprylate/chromatography albumin process.

Figure 2 shows preferred caprylate/chromatography albumin process conditions. BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a new process for the purification of albumin from human plasma, referred to herein as the Caprylate/Chromatography Albumin process. The starting material is effluent IN-1 from the Cohn fractionation process. The process includes caprylate treatment of IN-1 effluent (Cohn Method 9), followed by diafiltration. The resulting albumin solution is 96% pure (by immunonephelometry). The contaminants ceruloplasmin, alpha- 1 PI, alpha acid glycoprotein, prealbumin and antithrombin m, as well as caprylate, are removed by binding to an anion column. During development, various resins were screened and conditions were determined to obtain maximum purity

(100% by immunonephelometry) and maximum recovery (95%) of the albumin in the column flow through fraction. Focusing chromatography conditions to bind contaminants, instead of albumin (the bulk of the protein), resulted in a smaller column, fewer chromatography steps, and high albumin yield. Column resin cleaning and regeneration procedures were developed to remove the contaminant proteins, residual caprylate and lipids. The process was scaled to a 3.1 liter column (14 cm diameter) for processing 3.1 kg albumin (39 liters solution). Multiple runs have been completed at this scale, and the average albumin flow through was 100.0% pure by immunonephelometry and 99.7% monomer by high pressure liquid chromatography (HPLC). Average recovery of albumin over the chromatography step was 94%. The flow through was bulked, filled, pasteurized, and passed all standard product testing. Production scale is anticipated to require a 100 L column (80 cm diameter) to process 100 kg albumin (1250 liters of starting solution).

SPECIFIC EMBODIMENTS

MATERIALS AND METHODS

Materials:

Effluent IN-1 was prepared based on Cohn's method NI.

Methods : Caprylate Incubation: The pH of IN-1 effluent was adjusted to pH 5.55 with 0.5 M sodium carbonate and then 60 mM sodium caprylate was added. These steps were performed below 0°C due to 20% ethanol in IN-1 effluent. After caprylate addition, the solution was heated to 40°C. The caprylate treated effluent was incubated for 60 minutes before filtration. The incubated material was held at ambient or 5°C temperature before filtration. Incubation was performed in a 100 L jacketed tank. A heat exchanger was used to heat the effluent and a Lishtnin A310 blade design was used for mixing.

Filtration:

Incubated material was cooled to either 5°C or 20°C and filtered through a plate and frame filter press, either Sperry or JNK. All filtrations were performed at a constant pressure of 20 psi ⁱg< -

Ultrafiltration/Diafiltration fUF DF):

The process solution was adjusted to pH 6.60 to 6.90. Then ultrafiltered to 10% protein. The concentrated solution was diafϊltered against 5 volumes of 3% ΝaCl followed by 5 volumes WFI. The diafiltered solution was then concentrated to 12% protein.Νew membranes were rinsed with WFI per manufacture's instructions and then cleaned with 0.1Ν ΝaOH prior to first use. Membrane performance evaluation tests were performed, and after each lot, the cassettes were cleaned according to the manufacturer's recommendations.

Chromatography: The chromatography column (20 cm bed x 14 cm diameter) was used with a Pharmacia

Bioprocesssystem. 0.9 to 1.2 g albumin, pH 5.0 - 6.0 per mL of Pharmacia DEAE FF resin at 150 cm/h flow rate. The flow through was collected as the albumin fraction, then the column was cleaned and the cleaning fractions collected for mass balance. For 5% albumin final container, excipients were added to the flow through, followed by sterile filtration and final container filling. For 25% albumin final container, the flow through was concentrated by ultrafiltration to 30% albumin. Excipients were added to the concentrate, followed by sterile filtration and final container filling. The above example is intended to illustrate the invention and it is thought variations will occur to those skilled in the art. Accordingly, it is intended that the scope of the invention should be limited only by the claims below.

Claims

CLAIMSWhat is claimed:

1. A process of preparing albumin from a solution of plasma proteins that contain albumin, non-albumin proteins and contaminant manufacturing debris including metal ion contaminants, ethanol and salts, the improvement comprising the steps of: a) employing sodium caprylate in the solution of plasma protein at a pH of about 5.5 to 5.6; b) incubating the caprylate containing plasma protein solution between 30 and 50 degrees centigrade to separate the albumin from non-albumin proteins; c) separating the albumin from the non-albumin proteins; and d) diafiltering the separated albumin of step c) to remove caprylate, metal ion contaminants, ethanol and salts.

2. The method of claim 1 wherein diafiltration is done against a diafiltration buffer chosen from the group consisting of sodium chloride or WFI.

3. The method of claim 1 wherein the diafiltered albumin solution of step d) is processed through an anionic column to further purify the albumin product.

4. The method of claim 1 wherein the incubation step b) is carried out for one hour.