Preparation of protein formulations with reduced content of aggregates

0001The invention relates to a method for producing protein preparations reduced aggregate content, in particular for the preparation of medical preparations of blood plasma proteins such as albumin.

0002Human plasma proteins are large for some decades purified scale and made available for the therapy and prophylaxis. For the preparation of pure proteins or protein fractions from a complex plasma mixture, various methods used be such as fractionation by selective precipitation or Separation of protein mixtures by chromatographic methods such as ion exchange chromatography, gel filtration and Affinity chromatography. To obtain optimum results, these methods are often combined.

0003For the fractionation of plasma proteins on a large scale have long Z ince also precipitation methods with proven ethanol (Cohn et al., J. Am. Chem. Soc. 68 (1946), 459-475; Kistler and Nitschmann, Vox Sang. 7 (1962), 414-424). With this approach, even today most large amounts of plasma proteins, especially albumin, Immunoglobulins and coagulation factors, but also other proteins from human plasma, especially for medicinal purposes isolated. The However, ethanol fractionation of plasma proteins has some drawbacks. So can especially with high concentrations of alcohol and / or high Temperatures a partial denaturation of sensitive proteins done. Such denaturation can for partial or complete Loss of physiological function or structural changes lead, which in the activation of proenzymes or formation you express of new antigenic determinants or protein aggregates.

0004To prevent possible infectious contamination, including viruses and other pathogens, inactivate, may blood plasma preparations a final Pasteurization undergo. may albumin example by heating at 60 to 64 ° C for 10 h in the presence of stabilizers be such as N-acetyl tryptophan or sodium caprylate pasteurized (Gellis et al., J. Clin. Invest. 27 (1948), 239-244). More be pasteurization, for example in U.S. Patents 2,897,123; 3,227,626; 4,379,085; 4,440,679; 4,623,717 and 4,803,073 disclosed. Pasteurized preparations of plasma proteins have with respect to the transmission of viruses and pathogens to be very safe proved. Another advantage of the pasteurization is that the must accompany protein preparation, no toxic additives and thus in many cases, inactivation of pathogens in the is final container feasible. A disadvantage of pasteurization, however, that often found a significant increase in aggregate formation becomes.

0005This aggregation, subvisual to the forging of visual or lead particles in partly high amounts, however, is highly undesirable, particularly in medical and pharmaceutical Applications. Thus, large particles directly the function of affect capillaries. Adverse effects of subvisual particles or protein aggregates are for the most diverse known protein formulations and have been described. Therefore exist most regulatory authorities on limits for the aggregate content, example of albumin or immunoglobulin solutions. aggregates in Immunoglobulin solutions about an uncontrolled activation of trigger the complement system and cause serious side effects to lead. From protein aggregates is described as very fast from the circulation disappear, probably block the RES system and the body for shock states sensitize. Thus, Aggregates in protein solutions in extreme cases, life-threatening Situations result, which can be avoided under all circumstances have to.

0006is due to the very wide field of application of plasma proteins a very great need for methods by which the safety, tolerability and the biological activity of the proteins can be improved. In addition, such procedures should economical and easy to use be.

0007It was surprisingly found in the present invention that Proteinpäparationen be produced with reduced aggregate content can, for example, when a to eliminate possible infectious Contamination performed thermal treatment by a preceding or following separation step is improved, so that in the final product denaturation and / or aggregation significantly is reduced or prevented. The separation step is a protein or protein mixture which is then thermally treated shall, with respect to its desired active ingredient and native enriched. An object of the present invention is thus a A method for producing protein preparations with reduced Aggregate content comprising a thermal treatment, wherein the The method is characterized in that the before and / or after thermal treatment in a separation of the protein preparation existing aggregates, denatured proteins and / or Contamination occurs.

0008The inventive method is in particular for preparing Preparations of blood plasma proteins are suitable, but not on it limited. The blood plasma proteins are preferably selected from the group consisting of plasminogen, albumin, factor VIII, factor IX, Fibronectin, immunoglobulins, kininogen, AntithrombinIII, α-1-antitrypsin, Prekallikrein, fibrinogen, thrombin, (Apo -) - lipoproteins and Blood plasma protein fractions, which contain one or more of these proteins. Particularly preferred is a preparation of albumin, Immunoglobulins or (apo) lipoproteins, and most preferably a Albumin preparation forth.

0009An albumin preparation produced by the method according to the invention reduced aggregate content is significantly better for medical Applications suitable as preparations of the prior art. The Albumin preparations of the invention can be used as a plasma expander, but also be used for the therapy of burns. Furthermore Also other applications are possible, such as the Ultrasonic Imaging or the additive as a stabilizer to protein solutions, especially when are high-active proteins or peptides in low concentrations is such as hormones, chemokines, cytokines or enzymes that today are often produced by recombinant technology.

0010The inventive method is particularly well for the preparation of Preparations of chemically modified proteins suitable, the Modification of functional groups of the proteins, in particular to functional side chains is performed. Chemically modified Proteins such as albumin, for example, as a carrier of functional Molecules are used, which in vivo is not desired in the Compartments of the organism enter or not in the free form exhibit desired activity. Preferably, the chemical Modification selected from the group consisting of polyethylene glycol-modified, Iodination, acylation, eg, acetylation, oxidation, for example with Peroxides, dinitroxylation and crosslinking, for example with bifunctional Linkers.

0011The essential for the inventive step, ie a at least partial separation of aggregates, denatured proteins and / or contamination before and / or after a thermal Treatment, a precipitation, for example with ammonium sulfate, ethanol or Polyethylene glycol, connected to a separation of the precipitated Aggregate according to known methods, for example by centrifugation comprise. In addition, the separation can also include, for example, gel filtration with Fractogel EMD Bio SEC (Merck) or other gel filtration media such as Sephadex or Sepharose (Pharmacia). Preferably the However, separation by membrane filtration using Membranes of suitable pore size, for example, an exclusion size of 30 to 1000 kD, and especially 100 to 500 kD, whereby unwanted Components having a molecular weight above the cutoff size be separated. Separation may also nanofiltration, as used for depleting viruses include, for example, under Use of filtration materials DV20 or DV50 (Pall Corp., New York, USA) or Planova 15 N (Asaki, Tokyo, JP).

0012Carried out before or preferably after separation thermal Treatment preferably comprises a temperature increase over a prolonged period, as a pasteurization step, the known Manner can be carried out. Usually comprises a thermal treatment Heating the protein solution to at least 55 ° C for a sufficient Duration to infectious contaminants at least largely inactivate. The duration of heating is preferably at least 5 h, particularly preferably about 10 hours. The heating temperature is preferably in the range between 60 and 65 ° C. By inactivation of the To prevent proteins, the thermal treatment is preferably in The presence of known stabilizers such as N-acetyltryptophanate or sodium caprylate in the case carried out by albumin.

0013While many protein mixtures of the above-mentioned separation step already in itself a sufficient reduction in the formation of Aggregates, denatured proteins and / or contamination effects, may in other cases, the desired results obtained only by be that carried out a pre-treatment step prior to separation is where in the protein preparation aggregatable existing and / or transferred denaturable components in a separable state will. Preferably, the pretreatment step includes a change physico-chemical parameters in the protein preparation, in particular a stress treatment to existing in the preparation denatured, partially denatured or sensitive molecules or aggregate contamination or otherwise in a condition to bring, from which they removed with the subsequent separation step can be. This change of physicochemical parameters For example, a change in pH, a change, in particular an increase of temperature, a change in the Ionic strength or the dielectric constant, the supply of shearing forces or a combination of two or more of these measures include.

0014Particularly preferably this pretreatment step involves an increase the temperature of the protein preparation. The extent and duration of these Increase in temperature are of the respective protein or its preparation Sensitivity depends. On the one hand should the Temperaturerhöhrung and be their duration sufficient to the greatest degree of convert aggregierbarer components in a separable state; On the other hand, the conditions may not be too dramatic to inactivation of the desired proteins in the preparation possible to largely avoid. For a variety of proteins, for example, for albumin, it has proven to be favorable, the protein preparation for a Period of 30 minutes to 4 hours, especially for 1 hour to 2.5 h on a heating temperature of 40 to 70 ° C, especially from 45 to 65 ° C.

0015The inventive method is particularly suitable for the production of medical protein preparations with reduced aggregate content. Preferably, the aggregate content is compared with a - otherwise same - protein preparation without the separation step by at least 10%, particularly preferably at least 30% and most preferably reduced by at least 50%. In some cases, a Reduction of aggregate content by 90% or achieved it.

0016Finally, the invention also relates to a protein preparation with reduced aggregate content, which by the inventive method was produced. This protein preparation is particularly suitable for medical applications considerably better suited than preparations of Prior art, since it greatly in therapeutic use less Unverträglichkeitareaktionen triggers.

0017Finally, the invention is still by the following examples explained.

Examples

example 1

0018An albumin solution (10% in 10 mmol / l NaCl) was 90 min at 45 ° C incubated. Subsequently, the solution was filtered through a cassette with a Molecular weight cut off of 300 kD diafiltered. The ultrafiltrate contains predominantly monomeric albumin was in the presence of Stabilizers (16 mmol / l Na-caprylate, 16 mmol / l Acetyltryptophan) 10 h long pasteurized at 60 ° C.

results:

0019The aggregate content was after preincubation 2%, after ultrafiltration in (ultrafiltrate)> 0.1% and 0.6% after pasteurization. In the retentate Ultrafiltration was a heavy accumulation of aggregates measured (> 80%). Without pre-incubation and subsequent diafiltration was to the Pasteruisation an aggregate content of 6% was measured. The results were not significantly by the addition of stabilizers (N-acetyltryptophanate and sodium caprylate) during the pre-incubation of Albumin affected.

0020An analogous experiment was commercially available with a Albumin solution (20% in 140 mmol / l NaCl, 12 mmol / l N-acetyl tryptophan and Na-caprylate) performed. The behavior of the solutions during the Test and the results were the same as with the substantially 10% albumin solution as a starting material.

Determination of the aggregate content in solutions:

00211 mg protein was purified by high performance gel filtration on a Superose HR 10/30 column (Pharmacia) in 70 mmol / l potassium phosphate buffer, pH 7.0 separated with a flow of 1.0 ml / min. Detection was carried out by determining the absorbance at 280 nm. The monomer or Aggregate content of the samples was about the surfaces of the protein peaks calculated.

example 2

0022An albumin solution (10%, containing 8 per mmol / l stabilizers) was mixed with 1 mol / L NaOH to pH 10.5, and then 2 h at 65 ° C heated. Thereafter, the solution was cooled to room temperature and the pH with 1 mol / l HCl back-titrated on 7th

0023Additionally, selective precipitation were the aggregates formed as follows away:<sl><li>(A) The solution was washed with ammonium sulfate to a saturation of 25%, 30% or 35% was added. The haze after appearing was removed by centrifugation and the supernatant by Gel filtration on Sephadex G-25 (PD-10, Pharmacia) in 150 mmol / l NaCl umäquilibriert. Then, in the presence of Stabilizer (sodium caprylate, N-acetyl tryptophan, per 8 mmol / l) at 60 ° C is pasteurized for 10 hours.</li><li>(B) To the solution of polyethylene glycol (PEG) 3000 to a added final concentration of 6.1%, 7.1% or 8.3% and the resulting Turbidity caused removed by centrifugation. The supernatant was then umäquilibriert as described under (a) and obtaining the final product in the presence of stabilizers pasteurized.</li></sl>

results:

0024<tables><table><tgroup cols="4"><tbody><row><entry align="center" /><entry align="center" /><entry align="center">% <b>Aggregate content in the final product</b></entry><entry align="center">% <b>Decrease in aggregate formation bez. control</b></entry></row><row><entry align="left"><b>Albumin without pretreatment (control)</b></entry><entry align="right" /><entry align="center">22.9</entry></row><row><entry align="left"><b>ammonium sulfate</b></entry><entry align="center">25%</entry><entry align="center">15.8</entry><entry align="right">31</entry></row><row><entry align="left" /><entry align="center">30%</entry><entry align="center">3.7</entry><entry align="right">84</entry></row><row><entry align="left" /><entry align="center">35%</entry><entry align="center">2.8</entry><entry align="right">88</entry></row><row><entry align="left"><b>PEG</b></entry><entry align="center">6.1%</entry><entry align="center">20.5</entry><entry align="right">10</entry></row><row><entry align="left" /><entry align="center">7.1%</entry><entry align="center">16.8</entry><entry align="right">27</entry></row><row><entry align="left" /><entry align="center">8.3%</entry><entry align="center">9.3</entry><entry align="right">59</entry></row></tbody></tgroup></table></tables>

example 3

Activation of PEG:

00255.5 g of cyanuric chloride were in 400 ml of anhydrous benzene containing 10 dissolved g of sodium carbonate. 19 g PEG 1900 were added to the mixture and stirred overnight at room temperature. The solution was filtered and slowly 600 ml of petroleum ether were added. The suspension was filtered and the precipitate dissolved in 400 ml of benzene. The precipitation and Filtration process was repeated several times in order to free cyanuric chloride remove.

Binding of the activated PEG to albumin:

00261 g of albumin was dissolved in 100 ml pH 9.2 0.1 mol / l sodium tetraborate. At 4 ° C, 8 g of activated PEG was added, and the pH during maintained at 9.2 an hour. 80 to 90% of primary amino groups have been modified with PEG. After the reaction, excess PEG is removed by diafiltration (10 kD cut-off membrane) and the solution buffer exchanged against 10 mmol / l NaCl. The solution was then as in Example 1 pre-incubated at 45 ° C, diafiltered through a 300 kD cartridge and pasteurized for 10 h in the presence of stabilizers at 60 ° C.

Result:

0027Without pre-incubation was an in PEG-modified albumin Aggregate content of 10% were measured. Preincubation and subsequent diafiltration was the aggregate content reduced to 4.5% will.

example 4

0028A 20% albumin solution was adjusted to 0.1 mol / l borate buffer pH 9.5 10% diluted and cooled to 0 ° C. Then, 20% (v / v) were cold KJ₃ Solution was added and incubated at 0 ° C for 30 minutes. The reaction was prepared by adding a few drops of NaSO₃ (1 mol / l) is stopped, the Protein solution was incubated for 2 and cooled at 60 ° C h. An aliquot was used for 8 h at 60 ° C incubated (for the evaluation of aggregate formation).

0029The rest of the solution was passed through a 300 kD membrane against 4.5 volumes 140 mmol / l NaCl diafiltered. Finally, the retentate was a Protein concentration of 20% concentrated. The ultrafiltrate was determined by 10 kD diafiltration buffer exchanged: diafiltration against 10 volumes of 140 mmol / l NaCl and then concentration of the 10 kDa Diafiltration to a protein content of 15 to 20%. The resulting protein solution was as in Example 1 in the presence of Stabilizers pasteurized.

0030With pre-treatment (pre-incubation, 300 kD diafiltration) was in iodinated Albumin achieved an aggregate content of 5.2%, with no pretreatment an aggregate content of 12.7% was measured, ie it was a Reduction by 59% can be achieved.

example 5

0031Albumin (20%) was washed with saturated sodium acetate solution pH 7.5 1: 2 diluted and cooled to 0 ° C. was within 1 h in portions Acetic anhydride added (same weight as the presented protein). Subsequently, the pH was adjusted to 7.5, the sample through a filtered 1.2 micron filter and buffered by means of 10 kD diafiltration (against 30 Volumes of 140 mmol / l NaCl).

0032The protein solution was incubated for 2 h at 60 ° C, then cooled and a 300 kD membrane against 4 volumes of 140 mmol / l NaCl diafiltered. Finally, the retentate was concentrated to about 20%. The ultrafiltrate was by 10 kD diafiltration buffer exchanged (diafiltration against 10 volumes of 150 mmol / l NaCl) and on the same membrane to a protein content of 15-20% concentrated. The solution of the acetylated albumin was subsequently pasteurized as described in Example 1 in the presence of stabilizers.

0033With pre-treatment (pre-incubation, 300 kD, diafiltration) was in Final product an aggregate content of 62% measured without the Pretreatment gelled acetylated product, ie it was not soluble Protein longer exists.

example 6

0034An albumin solution (10%) with 0.5 mmol / L EDTA was mixed with 0.1 M Perchloric acid adjusted to pH 3.2 and heated to 30 ° C. Of the Protein solution was H₂O₂ ad 0.5 mmol / l added. Subsequently for 2 h incubated at 30 ° C. After the incubation, a 10 kD diafiltration against 4 volumes of 140 mmol / l NaCl. The retentate was a 300 kD membrane against 2 volumes of 140 mmol / l NaCl diafiltered, last the retentate was concentrated to 15 to 20% protein concentration.

0035The ultrafiltrate was buffered by means of 10 kD diafiltration (10 volumes 140 mmol / l NaCl) and the same membrane to a content of 10 to 15% protein concentrate. The solution of oxidized albumin was subsequently pasteurized as described in Example 1 in the presence of stabilizers.

0036With pretreatment (300 kD diafiltration) was a in the final product Unit content of 1.1% was measured. Without the pre-treatment showed the oxidized albumin an aggregate content to 11.1%. The aggregate content could thus be reduced by 90%.

example 7

0037An albumin solution (10%, 20 ml) was treated with NaOH to pH 8.5 to 9.0 set. To this was added 50 mg of dimethyl suberimidate, suspended in 2 ml Triethanolamine added at pH 9.7 and incubated for 2 h at room temperature. The reaction was stopped by adding 10% (v / v) 0.1 mol / l Tris pH 8.5 stopped. The protein solution was then incubated for 2 h at 60 ° C and then cooled. 15 ml of this solution over a 300 kD membrane diafiltered (against 12 volumes of 140 mmol / l NaCl). Last was the Retentate possible greatly narrowed.

0038The ultrafiltrate was kD by 10 diafiltration against 30 volumes 140 mmol / l NaCl and subsequently rebuffered by vacuum dialysis against 140 mmol / l NaCl at a level of 15 to 20% protein concentrated. The solution of the modified by the cross-linker albumin was subsequently as in Example 1 in the presence of stabilizers pasteurized.

0039With pretreatment (incubation, 300 kD diafiltration) was in the final product an aggregate content of 2.5% was measured. Without the pre-treatment showed the modified albumin an aggregate content to 20.5%. Of the Aggregate content could thus be reduced by 88%.

example 8

0040One albumin solution (10% protein, 10% ethanol) 4- (2-bromoacetamido) were 2,2,6,6-tetramethylpiperidine-1-oxyl (BrAcTPO dissolved in Of ethanol, 500 mg / ml) was added (0.177 g BrAcTPO / g protein). The solution was heated to 45 ° C, the pH was adjusted with NaOH to 9.5 and by continuous addition of alkaline solution for 2 to 3 hours in this Value held. Subsequently, the pH was titrated to 7.2 with HCl and the solution heated 90 min at 60 ° C. This was followed by a 300 kD Diafiltration with 15 volumes of 140 mmol / l NaCl. The ultrafiltrate was with 10 kD, from 5 to 10 volumes of 140 mmol / l NaCl diafiltered, and finally to the same membrane concentrated to a protein content of 20%. The Solution in the presence of Stabitisatoren as described in Example 1 pasteurized.

0041With pretreatment (incubation, 300 kD diafiltration) was in polynitroxylierten albumin an aggregate content of 0.1% was measured. Without pretreatment showed the modified albumin an aggregate content of 5.5%. The aggregate content was thus reduced by more than 90% will.

example 9

0042A solution of apolipoprotein Al (10 g / l in 10 mmol / NaCl) at pH 5.0 incubated at 60 ° C for 2 h. Subsequently, the pH value was on 7.5 adjusted and guanidine HCl was added to a concentration of 2 mol / l and incubated at 45 ° C for 2 h. This solution was passed through a 300 kD Membrane diafiltered (10 volume 10 mmol / l NaCl) and subsequently with a 10 kD membrane diafiltered and concentrated to 10 g / l.

0043Without pretreatment (4 tests) were in the apolipoprotein Al Solutions aggregate contents of 2.4% to 5.4% measured with Pretreatment (3 trials) was the aggregate content to 0.4 to 0.8% be lowered.

example 10

0044A solution of immunoglobulin G (20 g / l, NaCl ≤ 3 mmol / L) was added at pH 7.0, incubated at 45 ° C for 12 h. The aggregate content increased during the pretreatment of &lt;0.1 to 1.0%. Subsequently gel-filtered over a acrylic gel (Sephacryl S-300 HR), the pH is adjusted with HCl to 5.3 adjusted and concentrated with a 10 kD membrane to 120 g / l. The aggregate content was 0.2% and remained during storage while 6 months stable at 37 ° C. In an untreated control solution the content increased under the same conditions on 0,8%.