Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/16—Central respiratory analeptics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/10—Antioedematous agents; Diuretics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/829—Blood
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/829—Blood
  • Y10S530/83—Plasma; serum

Definitions

• the present invention relates to methods for provide a highly reliable method and commercially viable method of crystallizing human albumin. More specifically, the current invention provides a method to produce crystalline human albumin purified from various albumin sources, specifically including from transgenic animals or other recombinant sources.
• the present invention is directed to an improved method of crystallizing human serum albumin ("hSA")(herein, hSA will be used interchangeably with the term human serum albumin).
• hSA human serum albumin
• This process is preferably done to enhance purification procedures for recombinant hS A that can then be utilized in therapeutic applications or as an excipient in pharmaceutical preparations.
• human albumin as purified herein can be used as a therapeutic agent or as an excipient.
• suitable formulations can be found in REMINGTON'S PHARMACEUTICAL SCIENCES (16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990)), and in INTRODUCTION TO PHARMACEUTICAL DOSAGE FORMS (4th Edition, Lea & Febiger, Philadelphia (1985)), each of which is incorporated herein by reference.
• albumin administration is primarily to maintain circulating plasma volume by maintaining the plasma colloid oncotic pressure, and to treat otherwise resistant severe edema by making intracavital and interstitial fluids move into the blood vessels.
• Albumin products are used to achieve transient improvement of the condition by replenishing albumin in pathological conditions attributable to acute hypoproteinemia, and pathological conditions resulting from chronic hypoproteinemia which is resistant to other methods of treatment.
• Albumin was the first natural colloid composition for clinical use as a blood volume expander, and it is the standard colloidal agent for comparison with other colloid products.
• Some of the specific medical indications in which albumin may be used to increase intravascular oncotic pressure and thereby expand intravascular volume in patients include: hypovolemic shock; severe burn injury; adult respiratory distress syndrome (ARDS); ascites; liver failure; pancreatitis and in patients undergoing cardiopulmonary bypass. (Cochrane et al, 1998).
• Albumin may also be used to treat neonatal hyperbilirubinemia, hypoproteinemia, and nephrotic syndrome. (Nermeulen et al., 1995).
• the albumin portion of human blood serves three primary physiologic roles: (1) maintenance of plasma colloid osmotic pressure, (2) transport and sequestration of bilirubin, and (3) transport of fatty acids and other intermediate metabolites such as hormones and enzymes.
• the albumin accounts for approximately 80% of the oncotic pressure of plasma, a 50% reduction in serum albumin concentration consequently produces a 66% decrease in colloid oncotic pressure.
• risk of death is inversely related to serum albumin concentration. (Cochrane et al., 1998).
• co-variants e.g., renal function, serum trans-aminase, lactic acidosis
• hS A is perhaps the best known of all the plasma proteins judging both by the amount of scientific literature available describing it as well as through the number of industrial uses it enjoys.
• This abundant amount of knowledge is focused primarily on its physiology and the clinical use of albumin, not the methodology used to purify it or sourcing the molecule from anything other than plasma fractionation.
• the best-known and still widely used purification methods were developed by Cohn and co-workers 60 years ago (Cohn EJ. et al., 1947).
• the Cohn plasma fractionation method is primarily used to produce purified plasma products for a wide variety of clinical uses.
• Cohn also developed a widely used crystallization process which utilizes principles similar to those well-known from plasma fractionation processes for use with human serum albumin.
• the process has significant inefficiencies and often does not provide an adequate supply of highly purified hS A. Effect of pH
• Albumin has a varying isoelectric point depending on the chemical treatment that it has received. With a full complement of six bound fatty acids hS A' s pi is normally 4.6, however, when fully de-fatted its pi may be as high as 5.6. Therefore, the crystallization properties of hSA vary as between its "native" and defatted states and the reported optimum pH for the crystallization of hSA itself varies substantially in the literature from a low of pH 4.6 to a high of pH 8.0 and may be highly dependent upon the molecular state of hSA in a batch-by-batch basis.
• hSA has a very high solubility in varying salt concentrations. It can be precipitated or crystallized at low ionic strength with added ethanol (Cohn process; Cohn E.J. et al., 1947) or other solvents. Alternatively, salting out with very high salt concentration is possible, and the early literature mostly used ammonium sulfate or ethanol is described. In the more recent literature PEG solutions of various molecular weights have be utilized widely. However, the reagents present in the literature are unacceptable for the clinical use of the resulting hSA because of the remaining contaminants. Of the prior art precipitating reagents only ethanol and ammonium sulfate are useful in the production of has. However, they both have significant practical problems. Crystallization with ethanol requires the addition of toxic organic modifiers such as benzene or heavy metals. Ammonium sulfate is not a suitable salt for final albumin formulation, and would thus need to be removed.
• albumin In addition to its other characteristics albumin has an extraordinary capacity to combine and attach to a wide variety of smaller molecules and ions.
• reagents capable of significantly effecting the crystallization profile of hSA include: decanol, palmitic acid and caprylic acid.
• the time period needed for the crystallization of albumin in a given reaction to be complete can take up to several days.
• those employing ethanol may be the most rapid, requiring only 12 to 24 hours to initiate crystallization.
• the actual crystallization of albumin may not be possible at all without additives including seeding a reaction mixture with crystals formed from a prior reaction.
• the methods of crystallization relying on PEG may or may not utilize this type of additive. It should be noted that seeding does speed up the crystal growth significantly, though given the confusion in the art generally there are no references that can be utilized which give consistently reliable results or generate a high yield of crystal.
• the present invention provides improved methods for producing crystalline human albumin.
• the method is suitable for various albumin sources including: human plasma, and from recombinant albumin sources such as cultured mammalian cells, the milk or other bodily fluids of transgenic mammals, transgenic plants, transgenic avians, recombinant bacterial cell cultures, recombinant yeast cell cultures or recombinant insect cell cultures. That is, it is useful for the production of crystallized and pharmaceutically grade hSA regardless of the feedstream from which it comes.
• the method has a high purification power so that crystalline albumin can be effectively separated from other proteins, bacteria, fatty acids or other molecular species present in a particular starting material or feedstream.
• albumin can be dissolved and re-crystallized by heating and cooling cycle in the crystallizing medium. This, re-crystallization procedure can be repeated unlimited number of times according the needs of the user of the current invention. Purity of albumin is regularly improved in the re-crystallization procedure.
• the methods of the current invention also provide precise combinations of reagents and conditions that allow the optimization of the production of crystalline human albumin. In these methods important the process parameters such as pH and temperature are precisely manipulated.
• An additional embodiment of the current invention provides optimal concentrations of precipitating agents of sodium or potassium phosphates and/or caprylic acid or caprylate salts.
• the process of the current invention is based on certain key factors influencing the crystallization of human albumin.
• the process of the current invention optimizes the following variables in a specific manner so as to optimize the crystallization protocol parameters as follows:
• FIG. 1 shows a process flow diagram for crystallization of recombinant human serum albumin.
• FIG. 2 shows albumin solubility at different temperatures at pH 6.2, with exponential trendlines.
• FIG. 3 shows albumin solubility at different temperatures at pH 6.2, with linear trendlines
• FIG. 4 shows pH Solubility of albumin crystals at 15°C.
• FIG. 5 provides a solubility study of washed albumin crystals in different phosphate solution at a pH of 6.2.
• FIG. 6 provides a solubility study of washed albumin crystals in a 2.63M phosphate solution with varying caprylate concentration.
• FIG. 7 shows a heat precipitation study of hSA crystal slurry in 2.7 M phosphate pH 6.2.
• FIG. 8 shows a process flow diagram for preparative crystallization of albumin.
• FIG. 9 shows a process flow diagram for crystallization of recombinant human serum albumin.
• FIG. 10 shows a process flow diagram for the re-crystallization of recombinant human serum albumin.
• FIG. 11 shows crystallized human albumin in a solution mixture that is 2.3
• FIG. 12 shows crystallized human albumin, shows crystallized human albumin in a solution mixture that is 2.5 Molar Na-K-Phosphate with a pH of 6.2; saturated with Caprylate; hSA 46.5 mg/ml crystallized at 4°C overnight.
• FIG. 13 shows crystallized human albumin in a solution mixture that is 2.3 Molar Na-K-Phosphate with a pH of 6.2; Caprylate 1.4mg/ml; hS A 80 mg/ml crystallized at 4°C overnight, with 0 hours at room temperature (RT) in an air tight chamber.
• FIG. 14 shows, albumin crystals of the invention along with more amorphous precipitate.
• the typical crystal size is approximately 0.1 x 0.4 mm.
• the precipitate disappears over time becomes crystalline.
• the crystals were prepared at 4°C in 2.7 Molar ammonium sulfate containing 0.08 % decanol; 0.05 M Na-K phosphate atpH 7.4. Crystallized at 4°C hSA 58.7 mg/ml. DESCRIPTION OF THE PREFERRED EMBODIMENT
• pH A term used to describe the hydrogen-ion activity of a chemical or compound according to well-known scientific parameters.
• Colloids - refers to large molecules that do not pass readily across capillary walls. These compounds exert an oncotic (i.e., they attract fluid) load and are usually administered to restore intravascular volume and improve tissue perfusion.
• Diafiltration - An operation incorporating ultrafiltration membranes to efficiently remove salts or other small molecules from a macromolecular solution. The purpose is to remove small molecules from albumim in soltuon and adjust the buffer for the next procedure.
• Tissue Perfusion The amount of blood flow to tissue.
• Feedstream The raw material or raw solution provided for a process or method and containing a protein of interest.
• the methods of the current invention for the crystallization of hSA provide a highly desirable method to separate and purify albumin from a feedstream containing a variety of other protein components.
• Crystals are the most pure form of protein, once precipitated crystals have significantly better mechanical handling properties than amorphous precipitates and can be separated by a variety of methods known in the field. For example, crystals can be separated and washed efficiently on industrial filters. Crystallization is the most used final purification method of fine chemicals and pharmaceuticals.
• albumin is crystallized with a mixture of sodium and potassium phosphates. Crystallization is optimized by using the invented process conditions and methods in a systematic manner. Albumin may precipitate as amo ⁇ hous phase, liquid droplets or gel if the conditions are not adjusted optimally. Amorphous precipitate is very difficult to handle, it can not be separated and washed efficiently on filters. Amorphous phase does not readily convert to crystals. Crystals production is optimized when the process conditions are adjusted according to this invention. Various embodiments of the current invention are provided below.
• albumin is crystallized in relatively high phosphate concentration, typically around 2.7 M or higher.
• phosphate concentration typically around 2.7 M or higher.
• Figures 1 and 2 describe the solubility of albumin, crystals in phosphates with different molarities.
• the semi- logarithmic plot of Figure 2 shows that crystal solubility is systematically lowered by increasing phosphate molarities.
• Albumin yield can be adjusted in the process to a desirable level by using increasing molar concentrations of phosphates.
• Upper limits for usable phosphate concentrations are set by the solubility of phosphate salts.
• Phosphate solubility is lowered by lowering temperature and increasing albumin concentration.
• phosphate solubility is significantly lowered by reducing the temperature of the reagent to below 10°C.
• the available water concentration is also being lowered.
• most of the impurities precipitate effectively in starting material in the range of phosphate concentration 2.0 - 2.7 M, where albumin does not readily crystallize at room temperature (25 - 30 °C).
• the impurities are removed by being filtered (after adjusting phosphate concentration 2.0 - 2.7 M). Thereafter the filtrate is refrigerated to 10 °C where albumin is crystallized.
• phosphate concentration can be increased (utilizing the information of Figures 1 and 2) in order to increase albumin yield in the crystals, see also Table 2 and Figure 4. 2. Significance of pH:
• the pH of the crystallizing batch is fully controlled with the phosphate mixture.
• Examples of the mixtures with various pH values and the resulting effect on the albumin crystals are presented in the Table 1.
• the effect of pH on the albumin crystal solubility is presented graphically in the Figure 2, see also Table 2.
• Albumin crystal solubility is lowered by lowering pH from pH 5.6 to approximately 6.6.
• the crystal solubility remains at very low level up to at least pH 7.4. Crystals are completely dissolved below pH 5.5.
• the pH has a specific effect on the crystallization kinetics, thus the higher pH range 6.3 - 7.4 can not be used in a simple way.
• albumin precipitates as amo ⁇ hous (liquid droplet) phase if such pH is adjusted right in the beginning of the process.
• the crystallization process is preferably made initially with the phosphate pH 6.2 (see table 1 for mixing recipe of phosphates and Figures 2 and 3). Later on when albumin is mostly crystallized, phosphate concentration is increased and pH adjusted to higher value in order to increase the crystal yield. Higher pH is also used advantageously when washing crystals, since the loss of albumin is reduced.
• albumin crystal solubility is increasing at higher temperatures. Lowest solubility is found around 0°C. However, the preferred crystallizing temperature is around 10°C, since at lower temperature phosphate may crystallize and bring the process out of control. The example procedures around 0°C were possible to be performed, since phosphate crystallization is relatively slow.
• albumin solution is refrigerated to preferably 10°C.
• the solution is stirred and albumin is crystallized.
• Albumin can be re-crystallized by utilizing a heating cycle. The crystals are dissolved by heating to 45 °C. Albumin is crystallized again after refrigeration to 10 °C. Re-crystallization can be used to improve the crystal purity. After each crystallization, the mother liquor can be removed by being filtered. Crystals can be washed on the filter with cold 3 M phosphate. Re-crystallization cycle can be repeated unlimited number of times. Recrystallization can be used to improve albumin purity. For convenience please see Tables 7 and 8 and Figure 7.
• Albumin is relatively heat stable. It can be heated up to 65 - 70 °C for prolonged periods. Most other proteins denature and precipitate at such high temperatures. Thus heating treatment at 65 - 70 °C can be used to purify albumin solution prior to crystallization.
• the highest tolerable temperature is related to the composition and pH of albumin solution, hi high phosphate concentration pH 6.2 the highest temperature is 65 °C. hi low salt medium and pH 5.4 heating at 70 °C for 2 - 3 hours is possible. Examples of the effect of heat treatment are presented in the Tables 2 and 3 and Figure 6.
• Albumin needs to be saturated with caprylate to be able to crystallize.
• Caprylate has also a stabilizing effect on the albumin, specially on heat stability. Other long chain fatty acids and long chain alcohols are alternatively useful.
• Decanol is very effective and well known in prior art. Caprylate has dual effect depending of how it is used. It is beneficial when it is used only to saturate the binding sites of albumin. However, excess of caprylate will dissolve crystals and reduce albumin yield.
• caprylate is well revealed in the Figure 4. Addition of caprylate to the washed crystals clearly increased crystal solubility. Crystal solubility was rapidly increasing when caprylate was increasing up to 10 mM. The solubility increment was smaller but still significant when caprylate was increasing from 10 mM to 20 mM. At phosphate concentration 2.63 M and temperature 10°C, albumin solubility increased from 9 mg/ml in 10 mM caprylate to 21 mg/ml in 10 mM caprylate. At higher phosphate concentration 2.82 M and temperature 10 °C, albumin solubility increased from 2 mg/ml in 0 mM caprylate to 12 mg/ml in 10 mM caprylate.
• Dissolving effect of caprylate is so significant that concentration of free caprylate should be well controlled and maintained as low as possible in the crystallizing step. Small level of free caprylate, order of 1-2 mM may be acceptable when conditions are otherwise such that albumin crystal solubility is very low. Please see Figures 2 through 7.
• albumin concentration in the starting solution is set to a level that is higher than the solubility of crystals in the conditions utilized.
• initial clarification steps are typically used to provide a solution in with the concentration of albumin and other chemical parameters are adjusted or manipulated such that it is also higher than the solubility of hSA crystals.
• albumin recovery can be estimated by using the solubility information in the phase diagrams ( Figures 1-4).
• concentration levels of albumin in feedstream solutions are typically in the range of 15 - 300 grams of albumin in one liter of crystallizing batch. In feedstreams from biological sources and usable for the commercial or industrial production of hSA these same ranges are encountered.
• albumin need not be pure in the crystallizing process according to the preferred embodiments of the current invention.
• the processes developed - and provided by the current invention can be utilized to crystallize out albumin in source material wherein the level of purity is approximately 10%, that is, where albumin constitutes only 10% of the total protein of a given solution.
• the level of purity is approximately 10%, that is, where albumin constitutes only 10% of the total protein of a given solution.
• hSA sourced from either transgenic sources or cell cultures most of the impurities remaining after clarification procedures can be removed after precipitation with the first addition of phosphate up to 2.6 M concentration level.
• transgenic sources typcially milk, but also including other bodily fluids such as blood or urine may contain hSA as a consequence of the insertion of DNA constructs designed to cause the stable expression of hSA (or other protein of interest) in those bodily fluids or tissues.
• the filtrate is further concentrated by ultrafiltration in order to increase albumin concentration level.
• Phosphate stock solution containing 2.8 moles of NaH 2 PO 4 and 1.2 moles of K 2 HPO 4 dissolved in water and filled to 1.0 liter, was used in crystallization.
• This 4 M phosphate solution had pH 6.2 when measured after dilution to 0.5 M.
• 200 ml of a purified albumin solution was precipitated by adding 371 ml of 4.0M phosphate stock solution at a pH of 6.2. On the basis of the added volume, the phosphate concentration was 2.6 M.
• the solution was allowed to precipitate at room temperature for 4 - 18 hours, for this variation of the current invention the time period of interest is short relative to prior art methods, in all cases less than 24 hours.
• the amount of the produced precipitate is directly related to the amount of impurities in the albumin solution.
• the precipitated hSA was filtered through glass fiber or cellulose fiber paper having approximately 1 ⁇ m pore size.
• the filtered solution was then used in a preferred crystallization procedure according to the invention.
• Crystallization was performed in 10 °C thermostat incubator. The batch was stirred slowly (approximately 70 ⁇ m) with a top driven propeller. Phosphate concentration was increased gradually from 2.6 M to 3 M by adding 229 ml of 4M phosphate which was at a pH of 6.2. The crystallization batch was continued for a period of 4 days before harvesting and washing.
• hSA crystals were harvested by filtering the batch with glass fiber paper (1 ⁇ m pore size, 142 mm diameter). After being filtered, crystals were then washed with about 80 ml of cold 3 M phosphate pH 6.2. Crystals were then suspended in about 150 ml of cold 3 M phosphate pH 6.2.
• the mixing volume ratios are kept fixed.
• the pH should not be adjusted after making the buffer mixture.
• the pH values are approximate, since the value will change with the alteration in phosphate concentration.
• the buffers of the current invention were used in the study for effect of pH on crystal solubility.
• Buffer No: 10 provides conditions that are consistently optimized for crystal development and the conditions provided for this buffer are the preferred conditions for the standard buffer of the current invention. (See also, Figures 11-13).
• pH values are for diluted buffers in the range of 0.1 - 0.4 M.
• the buffers No: 1 - 10 have lower pH when M is higher.
• Buffers No: 12- 30 have higher pH when molarities are higher.
• Figure 6 of the current disclosure provides a profile of albumin solubility in 2.63 M phosphate as a function of caprylate concentration and temperature: open circles (o) mark samples incubated at 10 °C and black squares ( ⁇ ) mark samples incubated at 5°C.
• Crystallization examples from 48 samples were prepared according to a hanging drop microdiffusion method known in the art.
• the sample solution contained purified 198 mg/ml of albumin and 3.2 mg/ml sodium caprylate.
• the liquid solution of albumin was prepared by mixing 3 ⁇ l of albumin solution with 3 ⁇ l of 1.8 - 2.3 M phosphate.
• the drops were allowed to equilibrate in the closed microdiffusion wells and refrigerated at 5 °C. Crystals were produced in less than 24 hours according to this embodiment of the the current invention.
• feedstreams from other source material especially transgenic and cell culture sources, can also be utilized in conjunction with a microdiffusion hanging drop method.
• Crystallization of hSA is started by adding more of 4 M phosphate (pH 6.2) in the filtrate until concentration is 2.8 M. Crystallization is performed at refrigerated temperatures preferably around 5.0°C. Crystallization of the hSA, according to this embodiment of the current invention starts spontaneously within 24 hours. In an alternate embodiment the addition of seed crystals will make the process more rapid.
• crystals are washed either by centrifugation or preferably by being filtered.
• the washed crystals are dispersed in a small volume of the 2.8 M phosphate buffer. Crystals can be stored in this until formulated for the next step.
• the feedstream material used for this example had had a significant amount of proteins as impurities. Albumin was approximately 30% of the protein present. As already stated this is within the typical range of feedstream materials supplied from clarified or partially purified transgenic or cell culture sources.
• the solutions used included 107 ml of 4 M phosphate pH 6.2 was added into 200 ml of starting material solution (which was in 2 M phosphate before this step) to get 2.70 M solution. According to the current invention this precipitated solution was used as starting solution for crystallization.
• the precipitated starting solution (in 2.70 M phosphate) was incubated at 55 °C for 90 minutes. A substantial amount of rod-like crystals were formed along with an amo ⁇ hous precipitate as a result of this heat treatment. Approximately 75% to 80% of total protein was crystallized or precipitated. The crystals and precipitate were removed by filtering the slurry through glass fiber filter when it was still hot. Some diatomaceous earth was used as a filter aid. Only the supernatant filtrate containing albumin was taken for the next step. Concentration and Filtration
• Genzyme Transgenics hSA albumin concentrate lot # XI 13 IFF
• protein concentration assays TP 84 g/1 and 79 g/1, ALB 28.58 g/1 and 28.75 g/1.
• albumin was crystallized very effectively when pH was increased to the values 6.4 and 6.5. Albumin solubility was very low above pH 6.4. Unfortunately, at the higher pH levels albumin was crystallized as very small needles (See Figures 10 - 13). It is likely that the crystal size could be developed larger by starting the crystallization at lower pH 6.2 or 6.3. After achieving near equilibrium, pH could be adjusted to higher level by gradually adding 4M K 2 HPO 4 .
• the washed crystals (102-12) were heated 5 minutes at 45 °C. The crystals dissolved rapidly. The solution was filtered with 0.45 ⁇ m syringe filter while still warm. The filtered solution was agitated at 2 °C until crystallized (3 days). The batch was crystallized well ( Figure 2). The crystals were harvested by being filtered and washed with 2.82 M pH 6.2 phosphate on a 0.45 ⁇ m (50 mm diameter) Sartorius membrane. The washed crystals were dispersed in 2.82 M phosphate buffer.
• Hypovolemia is a possible indication for albumin purified and made available by the method of the current invention, 25% Solution, Buminate 25%. Its effectiveness in reversing hypovolemia depends largely upon its ability to draw interstitial fluid into the circulation. It is most effective with patients who are well hydrated. When hypovolemia is long standing and hypoalbuminemia exists accompanied by adequate hydration or edema, 25% albumin is preferable to 5% protein solutions. However, in the absence of adequate or excessive hydration, 5% protein solutions should be used or 25% albumin should be diluted with crystalloid. Although crystalloid solutions and colloid-containing plasma substitutes can be used in emergency treatment of shock, albumin has a prolonged intravascular half-life. When blood volume deficit is the result of hemorrhage, compatible red blood cells or whole blood should be administered as quickly as possible.
• Hypoalbuminemia is another possible indication for use of albumin purified and made available by the method of the current invention, 25% Solution, Buminate 25%.
• Hypoalbuminemia can result from one or more of the following: Inadequate production (malnutrition, bums, major injury, infections, etc.); Excessive catabolism (burns, major injury, pancreatitis, etc.); Loss from the body (hemorrhage, excessive renal excretion, bum exudates, etc.); and Redistribution within the body (major surgery, various inflammatory conditions, etc.).
• albumin deficit is the result of excessive protein loss, the effect of administration of albumin will be temporary unless the underlying disorder is reversed. In most cases, increased nutritional replacement of amino acids and/or protein with concurrent treatment of the underlying disorder will restore normal plasma albumin levels more effectively than albumin solutions. Occasionally hypoalbuminemia accompanying severe injuries, infections or pancreatitis cannot be quickly reversed and nutritional supplements may fail to restore serum albumin levels. In these cases, albumin (Human), 25% Solution, Buminate 25% might be a useful therapeutic adjunct.
• ARDS Adult Respiratory Distress Syndrome
• a characteristic of ARDS is a hypoproteinemic state which may be causally related to the interstitial pulmonary edema. Although uncertainty exists concerning the precise indication of albumin infusion in these patients, if there is a pulmonary overload accompanied by hypoalbuminemia, 25% albumin solution may have a therapeutic effect when used with a diuretic.
• Albumin (Human), 25% Solution may be a useful aid in treating edema in patients with severe nephrosis who are receiving steroids and/or diuretics.
• Albumin (Human), 25% Solution, Buminate 25% may be administered in an attempt to bind and detoxify unconjugated bilirubin in infants with severe HDN.
• albumin purified by means of the current invention as an excipient for the delivery of pharmaceuticals.
• the hanging drop screens are made in boxes with 24 wells divided in 4 rows (A, B, C, D) and 6 columns.
• the screens are presented in the preliminary report only as lists of the reagents as follows. A brief comment to the results is provided. In the final report the results are provided in tables with complete details similar to the following example tables.
• AA A significant amount of amo ⁇ hous precipitate
• L liquid phase separation, spherical transparent droplets, looking like oil in water
• G gel lumps, irregular glassy transparent particles of several micrometers in diameter GG — A significant amount of gel
• Row D like row C, but add 50 ⁇ l 4% decanol to each 500 ⁇ l of A reagents to get final 0.36 % decanol.
• Results Crystals of high quality ( Figure 14) were produced with 2.4M and 2.7M ammonium sulfate-decanol combinations Crystals of a different habit were produced with 19% PEG, 0.08% decanol. Amo ⁇ hous or gel precipitates were also produced with both reagents. Crystallization is very critically related to the reagent concentration and temperature.
• a growing number of recombinant proteins are being developed for therapeutic and diagnostic applications. However, many of these proteins may be difficult or expensive to produce in a functional form and/or in the required quantities using conventional methods.
• Conventional methods involve inserting the gene responsible for the production of a particular protein into host cells such as bacteria, yeast, or mammalian cells, e.g., COS or CHO cells, and then growing the cells in culture media. The cultured cells then synthesize the desired protein.
• Traditional bacteria or yeast systems may be unable to produce many complex proteins in a functional form. While mammalian cells can reproduce complex proteins, they are generally difficult and expensive to grow, and often produce only mg/L quantities of protein.
• non-secreted proteins are relatively difficult to purify from procaryotic or mammalian cells as they are not secreted into the culture medium.
• the transgenic technology features, a method of making and secreting a protein which is not normally secreted (a non-secreted protein).
• the method includes expressing the protein from a nucleic acid construct which includes:
• a promoter e.g., a mammary epithelial specific promoter, e.g., a milk protein promoter
• a signal sequence which can direct the secretion of a protein e.g. a signal sequence from a milk specific protein
• a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein e.g., a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the non-secreted protein
• a sequence which encodes a non-secreted protein wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited.
• elements a, b, c (if present), and d are from the same gene; the elements a, b, c (if present), and d are from two or more genes.
• the secretion is into the milk of a transgenic mammal.
• the signal sequence is the ⁇ -casein signal sequence; the promoter is the ⁇ -casein promoter sequence.
• the non-secreted protein-coding sequence is of human origin; codes for a truncated, nuclear, or a cytoplasmic polypeptide; codes for human serum albumin or other desired protein of interest.
• the transcriptional promoters useful in practicing the present invention are those promoters that are preferentially activated in mammary epithelial cells, including promoters that control the genes encoding milk proteins such as caseins, beta lactoglobulin (Clark et al., (1989) Bio/Technology -_7: 487-492), whey acid protein
• Casein promoters may be derived from the alpha, beta, gamma or kappa casein genes of any mammalian species; a preferred promoter is i derived from the goat beta casein gene (DiTullio, (1992) Bio/Technology 10:74-77).
• the milk-specific protein promoter or the promoters that are specifically activated in mammary tissue may be derived from either cDNA or genomic sequences. Preferably, they are genomic in origin. [0098] DNA sequence information is available for all of the mammary gland specific genes listed above, in at least one, and often several organisms. See, e.g., Richards et al., J. Biol.
• the signal sequences that are useful in accordance with this invention are milk-specific signal sequences or other signal sequences which result in the secretion of eukaryotic or prokaryotic proteins.
• the signal sequence is selected from milk-specific signal sequences, i.e., it is from a gene which encodes a product secreted into milk.
• the milk-specific signal sequence is related to the milk-specific promoter used in the expression system of this invention.
• the size of the signal sequence is not critical for this invention. All that is required is that the sequence be of a sufficient size to effect secretion of the desired recombinant protein, e.g., in the mammary tissue.
• signal sequences from genes coding for caseins e.g., alpha, beta, gamma or kappa caseins, beta lactoglobulin, whey acid protein, and lactalbumin are useful in the present invention.
• the preferred signal sequence is the goat ⁇ -casein signal sequence.
• Signal sequences from other secreted proteins e.g., proteins secreted by liver cells, kidney cell, or pancreatic cells can also be used.
• Transgenic Mammals [00101]
• the DNA constructs of the protein of interest in this case human serum albumin, are introduced into the germ line of a mammal.
• one or several copies of the construct may be inco ⁇ orated into the genome of a mammalian embryo by standard transgenic techniques.
• Any non-human mammal can be usefully employed in this invention.
• Mammals are defined herein as all animals, excluding humans, that have mammary glands and produce milk. Preferably, mammals that produce large volumes of milk and have long lactating periods are preferred. Preferred mammals are cows, sheep, goats, mice, oxen, camels and pigs. Of course, each of these mammals may not be as effective as the others with respect to any given expression sequence of this invention. For example, a particular milk-specific promoter or signal sequence may be more effective in one mammal than in others. However, one of skill in the art may easily make such choices by following the teachings of this invention.
• albumin is crystallized with various compounds, ethanol and mineral salts including phosphates industrial methods for crystallization with phosphates are not found in the literature.
• human albumin can be crystallized advantageously with phosphate salts by utilizing in full extent the invented key process parameters and/or conditions of the current invention. The invented parameters and some variations thereof are listed and described above.
• Carter DC et al., Preliminary Crystallographic Studies of Four Crystals Forms of Serum Albumin, EUR JBlOCHEM (1994); 226: 1049-1052. 5. Carter DC, et al., Three-Dimensional Structure of Human Serum Albumin,

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