WO1989006976A1 - Method of drying macromolecules - Google Patents

Method of drying macromolecules Download PDF

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Publication number
WO1989006976A1
WO1989006976A1 PCT/GB1989/000093 GB8900093W WO8906976A1 WO 1989006976 A1 WO1989006976 A1 WO 1989006976A1 GB 8900093 W GB8900093 W GB 8900093W WO 8906976 A1 WO8906976 A1 WO 8906976A1
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WO
WIPO (PCT)
Prior art keywords
dried
sulphate
salt
water
efflorescent
Prior art date
Application number
PCT/GB1989/000093
Other languages
English (en)
French (fr)
Inventor
Bruce Joseph Roser
Original Assignee
Quadrant Bioresources Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Quadrant Bioresources Limited filed Critical Quadrant Bioresources Limited
Publication of WO1989006976A1 publication Critical patent/WO1989006976A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • This invention relates to an improved process for drying biological materials, e.g. proteins and other macromolecules, viruses etc.
  • biological materials e.g. proteins and other macromolecules, viruses etc.
  • the invention concerns a method of obtaining a dried product which will remain dry without being hermetically sealed or otherwise carefully stored.
  • the invention also relates to dried products produced by such a method.
  • a method of drying a biological macromolecular substance by drying an aqueous solution or suspension thereof, optionally in the presence of stabilising material, characterised in that the aqueous solution or suspension is formulated in the presence of one or more efflorescent alkali metal, ammonium or alkaline earth metal salts and then dried.
  • the method depends on replacement of the deliquescent salts, such as sodium chloride, in conventional buffers/osmotic pressure regulators with efflorescent salts such as sodium sulphate
  • efflorescent is used herein to refer to salts which lose water under ambient humidity, e.g. those salts having a vapour pressure of water of crystallization of at least 15mm Hg (2000 Pa).
  • Various salts can be used depending on the nature of the material to be dried. Naturally, the salt must be compatible with the material to be dried. For food applications the approved additive calcium lactate is efflorescent and suitable.
  • Other efflorescent salts which might find application in certain formulations include: 1.
  • Sodium salts acetate trihydrate, tetraborate decahydrate (borax), bromoiridite dodecahydrate, carbonate heptahydrate, metaperiodate trihydrate, metaphosphate(tri)hexahydrate, hydrogen orthophosphate dodecahydrate, sulphite heptahydrate, thiosulphate pentahydrate (hypo).
  • Non-sodium salts (where applicable): calcium lactate hydrate, magnesium salicylate tetrahydrate, magnesium sulphate heptahydrate (Epsom salts) and ammonium sulphate.
  • the efflorescent salt can be added as such, without the need to replace the buffer salt, provided that care is taken to maintain appropriate molarities.
  • sodium sulphate When dissolved in an excess of water, sodium sulphate gives a solution of neutral pH and is non reactive in solution and non toxic to cells or tissues.
  • a 0.12M solution is roughly isotonic with mammalian serum. It substitutes in all significant respects for the physiological salt sodium chloride.
  • Example 1 Quantitation of water adsorption by dried protein films under controlled humidity in the presence of hygroscopic and efflorescent buffers.
  • a 10% w/v solution of salt-free bovine plasma albumin (BSA) was made up in either physiological Dulbecco's phosphate-buffered saline (PBS) or in 0.12M sodium sulphate solution. Samples of about 1ml of solution were spread in pre-weighed sterile polystyrene tissue culture grade Petri dishes (Nunclon 60 ⁇ 15mm Cat No.1-50288) and dried by being left open in a warm room (37oC) for 48hr. They were then weighed on a 5 decimal place Mettler HK60 electronic microbalance to obtain the dry weight of protein + buffer.
  • PBS physiological Dulbecco's phosphate-buffered saline
  • 0.12M sodium sulphate solution 0.12M sodium sulphate solution.
  • RPE fluorescent reagent R-Phycoerythrin
  • the reagents consisted of a conjugate of the biotin-binding protein Streptavidin, (purified from Streptomyces avidinii), with the fluorescent phycobiliprotein R-Phycoerythrin. This conjugate was dried in flat bottomed wells of a 96-well microtitre plate in buffers of various formulations containing the disaccharide trehalose as a means of completely preventing the loss of conjugate activity on drying.
  • the buffer formulations used were various ratios of standard Phosphate-buffered saline and 0.1M sodium sulphate ranging from 90% of the former plus 10% of the latter to 90% sodium sulphate buffer and 10% PBS.
  • Plates containing these preparations were dried in a warm room at 37oC for 24 hr. Samples of these plates were then stored at 37°C or at room temperature or at 4-10°C in a standard refrigerator for 2 months.
  • the dried preparations were tested for their ability to stain rat lymphocytes labelled with saturating amounts of a biotin-labelled monoclonal mouse anti-rat CD4 antibody W3/25. (D.W.Mason. R.P.Arthur, M.J.Dallman, J.R.Green, G.P.Spickett & M.L.Thomas. 1983, Immunological Reviews, 74 pp.57-82).
  • the stained preparations were produced by rehydrating the wells of the microtitre plates with 50 ⁇ l distilled water and then adding a suspension of 10 6 rat lymph-node lymphocytes to each well.
  • the lymphocytes were suspended in complete RPMI 1640 tissue culture medium containing 10% Haemaccel as a protein supplement (TCM).
  • the intensity of staining of these lymphocytes with the R-Phycoerythrin/Streptavidin conjugate is a direct measure of the activity of the conjugate.
  • the fluorescence intensity is recorded as the logarithm of the brightness on the X axis against the number of cells on the Y axis. Thus any decrease in activity is revealed as a shift to the left of the modal value (peak) of the brightly fluorescent cells.
  • the preservation of activity in the conjugates was dependent on preservation of the dry state. This could be achieved by maintaining the conjugates in a dry environment such as the warm room or by using a high concentration of sodium sulphate buffer which maintained the dried conjugate in the dry state by efflorescence even in humid conditions.
  • an instant two colour immunofluorescence assay was formulated in the dry state by mixing 25 ⁇ l of a solution of R-Phycoerythrin conjugated to the mouse IgGi monoclonal anti rat CD4 antibody W3/25 and 25 ⁇ l of a solution of fluorescein conjugated to the mouse IgGi anti rat CD8 antibody MRC OX-8 (both dissolved in 0.1M sodium sulphate solution) was 50 ⁇ l of 10% trehalose in distilled water in the wells of a microtitre plate and drying at 37°C.
  • SA/RPE conjugates were dried at 37°C in the wells of flat-bottomed microtitre plates in buffers which consisted of various mixtures of 0.1M sodium sulphate and PBS. Thereafter they were stored either at room temperature, 4-10°C in a refrigerator or at 37oC in a warm room for 2 months before use for staining.
  • F1 2 red fluorescence on RPE
  • F1 1 green fluorescence of FITC
  • Middle conjugate dried in 0.1M sodium sulphate containing 10% trehalose and stored for 1 month at RT O before staining
  • Bottom conjugate dried in 0.1M sodium sulphate buffer without trehalose showing loss of staining potency.
  • Sodium sulphate buffer maintains trehalose-dried conjugate with full potency when stored at room temperature but does not substitute for trehalose as a preservative of conjugate function.
  • the model system consisted of 1ml 1% bovine serum albumin plus 10% trehalose in 10mM Trishydroxymethylaminomethane (Tris) chloride buffer pH 7.4 to which was added the relevant concentration of the test salts.
  • Tris Trishydroxymethylaminomethane
  • the mixture was dried at 3° for 24hr in hydrophobic plastic dishes preweighed on a top-pan balance to an accuracy of 1 milligram, then reweighed and transferred to sealed chambers in which the relative humidity was controlled at 20-22°C by saturated solutions of various salts as described in "Handbook of chemistry and physics" 67th edition p E-42 R.C. Weast editor (1986) CRC press Paton Fla. USA.
  • the plastic dishes were removed and immediately reweighed.. After about 120 hr they were returned to ambient temperature and relative humidity and reweighed at intervals thereafter until their weight was stable.
  • Zinc sulphate heptahydrate (ZnSO 4 .7H 2 O)
  • BSA readily dissolved in 200mM and 300mM solution. Solutions were dried at about 47% RH in an incubator room at 37° and then transferred at 24hr from the 37° room to humidity chambers at 51% RH, 80% RH or 90% RH. There was uptake of water at all 3 RH. This is probably due to the fact that the dodecahydrate crystals lose water at 37° to the dihydrate which then absorbs water to the heptahydrate form in more humid environments. This is the form that is stable at ambient temperature and humidity. This is supported by the fact that 1ml
  • 300mMolar disodium hydrogen orthophosphate dihydrate theoretically absorbs 1.5 ⁇ 10 -3 gm moles of water to form the heptahydrate. This amounts to 27mg which is precisely the increase in weight of the phosphate-dried mixture on equilibration with ambient humidity.
  • BSA readily dissolves in 200mM or 300mM sodium sulphate. When transferred from the 37° room RH to humidity chambers, there was no water uptake at 51% or at 80% RH. There was uptake of 50% of the initial weight only at 90% RH. This water was promptly lost on return to roon air. Fig. 4. 5. Sodium chloride (NaCl). [control, non efflorescent]
  • Both disodium hydrogen orthophosphate and sodium sulphate prevent uptake of water by the hygroscopic mixture of BSA and trehalose at 80% RH and reduce the water uptake by 50% at RH of 90% when compared with the neutral (non hygroscopic, non efflorescent) salt sodium chloride.
  • the phosphate buffer is as effective limiting the uptake of water at 90% RH when the water of hydration of the heptahydrate (stable) form of the salt is taken into account.
  • Na 2 SO 4 is the ideal salt as it is neutral in solution, non toxic, nonreactive with biological molecules, has a normal salty taste and will protect trehalose-dried biological molecules from moisture damage at relative humidities up to between 80 and 90%.
  • Disodium hydrogen orthophosphate is another example of an effective salt with a potentially slightly higher protective effect. It suffers from the disadvantage that reducing the pH will lead to the formation of sodium dihydrogen orthophosphate which is slightly deliquescent. If the pH is not adjusted a solution of disodium hydrogen phosphate in water is alkaline with a pH of about 9.5. This is thus not a suitable salt for biological molecules which require a physiological pH of 7.4.

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  • Health & Medical Sciences (AREA)
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PCT/GB1989/000093 1988-02-01 1989-02-01 Method of drying macromolecules WO1989006976A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB888802174A GB8802174D0 (en) 1988-02-01 1988-02-01 Method of drying macromolecules
GB8802174 1988-02-01

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WO1989006976A1 true WO1989006976A1 (en) 1989-08-10

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EP (1) EP0353281A1 (cs)
JP (1) JPH02504312A (cs)
CN (1) CN1037518A (cs)
AU (1) AU3054889A (cs)
CS (1) CS68889A2 (cs)
ES (1) ES2018102A6 (cs)
GB (1) GB8802174D0 (cs)
WO (1) WO1989006976A1 (cs)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0415567A3 (en) * 1989-07-31 1991-08-21 Quadrant Bioresources Limited Composition and method for stabilising organic compounds
US5422254A (en) * 1992-02-14 1995-06-06 Oy Alko Ab Method to increase the trehalose content of organisms by transforming them with the structural genes for the short and long chains of yeast trehalose synthase
WO1995021251A1 (en) 1994-02-04 1995-08-10 Cantab Pharmaceuticals Research Limited T-cell antigens, and their use in diagnosis and treatment of t-cell mediated conditions
US5773222A (en) * 1992-05-27 1998-06-30 National Blood Authority Solid phase immunological assay
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6225289B1 (en) 1998-12-10 2001-05-01 Genvec, Inc. Methods and compositions for preserving adenoviral vectors
US6586006B2 (en) 1994-08-04 2003-07-01 Elan Drug Delivery Limited Solid delivery systems for controlled release of molecules incorporated therein and methods of making same
US7300919B2 (en) 1992-09-29 2007-11-27 Nektar Therapeutics Pulmonary delivery of active fragments of parathyroid hormone
US7306787B2 (en) 1997-09-29 2007-12-11 Nektar Therapeutics Engineered particles and methods of use
US7521069B2 (en) 1994-03-07 2009-04-21 Novartis Ag Methods and compositions for pulmonary delivery of insulin
US7628978B2 (en) 1997-09-29 2009-12-08 Novartis Pharma Ag Stabilized preparations for use in metered dose inhalers
WO2011048379A2 (en) 2009-10-21 2011-04-28 Innovata Limited Composition
US8877162B2 (en) 2000-05-10 2014-11-04 Novartis Ag Stable metal ion-lipid powdered pharmaceutical compositions for drug delivery
US9421166B2 (en) 2001-12-19 2016-08-23 Novartis Ag Pulmonary delivery of aminoglycoside
US9554993B2 (en) 1997-09-29 2017-01-31 Novartis Ag Pulmonary delivery particles comprising an active agent

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110027129A1 (en) * 2006-09-26 2011-02-03 Arkray, Inc. Method for formation of reagent layer in analysis apparatus, method for manufacture of anlalysis apparatus, and analysis apparatus
WO2016009971A1 (ja) * 2014-07-14 2016-01-21 国立大学法人名古屋大学 抗ホスホリパーゼａ２レセプター抗体の簡易測定

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US3539450A (en) * 1966-06-30 1970-11-10 Calbiochem Stabilization of enzymes
US3862302A (en) * 1969-03-20 1975-01-21 Akzona Inc Pelletized pregnancy test reagents
GB1575155A (en) * 1978-04-11 1980-09-17 Merck & Co Inc Vaccine stabilizer
CH622672A5 (cs) * 1975-10-15 1981-04-30 Behringwerke Ag
EP0030199A2 (en) * 1979-12-03 1981-06-10 Merck & Co. Inc. Lyophilization process for live viral compositions
CA1153314A (en) * 1980-03-17 1983-09-06 American Home Products Corporation Method of obtaining a lyophilized product
EP0192320A1 (en) * 1985-01-11 1986-08-27 Unilever Plc Preparation of reagents

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DE2640517C2 (de) * 1976-09-09 1984-10-04 Rollei Fototechnic GmbH, 3300 Braunschweig Rollfilmkassettenkamera
JPS61189454A (ja) * 1985-02-18 1986-08-23 Fujisawa Pharmaceut Co Ltd 安定化された固定化抗体

Patent Citations (7)

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Publication number Priority date Publication date Assignee Title
US3539450A (en) * 1966-06-30 1970-11-10 Calbiochem Stabilization of enzymes
US3862302A (en) * 1969-03-20 1975-01-21 Akzona Inc Pelletized pregnancy test reagents
CH622672A5 (cs) * 1975-10-15 1981-04-30 Behringwerke Ag
GB1575155A (en) * 1978-04-11 1980-09-17 Merck & Co Inc Vaccine stabilizer
EP0030199A2 (en) * 1979-12-03 1981-06-10 Merck & Co. Inc. Lyophilization process for live viral compositions
CA1153314A (en) * 1980-03-17 1983-09-06 American Home Products Corporation Method of obtaining a lyophilized product
EP0192320A1 (en) * 1985-01-11 1986-08-27 Unilever Plc Preparation of reagents

Non-Patent Citations (1)

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0415567A3 (en) * 1989-07-31 1991-08-21 Quadrant Bioresources Limited Composition and method for stabilising organic compounds
US5422254A (en) * 1992-02-14 1995-06-06 Oy Alko Ab Method to increase the trehalose content of organisms by transforming them with the structural genes for the short and long chains of yeast trehalose synthase
US5773222A (en) * 1992-05-27 1998-06-30 National Blood Authority Solid phase immunological assay
US7300919B2 (en) 1992-09-29 2007-11-27 Nektar Therapeutics Pulmonary delivery of active fragments of parathyroid hormone
WO1995021251A1 (en) 1994-02-04 1995-08-10 Cantab Pharmaceuticals Research Limited T-cell antigens, and their use in diagnosis and treatment of t-cell mediated conditions
US7521069B2 (en) 1994-03-07 2009-04-21 Novartis Ag Methods and compositions for pulmonary delivery of insulin
US6586006B2 (en) 1994-08-04 2003-07-01 Elan Drug Delivery Limited Solid delivery systems for controlled release of molecules incorporated therein and methods of making same
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6294365B1 (en) 1996-07-03 2001-09-25 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US7306787B2 (en) 1997-09-29 2007-12-11 Nektar Therapeutics Engineered particles and methods of use
US7628978B2 (en) 1997-09-29 2009-12-08 Novartis Pharma Ag Stabilized preparations for use in metered dose inhalers
US9554993B2 (en) 1997-09-29 2017-01-31 Novartis Ag Pulmonary delivery particles comprising an active agent
US6514943B2 (en) 1998-12-10 2003-02-04 Genvec, Inc. Method and composition for preserving viruses
US6225289B1 (en) 1998-12-10 2001-05-01 Genvec, Inc. Methods and compositions for preserving adenoviral vectors
US8877162B2 (en) 2000-05-10 2014-11-04 Novartis Ag Stable metal ion-lipid powdered pharmaceutical compositions for drug delivery
US9439862B2 (en) 2000-05-10 2016-09-13 Novartis Ag Phospholipid-based powders for drug delivery
US9421166B2 (en) 2001-12-19 2016-08-23 Novartis Ag Pulmonary delivery of aminoglycoside
WO2011048379A2 (en) 2009-10-21 2011-04-28 Innovata Limited Composition
EP2490670B1 (en) * 2009-10-21 2019-04-17 Innovata Limited Composition for inhalation

Also Published As

Publication number Publication date
GB8802174D0 (en) 1988-03-02
CN1037518A (zh) 1989-11-29
ES2018102A6 (es) 1991-03-16
AU3054889A (en) 1989-08-25
JPH02504312A (ja) 1990-12-06
CS68889A2 (en) 1991-07-16
EP0353281A1 (en) 1990-02-07

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