CN100471866C

CN100471866C - Method and apparatus for continuous large-scale radiolabeling of proteins

Info

Publication number: CN100471866C
Application number: CNB200480017742XA
Authority: CN
Inventors: R·佩利卡; S·W·金; P·布劳恩斯泰因; P·A·舒比格
Original assignee: Peregrine Pharmaceuticals Inc
Current assignee: Avid Bioservices Inc
Priority date: 2003-06-25
Filing date: 2004-06-25
Publication date: 2009-03-25
Anticipated expiration: 2024-06-25
Also published as: CN1829733A

Abstract

Disclosed are improved methods and apparatus for radiolabeling, particularly methods and apparatus for large scale in-line radiolabeling of products, such as proteins and antibodies.

Description

The method and apparatus of continuous large-scale radiolabeling of proteins

Background of invention

The present invention requires first U.S. Provisional Application series No.60/482 of common unsettled application in 25 days June in 2003,387, second U.S. Provisional Application series No.60/493 of application on August 7th, 2003,121, the 3rd U.S. Provisional Application series No.60/493 with application on August 8th, 2003,689 right of priority, wherein each application comprises that specification sheets, claim and accompanying drawing specially are incorporated herein by reference with its integral body without reservation at this.

1. invention field

The present invention generally relates to the chemically modified field of molecule, and the method and apparatus of the multiple material of radio-labeling especially is provided, and these materials include but not limited to be used for the protein and the peptide of pharmaceutical industry.More specifically, but not to limit after this described particular, the present invention relates to the method and apparatus of continuous large-scale radiolabeling of proteins and peptide.

2. description of related art

Biotechnology plays an important role in medicine, agricultural and other technical field.The sustainable development in this field needs continuous progressive development of biology, comprise for example protein such as antibody and peptide growth and effective production as the radioactive marker substance, and need improved other medicines, comprise the electrophilic addition of desired molecule or element, as radio-labeling.

At the research well afoot that improves Radiolabelling method.Yet, but prior art and present preparation method, all have its specific deficiency and defective.These defectives comprise low relatively gross activity, the radiolysis of product and mark batch relative low volume.

Therefore, for the kind of the radio-labeling material that promotes that ongoing biotechnology and Biomedical Development need is provided, this area needs the especially method new and improvement of protein and antibody of radio-labeling material.Especially seek the development of the extensive Radiolabelling method that is used for pharmaceutical industry.

Summary of the invention

By radiolabeled modification method and device are provided, in particular for pharmacy, biomedicine and other industrial in extensive radiolabeled method and apparatus, the invention solves needs for a long time in the art, and overcome existing difficulty.Enough several GBq (the gigabit Becquerels of this method energy; 37GBq=1 Curie=3.7 * 10 ¹⁰The decay per second) radiolabeled protein in the short production time is as antibody.

Therefore the present invention is suitable on a large scale, produces cheaply the radiopharmaceuticals as diagnostic reagent and therapeutical agent.Method and apparatus of the present invention, and various example and certain preferred embodiment are described in specification sheets of the present invention, claims and the relevant accompanying drawing, and in work embodiment illustrated.

Example device of the present invention or equipment are to use first radio-labeling at least, preferred oxidable radio-labeling comes mark biotic component or molecule, as the device or the equipment of protein, polypeptide, peptide, antibody or antibody molecule, the radio-labeling device or the equipment of preferred continuous mode.Such device or equipment can comprise:

At least first reaction chamber or the reaction tubes at least that comprise first end and second end;

At least first and second reagent container, with first end of reaction chamber or reaction tubes operationally (operatively) is connected i.e. fluid connection (fluidly connected); More particularly:

First reagent container is operably connected with first end of reaction chamber or reaction tubes by first reagent pipe or transfer lime or connects, and promptly fluid connects, and first reagent pipe coupling is in first pump at least; With

Second reagent container is operably connected with first end of reaction chamber or reaction tubes by second reagent pipe or transfer lime or connects, and promptly fluid connects, and second reagent pipe coupling is in pump; With

At least first purification unit at least that comprises import and outlet, the import of purification unit is operably connected with second end of reaction chamber or reaction tubes or connects, and promptly fluid connects.

At least " first end and second end " of first reaction chamber or reaction tubes can be described as " entrance end and exit end ".At least " first and second reagent container " and any other reagent container can be described as " first and second holder " and any other " holder ".The example of reagent container and holder is " bottle ".Any such " bottle " is called " reagent bottle ", makes a distinction with any " receiving flask " that be used for being connected with device or equipment.

Therefore, the radio-labeling device or the equipment of device or continuous mode can comprise first reaction chamber or reaction tubes at least with entrance end and exit end; At least first and second holder, each operationally connects the entrance end of reaction tubes; Operationally connect first purification unit at least of reaction tubes exit end.

At least one reagent container or holder can be contained in " radiation shielding ", in lead shielding.Usually, comprise or plan or be designed to comprise at least a radio-labeling, preferred oxidable radio-labeling, or the reagent container or the storer that comprise at least a radioactivity or oxidable radiolabeled at least a component, fluid or solution can be reagent container or the holder that is contained in any radiation shielding or the lead shielding.

" exercisable " connects or connects and can be described as " fluid ", " fluidic " or " fluidity " and connect or connect, make that composition " be operably connected or connect " is " fluid ground or fluidity ground connect or connects ", make fluid between the parts that connect or connect, can move effectively, transmit, by or flow.

Being appreciated that exercisable, fluid is connected with fluidity with connecting is that " controllable " is exercisable, fluid is connected with fluidity and connects.Promptly, exercisable, fluid and fluidity connect and connect " permissions " fluid " under controlled condition " between the parts that connect or connect, move, transmit, by or mobile, promptly the condition of directly or indirectly controlling by the operator is included under the condition of operator, computer or computer program control.Therefore, exercisable, fluid or fluidity connect or connect do not mean that fluid between free that connecting or that connect the under all conditions parts of institute, move naturally, transmit, by or flow, only be as controlling by operator, computer or computer program.

In the operation of apparatus of the present invention or equipment, operate or control exercisable between each parts, fluid or fluidity usually and connect and connect, make fluid between the parts that connect or connect with " directed " mode move, transmit, by or flow.That is, fluid usually " from " point move, transmit, by or flow " extremely " another point and/or " by " parts and/or " by " as a whole device or equipment moves, transmits, passes through or mobile.However, exercisable, fluid or fluidity connect with connecting and do not need " unidirectional " to be connected or connect, because can give or obtain directed control in the operation of device or equipment.

In device or the equipment, and in the operation of the inventive method, first " reaction chamber or reaction tubes " is any reaction chamber or pipe at least, can move, transmit, pass through or flow along its fluid and solution, and common " reacting ".Preferably, reaction chamber or reaction tubes are " reaction tubess ".

Reaction chamber or reaction tubes, the preferred reaction pipe is predetermined or preselected length and predetermined or preliminary election diameter.Usually determine, select or select in such " predetermined or preselected length and diameter " feasible operating gear in the methods of the invention or equipment, the length of reaction tubes can make effectively that with diameter used composition contacts, to produce the radiolabeled molecule of aequum at time per unit.

In device or the equipment, and in operation as the inventive method, device or equipment can comprise and use first and second reagent container or holder, with first, second and the 3rd reagent container or holder, or with the 4th, the 5th or more reagent container or holder operation.Usually embodiment preferred is wherein to install or equipment comprises and/or operate with first and second reagent container or holder; And wherein device or equipment comprises and/or with first, second and the 3rd reagent container or holder operate.Comprise first, the device of second and the 3rd container or holder or equipment still can be only to use its first and the pattern of second reagent container or holder.

No matter install or whether equipment comprises first and second reagent container or holder, or first, second and the 3rd reagent container or holder or more, each in reagent container or the holder use single pump or use more than a pump operationally or fluid connect or connect first end of reaction chamber or reaction tubes.Preferably, reagent container or holder use " one or more reagent pipe " in conjunction with single pump or two or more pump operationally or fluid connect or connect first end of reaction chamber or reaction tubes.

Therefore, in device or the equipment, and in the operation of the inventive method, first reagent container by first reagent pipe operationally or fluid connect or connect first end of reaction chamber or reaction tubes, first reagent pipe coupling is in first pump at least; Second reagent container by second reagent pipe operationally or fluid connect or connect first end of reaction chamber or reaction tubes, second reagent pipe coupling is in pump, such as being connected in first pump at least.

First with second reagent container or holder in each can be operationally or fluid be connected or connect first end of reaction chamber or reaction tubes, method is by they first and second reagent pipe separately, use identical pump, that is, and first or first pump at least.In this embodiment, first or at least first pump be preferably multi-channel peristaltic pump.

Perhaps, first with second reagent container or holder in each can be operationally or fluid be connected or connect first end of reaction chamber or reaction tubes, method is by they first and second reagent pipes separately, uses different pumps, that is, use first and second pump at least.In this embodiment, first and second pump are preferably peristaltic pump.

Randomly, the 3rd reagent container by the 3rd reagent pipe operationally or fluid connect or connect first end of reaction chamber or reaction tubes, the 3rd reagent pipe is connected with pump.In such device, first, second can pass through identical pump with the 3rd reagent container, operationally or fluid connect or connect first end of reaction chamber or reaction tubes, described pump is generally the peristaltic pump of three passages, and it operationally is positioned between first end of reagent container and reaction chamber or reaction tubes.First, second with the 3rd reagent container or holder in each can be operationally or fluid be connected or connect first end of reaction chamber or reaction tubes, method be by they separately first, second and the 3rd reagent pipe, use different pumps, that is, use at least first, second and the 3rd pump.In this embodiment, first, second pump and the 3rd pump be preferably peristaltic pump.

Irrelevant with being connected between the quantity of pump and reagent container or holder and reaction chamber or the reaction tubes, preferably by the computer control pump.In device and the equipment, and in the inventive method in device or the operation of equipment, the preferred use passed through computer-controlled one or more multi-channel peristaltic pumps.

Except or replace first pump at least, device of the present invention or equipment can comprise at least one other unit, equipment or device and be used to make fluid to pass through device or equipment, fluid is moved by device or equipment to be obtained basically successive and flows.Such unit, equipment and device comprise by utilization pressure and/or vacuum or other power (as revolving force or centrifugal force) those of moving liquid effectively.Therefore at least one, two, three or more such " controlled flowing unit " can be included in, that is, operationally be positioned in device of the present invention or the equipment.

In the particular of the present invention, include but not limited to, wherein device or equipment comprise two reagent containers and holder and/or use in conjunction with two reagent containers and holder, and reaction chamber or reaction tubes can comprise or comprise first aid mark molecule or reagent at least." aid mark molecule or reagent " is material, composition, molecule or the reagent of assisting, improving or help mark to handle as used in this.Example comprises sequestrant, and preferred examples is at least the first kind of oxidation or " oxygenant ", and the oxidable radio-labeling of its preferred combination uses.

Such reaction chamber or reaction tubes be " comprise or comprise " at least the first kind of aid mark molecule or reagent usually, preferred at least the first kind of oxygenant, enter with " effectively contact ", along or pass reaction chamber or reaction tubes moves, transmits, by or the mode of mobile fluid or solution.Usually, " effectively contact " significantly do not hinder hinder fluid or solution enters, along, by or flow out passing through, advance or flowing of reaction chamber or reaction tubes.Contact also is " effectively " for the composition " reaction " in aid mark molecule, reagent or oxygenant and fluid or the solution, and preferably along with they enter, along with by the moving of reaction chamber or reaction tubes, transmit, by and flow, the composition that is used for aid mark molecule, reagent and oxygenant and fluid or solution reacts.

The embodiment of this reaction chamber or reaction tubes is such reaction chamber or reaction tubes, wherein to small part, preferably basically or quite most of, the internal surface of reaction chamber or reaction tubes applies at least the first kind of aid mark molecule or reagent, preferably at least the first kind of oxygenant.Therefore, the method that the invention provides device or equipment and use this device or equipment, wherein first reaction chamber or reaction tubes comprise first end, second end, outside surface and internal surface at least; And wherein to small part, basically or quite most internal surface apply at least the first kind of aid mark molecule or reagent, preferred oxidant.

Therefore herein " coatings " to look like be wherein to small part, and preferably basically or quite most internal surface be " operationally being attached to " at least the first kind of aid mark molecule or reagent, preferred oxidant.The oxygenant example that can coated or operationally be attached on such reaction chamber or the reaction tubes internal surface is 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril (diphenylglycouril).

Another embodiment of such reaction chamber or reaction tubes is reaction chamber or the reaction tubes that comprises or comprise immobilized aid mark molecule or reagent, and the aid mark molecule is at least the first kind of oxygenant preferably, is not attached to the internal surface of reaction chamber or reaction tubes." immobilized " aid mark molecule, reagent or oxygenant comprise and being attached to like this, promptly " operationally are attached to " aid mark molecule, reagent or the oxygenant of one or more anchoring base." one or more anchoring base " comprise can operationally be attached to selected aid mark molecule, reagent or oxygenant and be inserted into reaction chamber or reaction tubes in and significantly do not hinder hinder fluid or solution enters, along, by or flow out the passing through of reaction chamber or reaction tubes, any solid support material of progressive.

Such anchoring base example is grid, fiber or porosu solid upholder and particulate or pearl; or its set; wherein the constituent of grid, fiber, particulate or pearl operationally is attached at least the first kind of aid mark molecule or reagent, preferably is attached at least the first kind of oxygenant.Porous anchoring base or solid support can comprise natural fiber, comprise cotton or synthon and/or polymkeric substance or its mixture.The oxygenant example that can operationally be attached to grid, fiber, particulate and pearl in this mode is a N-toluene(mono)chloride sulphonamide.

Therefore device that the present invention is exemplary and equipment are such device and equipment, comprise:

The reaction chamber or the reaction tubes that contain first end and second end;

By first reagent container that first reagent pipe is connected with the first end fluid of reaction chamber or reaction tubes, first reagent pipe coupling is in first pump at least;

By second reagent container that second reagent pipe is connected with the first end fluid of reaction chamber or reaction tubes, second reagent pipe coupling is in pump; With

First purification unit at least that comprises import and outlet, the import of purification unit is connected with the second end fluid of reaction chamber or reaction tubes.

Another example device of the present invention or equipment are such device or equipment, comprise:

Reaction chamber with first end and second end,

By first reagent container that first reagent pipe is connected with the reaction chamber first end fluid, first reagent pipe coupling is in pump;

By second reagent container that second reagent pipe is connected with the reaction chamber first end fluid, second reagent pipe coupling is in pump;

By the 3rd reagent container that the 3rd reagent pipe is connected with the reaction chamber first end fluid, the reagent pipe coupling is in pump;

Purification column with import and outlet, the import of purification column is connected with the second end fluid of reaction chamber; With

Be connected in the collection container of purification column outlet.

Further example device of the present invention or equipment are such device or equipment, comprise:

By first reagent container that first reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, first reagent pipe coupling is in pump;

By second reagent container that second reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, second reagent pipe coupling is in identical pump;

By the 3rd reagent container that the 3rd reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, the 3rd reagent pipe coupling is in identical pump;

Still an example device more of the present invention or equipment are this sampling device or equipment, comprise:

By first reagent container that first reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, first reagent pipe coupling is in first pump;

By second reagent container that second reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, second reagent pipe coupling is in second pump;

By the 3rd reagent container that the 3rd reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, the 3rd reagent pipe coupling is in the 3rd pump;

In said apparatus or the equipment first, second and the 3rd pump are preferably peristaltic pump.In said apparatus or the equipment first, second and the 3rd pump also preferred each by computer control.

Still further example device of the present invention or equipment are such device or equipment, comprise:

The reaction chamber or the reaction tubes that contain first end, second end, outside surface and internal surface;

By first reagent container that first reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, first reagent pipe passes first pump at least;

By second reagent container that second reagent pipe is connected with reaction chamber or the reaction tubes first end fluid, second reagent pipe passes pump; With

About above-mentioned device or equipment, at least the first kind of oxygenant can the association reaction chamber or reaction tubes to the internal surface of small part.Oxygenant can be 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril.Such device or equipment can also further comprise the particulate that is attached at least the first kind of oxygenant, and wherein particulate is contained in reaction chamber or the reaction tubes.The example particulate is to be attached to those of oxygenant N-toluene(mono)chloride sulphonamide.

In device of the present invention or the equipment, and in the operation of the inventive method, " purification unit " can be any or multiple purifying parts or the device of the radiolabeled composition of purifying or molecule basically.Promptly, at least first purifying parts or device can or effectively from any free radio-labeling, radio-labeling handle or reaction any residual component, in aid mark molecule or reagent (for example oxygenant) and any other unlabelled composition and molecule, isolate radiolabeled composition or molecule basically.

For example, one or more purifying parts or device can be isolated radiolabeled proteins, polypeptide, peptide, antibody or antibody molecule basically from the oxidable radio-labeling of any free, any residual oxygenant and any unlabelled protein, polypeptide, peptide, antibody and antibody molecule.In the particular of apparatus of the present invention, equipment and method, purification unit comprises at least the first kind of material in conjunction with iodine, mixing halogen species and iodine oxide kind.

The purification unit of apparatus of the present invention, equipment and method can comprise first parts and equipment at least, and wherein purification mechanisms comprises the separation based on electric charge, as first ion exchange column or anion-exchange column at least.Purification unit can also comprise first parts or device at least, and wherein purification mechanisms comprises the separation based on volume, as first volume-exclusion post or gel-filtration column at least.Can also use affinity column and other tools for purification or equipment.In addition, one or more purification unit can comprise first dialysis unit at least.Such dialysis unit comprises import and outlet usually, and the unitary import of dialysis is connected with the second end fluid of reaction chamber or reaction tubes.

Although for of the present invention successfully operate optional, device, equipment and method can be utilized and comprise two and a plurality of purification devices or unitary purification unit, as first and second at least who connects successively, or at least first, second and the 3rd purifier apparatus or post. for example, mainly can be used in combination with main parts, or mainly can be used in combination with the dialysis unit based on the parts of charge separation based on size separation based on the parts of charge separation.

In apparatus of the present invention or the equipment, and in the operation of the inventive method, device or equipment can also comprise first allocation units or collection container at least.Any allocation units or collection container directly or indirectly connect, and promptly operationally are connected in the outlet of purification unit with fluid.If there is the dialysis unit, the common fluid of the unitary import of dialysis is connected in second end of reaction chamber or reaction tubes, and the unitary outlet of dialysis is connected in collection container then.

Can also use first filter at least in device of the present invention, equipment and the method, preferred sterile filters.Preferably, such filter also operationally or fluid be connected in the outlet of purification unit.In the specific preferred embodiment, filter is placed at least between first purification unit and any allocation units or collection container.For example, the invention provides such device, equipment and method: used the filter that comprises entrance end and exit end; The entrance end fluid of filter is connected in the outlet of purification unit; And the exit end of filter is connected in collection container.

Device of the present invention and equipment, method especially of the present invention can be used for any material of radio-labeling or molecule basically.That is, can stand any material or the molecule of radio-labeling reaction.As used in this, " target " material, composition or the molecule meaning are " treating radiolabeled material, composition or molecule ".The example of preferred radio-labeling target material is biotic component and molecule according to the present invention, as but be not limited to protein, polypeptide, peptide, antibody and antibody molecule.

Term " antibody ", " antibody molecule " and " immunoglobulin (Ig) " always refer to any wedding agent based on antibody as used in this, comprise polyclone and monoclonal antibody.Antibody comprises five main kinds, and IgA, IgD, IgE, IgG and IgM further comprise any subclass or isostructural antibody, as IgG1, IgG2, IgG3, IgG4 or the like.Therefore, comprise that wherein CH is α, δ, ε, γ and μ; And wherein light chain is the antibody of kappa (κ) or lambda (λ).

Unit of the present invention and method especially can be used for radio-labeling monoclonal antibody (MAb) and derivative thereof, comprise the monoclonal antibody in mouse, people, monkey, rat, hamster, rabbit, frog and chicken source.Usually preferred radio-labeling mouse, people or peopleization monoclonal antibody.Especially comprise radio-labeling peopleization, groups of peopleization or people's antibody.

Radio-labeling " antibody " or " antibody molecule " have been contained all antibody of all species of radio-labeling as used in this, and Fab, comprise dimerization, trimerization and poly antibody; Bi-specific antibody; Chimeric antibody; People and humanized antibodies; Reorganization, through engineering approaches and camelization (camelised) antibody and fragment thereof.Especially comprise radiolabelled antibody fragment such as Fab ', Fab, F (ab ') ₂, single domain antibody (DAB), complementary determining region (CDR), CDR1-3, Fv, scFv (strand Fv), straight chain antibody, two anti-, camelization antibody or the like.

Only be for example, device of the present invention and equipment, and method especially of the present invention, can be used for radio-labeling target reagent or composition, as protein, polypeptide, peptide, antibody or antibody molecule, its in conjunction with express, be easy in conjunction with or be positioned composition on the tumor cell surface, or in conjunction with the composition that discharges from downright bad tumour cell.As more example, device of the present invention or equipment, especially method of the present invention, can be used for radio-labeling target reagent or composition, as protein, polypeptide, peptide, antibody or antibody molecule, its in conjunction with express, be easy in conjunction with or be positioned vascular or the lip-deep composition of tumor stroma in tumor vascular system, the tumour.Only be that such target material is disclosed in U.S. Patent No. 6,093 for example, 399,6,004,555,5,877,289,6,036,955 and 6,749,853; With 5,019,368,4,861,581 and 5,882,626; With 6,004,554,5,965,132,5,855,866,5,776,427,5,863,538,6,051,230,6,261,535 and 5,660,827; With 6,406,693; With 6,312,694; With 6,342, in 219,6,342,221,6,416,758,6,524,583,6,676,941 and 6,703,020; Each is incorporated herein by reference hereby.

Except protein, polypeptide, peptide and antibody molecule, other molecules that can stand electrophilic substitution can be labeled in the present invention.Comprise that maybe can be modified to the biotic component and the molecule that comprise first group at least that can stand electrophilic substitution especially comprises.Therefore, use the present invention can the radio-labeling lipid, carbohydrate, glycoprotein, protein-polysaccharide and multiple small molecules.

Although be particularly suited for using iodine to carry out mark, radioiodination for example, device of the present invention, equipment and method can use various other marks to come mark substance, composition and molecule, especially when and any required aid mark reagent when being used in combination.Therefore, for example, technetium ⁹⁹M, indium ¹¹¹, rhenium ¹⁸⁸, rhenium ¹⁸⁶, copper ⁶⁴, copper ⁶⁷, yttrium ⁹⁰, astatine ²¹¹And gallium ⁶⁷Can be used for the present invention.

Can oxidation forming the isotropic substance that mixes halogen species is preferred among the present invention.With regard to iodine, device of the present invention, equipment and method can be in conjunction with iodine ¹²³, iodine ¹²⁴, iodine ¹²⁵And iodine ¹³¹Use.Preferably, wherein radio-labeling is iodine-131 or iodine-125; And most preferably, wherein radio-labeling is an iodine-131.

Method of the present invention again the invention provides the method for preparation at least the first kind of radiolabeled material, composition and molecule, as biological substance, composition and molecule, for example, one or more protein, polypeptide, peptide, antibody and antibody molecule.This method always comprise make significant quantity treat radiolabeled material, composition and molecule and radio-labeling, and the reaction needed of significant quantity or preferred any aid mark molecule and reagent together, by device according to the present invention or equipment.Under the condition and time that effectively produce radiolabeled material, composition and molecule " by device or equipment ".From device or equipment, collect radiolabeled material, composition and the molecule that obtains that produce then.

Usually in the embodiment preferred, the oxygenant that method is included in significant quantity exists down, and significant quantity treated that radiolabeled material, composition or molecule and oxidable radio-labeling are by according to device of the present invention or equipment.Produce " passing through device or equipment in the presence of the oxygenant in the significant quantity " for some time under the mode of radiolabeled material, composition or molecule in the oxidable radiolabeled condition of the efficient oxidation and being suitable for.From device or equipment, collect radiolabeled material, composition or the molecule that obtains that produce then.

Therefore, prepare the method for at least the first kind of radiolabeled material, composition or molecule, comprising:

In the presence of at least the first kind of oxygenant of significant quantity, at least the first kind of significant quantity treated radiolabeled material, composition or molecule, or contain at least the first kind of composition, fluid or the solution for the treatment of radiolabeled material, composition or molecule of significant quantity, at least a oxidable radio-labeling with significant quantity, or contain oxidable radiolabeled at least the second kind of composition, fluid or the solution of significant quantity, by first reaction chamber or reaction tubes at least;

At least the first kind of oxygenant, at least a oxidable radio-labeling and at least the first kind of material, composition or molecule are reacted in the process of passing reaction chamber or reaction tubes, therefore produce radiolabeled molecule; With

Collect radiolabeled molecule after passing reaction chamber.

Such method can be described as those, comprising:

In the presence of the oxygenant of significant quantity, with the oxidable radio-labeling for the treatment of radiolabeled material, composition or molecule and significant quantity of significant quantity by reaction chamber or reaction tubes;

Oxygenant, oxidable radio-labeling and material, composition or molecule are reacted in the process of passing reaction chamber or reaction tubes, therefore produce radiolabeled molecule; With

Radiolabeled molecule is collected in the back after passing reaction chamber.

" passing " reaction chamber or reaction tubes and " making " oxygenant, oxidable radio-labeling and material, composition or molecule " passing " reaction chamber or reaction tubes " process in the react " meaning is that composition passes through reaction chamber or reaction tubes, or is effectively producing the condition of radioactive marker substance, composition or molecule and carrying out under the time period by device or equipment process as a whole.Therefore pass or be applied to " enter and flow out " or " passing " reaction chamber or reaction tubes, or device as a whole, be the active process, rather than static state " batch processing mode " process in prior art and usual means, equipment and the method.

The oxygenant that Radiolabelling method is preferably included in significant quantity exists down, in the mode of " being enough to mix continuously basically " each composition, " application " or " by " significant quantity treat that radiolabeled material, composition or molecule and oxidable mark enter or by reaction chamber or reaction tubes.Promptly, with suitable manner and continue for some time and be enough to basically to continue to mix treat radiolabeled material, composition or molecule, oxidable radio-labeling and oxygenant, promptly, between these compositions, " keep effective contact basically ", passing or passing through in the process of reaction chamber or reaction tubes." effectively contact " between these compositions is " effective, reactive contact " or " reactive contact ", that is, contact can allow or help radio-labeling to react effectively, so the described material of radio-labeling, composition or molecule.

Therefore Radiolabelling method of the present invention can comprise:

In the presence of the oxygenant of significant quantity, significant quantity treated radiolabeled material, composition or molecule, or contain and remain at least the first kind of composition, fluid or the solution of radiolabeled material, composition or molecule, at least a oxidable radio-labeling with significant quantity, or at least the second kind of composition, fluid or the solution stream that contain the oxidable substance markers of significant quantity cross reaction chamber or reaction tubes, therefore prepares radiolabeled molecule; With

From the effluent of reaction chamber or reaction tubes, obtain radiolabeled molecule.

Similarly, radiolabeled method of the present invention can comprise:

In the presence of the oxygenant of significant quantity, basically constantly significant quantity treated radiolabeled material, composition or molecule, or contain at least the first kind of composition, fluid or the solution for the treatment of radiolabeled material, composition or molecule of significant quantity, with at least a oxidable radio-labeling of significant quantity, or at least the second kind of composition, fluid or the solution that contain the oxidable substance markers of significant quantity flow into reaction chamber or reaction tubes;

Oxygenant, oxidable radio-labeling and material, composition or molecule are reacted in reaction chamber or reaction tubes, therefore produce radiolabeled molecule; With

From reaction chamber or reaction tubes, flow out radio-labelled molecule basically constantly, therefore obtain radiolabeled molecule.

About reaction chamber or reaction tubes, the preferred usually reaction tubes that uses.The preferred reaction pipe is predetermined or preselected length and predetermined or preliminary election diameter.Usually decision, selected or select that the length of predetermined like this or preliminary election and diameter make that the length of reaction tubes and diameter be suitable for making significant quantity treat radiolabeled material, composition or molecule, oxidable radio-labeling contacts in the mode that time per unit effectively produces the aequum radio-labelled molecule with oxygenant.

The present invention can use any effective means, use, enter and flow out, pass, mix continuously basically, keep basically effectively or reactively contact, flow into, flow out and pass, with influent stream with go out stream, as long as at least the first kind of composition, fluid or solution continue to pass through basically in the operation of this method or flow through or along reaction chamber or reaction tubes, or device as a whole or equipment.Usually the preferred method that obtains continuous flow basically is to use first pump at least.

Perhaps, can applying pressure and/or vacuum or other power (as revolving force or centrifugal force) make fluid pass through radio-labeling device of the present invention.Therefore, one or more reactants can pneumatically move through reaction chamber or reaction tubes and device as a whole or equipment, and use is at reaction chamber or reaction tubes second end or export applied pressure (being applied to reagent container) or vacuum.Method that all are such and operator scheme are included in the method for the present invention, and in corresponding device thereof and the equipment.

Therefore, the certain preferred Radiolabelling method of the present invention is that those wherein pass reaction chamber or reaction tubes with significant quantity to wait a little while,please radiolabeled material, composition or molecule and oxidable radio-labeling pumping, and preferred, reaction chamber or reaction tubes are passed in uninterrupted pumping basically.More preferably, with passing to wait a little while,please radiolabeled material, composition or molecule and oxidable radio-labeling pumping of significant quantity, preferably basically continuously pumping pass reaction tubes.

In the method operation, and use in the reaction tubes of predetermined or preselected length and diameter, preferably every kind of flow velocity with predetermined or preliminary election at least the first kind of target material, composition or molecule of significant quantity and at least the first kind of oxidable radio-labeling is pumped in reaction chamber or the reaction tubes.These methods can be described as those wherein will contain at least the first kind of composition, fluid or the solution for the treatment of radiolabeled material, composition or molecule of significant quantity and every kind of flow velocity with predetermined or preliminary election containing in oxidable radiolabeled at least the second kind of composition, fluid or the solution of significant quantity pumps into reaction chamber or reaction tubes.

The flow velocity of preliminary election " predetermined or " normally determine, flow velocity selected or control makes the oxidable radio-labeling of oxygenant oxidation, form the reactive intermediate product with the substitution reaction that can stand target material, composition or molecule that electrophilic replaces, time per unit produces radiolabeled material, composition or the molecule of aequum thus.Therefore, the predetermined flow velocity of control makes the oxidable radio-labeling of oxygenant oxidation, forms the reactive intermediate product with the substitution reaction that can stand target material, composition or molecule that electrophilic replaces, produces the mixture that contains radiolabeled molecule thus.Preferably, select predetermined flow velocity to produce the mixture that contains the radiolabeled molecule of aequum, and do not destroy radiolabeled molecule basically, for example pass through radiolysis.

In the particular, predetermined flow velocity provides the flow velocity of enough or sufficient time, make the oxidable radio-labeling of oxygenant oxidation, formation can be reacted the halogen species that mixes that produces radiolabeled protein's material, composition or molecule with the tyrosine and the histidine residues of target protein material, composition or molecule.At this, the preferred flow velocity of selecting to be scheduled to forms and can react the halogen species that mixes that produces the radiolabeled protein with the tyrosine and the histidine residues of target protein, and do not have the substantive radio-labelled molecule that destroys, for example, do not cause substantive radiolysis.

Can control predetermined flow velocity by controlling one or more pumps.Therefore " pumping " refer to that preferably moving one or more pumps with set rate enters oxygenant, target material, composition or molecule and oxidable radio-labeling and preferably pass through reaction chamber or reaction tubes.

One or more pumps in the method in the operation are preferably one or more peristaltic pumps.Irrelevant with the quantity of pump, at least first, second, the three or more, and preferably each, pump be by computer-controllable system or control.Method is preferably used by computer-controlled first multi-channel peristaltic pump at least.

The quantity that is used for the pump of the inventive method, device and equipment can change according to selected embodiment.As, in using oxidable radiolabeled Radiolabelling method, the position of at least the first kind of oxygenant can influence the selection of reagent container or holder quantity and the selection of pump quantity.

In using the method for first and second reagent container or holder, first and second reagent container or holder can be operationally or fluid be connected in pump separately, promptly connect first and second pump separately.Perhaps, first can be operationally with second reagent container or holder or fluid be connected in identical pump, that is, be connected at least first or first pump.To use first, the method for second and the 3rd reagent container or holder also is same.Therefore, first, second and the 3rd reagent container or holder can be operationally or fluid be connected in pump separately, promptly be connected in separately first, second and the 3rd pump.On the other hand, first, second can be operationally with the 3rd reagent container or holder or fluid be connected in identical pump, promptly be connected at least first or first pump.Usually, operationally or fluid connects or the pump that connects two or more reagent containers or holder is a multi-channel pump, preferred multi-channel peristaltic pump.

Preferably, material, composition or molecule and radio-labeling to be marked at least with significant quantity, preferred oxidable radio-labeling, reaction chamber or reaction tubes are passed in pumping, more preferably be maintained in reagent container separately or the holder, and by passing reaction chamber or reaction tubes from the reagent container or the holder extraction pumping that separate.Can use reagent container that single pump or two pumps that separate will treat that radiolabeled material and radio-labeling separate from them or the holder and extract out or pump.

For with aid mark molecule or reagent, particularly oxygenant, be applied to reaction chamber or reaction tubes and have more multiselect item usually.In the process of implementation method, such molecule or reagent, preferred oxidant can be positioned at least one reagent container or the holder and with its significant quantity and pumps in reaction chamber or the reaction tubes.The oxygenant example that has suitable solubility in such purposes is a N-toluene(mono)chloride sulphonamide.

Therefore, with significant quantity treat radiolabeled every kind of material, composition or molecule, reaction chamber or reaction tubes are passed in every kind of pumping in oxidable radio-labeling and the oxygenant.Can be from three isolating reagent containers or holder every kind of this three kinds of basic ingredients of significant quantity be pumped into reaction chamber or reaction tubes.If expectation can be positioned over the mixture for the treatment of radiolabeled molecule and oxygenant or the mixture of significant quantity in first reagent container or the holder, and pumps in reaction chamber or the reaction tubes as pre-composition.In this case, oxidable radio-labeling can be positioned in second reagent container or in the holder, and in an identical manner significant quantity is pumped in reaction chamber or the reaction tubes.

In addition, before this method of enforcement, with the aid mark molecule or the reagent of significant quantity, preferably at least the first kind of oxygenant is positioned over, is positioned or be included in reaction chamber or the reaction tubes.Wherein at least the first kind of oxygenant has been arranged in reaction chamber or reaction tubes, in the process that enters and pass reaction chamber or reaction tubes, treat radiolabeled material, composition or molecule and the agent of oxidable radio-labeling catalytic oxidation and with its reaction.

Be used for will at least the first kind the oxygenant option that is positioned reaction chamber or reaction tubes comprise the oxygenant of significant quantity wherein be present in reaction chamber or reaction tubes to the small part internal surface, and preferably, be present in basically or quite most this internal surface on.The exemplary oxygenant that is used for this " coating " is 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril.

It is that the oxygenant of wherein significant quantity is present on the anchoring base that at least the first kind of oxygenant is positioned another option in reaction chamber or the reaction tubes, as is contained in grid, fiber, porosu solid upholder in reaction chamber or the reaction tubes, or in the particulate set.The exemplary oxygenant that is used for this " packing " is a N-toluene(mono)chloride sulphonamide.

Because use device of the present invention and equipment, method of the present invention is used at least one first purification unit of the radiolabeled material of purifying, composition or molecule basically usually.Therefore the radiolabeled molecular application of wash-out in reaction chamber or the reaction tubes is separated the radiolabeled molecule of purifying basically to first purification unit at least and from it.

Exemplary purification unit comprises one or more ion exchange columns, anion-exchange column, gel-filtration column, affinity column, dialysis unit and combination thereof.With reaction chamber or reaction tubes pumps or effusive composition, fluid or the solution that contains radiolabeled product, be applied to first purification unit at least, from any other composition wherein, isolate radiolabeled product basically thus.Especially, thus from any residual component of radioreaction such as oxygenant, oxidable radio-labeling, reaction intermediate and any unlabelled raw material, isolate radiolabeled product.In being applied to the dialysis unit, this method dialysis goes out any residual component of radio-labeling reaction, produces the radiolabeled product of purifying basically thus.

Case method among the present invention is to use and comprises at least first and the device of second reagent container or the method for equipment, and wherein such method comprises:

The first kind of solution or the mixture that will contain target molecule and oxygenant are put into first reagent container;

To contain oxidable radiolabeled second kind of solution and put into second reagent container;

Reaction chamber or reaction tubes are passed in first kind of solution and second kind of solution pumping of significant quantity, wherein target molecule, oxygenant and oxidable radio-labeling react in the process of passing reaction chamber or reaction tubes, therefore produce the mixture that contains the radiolabeled molecule of significant quantity;

Purification unit is passed in the mixture pumping; With

Collect the radiolabeled molecule of purifying basically from purification unit.

In the aforesaid method, preferably under predetermined flow velocity, use pump to carry out " pumping ".The predetermined flow velocity of control makes the oxidable radio-labeling of oxygenant oxidation form reaction intermediate in entire method." reaction intermediate " be can be incorporated into can experience target molecule that electrophilic replaces substituent those." be incorporated into and experience the substituting group that replaced by electrophilic " effectively making reaction intermediate and substituting group " reaction " come the condition of labels targets molecule and taking place under the time, by the radiolabeled molecule or the product that produce significant quantity.

Preferably will contain the mixture of active marked product or solution and pump into purification unit (as purification column) from reaction chamber or reaction tubes and come purifying radiolabeled " product ", to produce the radiolabeled product of purifying basically.More preferably, filter (preferred sterile filters) is passed in the radiolabeled product pumping of purifying basically filter the radiolabeled product of purifying basically, and collect purifying basically, filtering radiolabeled product.

Remaining among the present invention further, illustrative methods is such method: use the device or the equipment that comprise at least first and second reagent container, and be included at least the first kind of oxygenant of significant quantity in reaction chamber or the reaction tubes.Such method comprises:

First kind of solution that will contain target molecule is put into first reagent container;

First kind of solution and second kind of solution pumping are passed reaction chamber or reaction tubes, wherein target molecule and oxidable radio-labeling and oxygenant react in reaction tubes or reaction chamber in the process of passing reaction chamber or reaction tubes, therefore produce the mixture that contains radiolabeled molecule;

Purification unit is passed in the mixture pumping; With

Remain among the present invention further illustrative methods and be used to use comprise first, the device of second and the 3rd reagent container or the method for equipment, wherein such method comprises:

Oxygenant is put into first reagent container;

Target molecule is put into second reagent container;

Oxidable radio-labeling is put into the 3rd reagent container;

Oxygenant, target molecule and the oxidable radio-labeling of significant quantity are passed through reaction chamber or reaction tubes with set rate, it is enough to make the oxidable radio-labeling of oxygenant the efficient oxidation, form the reaction intermediate with the substitution reaction that can stand the target molecule that electrophilic replaces, produce the radiolabeled molecule of significant quantity thus;

Radiolabeled molecule is passed purification unit come the radiolabeled molecule of purifying; With

If select, the radiolabeled molecule of the purifying basically that can obtain from purification unit filters by the filter (preferred sterile filters) that is connected in purification unit, so obtains radiolabeled molecule filtering, purifying basically.Such filter can be placed between purification unit and any allocation units or the collection container.When using in conjunction with first allocation units at least or collection device and container, such allocation units directly or indirectly are connected in purification unit, make the operation of this method will be basically purifying or basically purifying and filtering radiolabeled material, composition or molecule be dispensed in selected collection container or a series of container or the susceptor.

Method of the present invention may further include at least the first kind of radio-protector with significant quantity add purifying basically or basically purifying and filtering radiolabeled material, composition or molecule in." radio-protector " be can absorbing radiation composition, molecule or reagent.Preferably, radio-protector is absorbing radiation and other composition of contacting with radio-protector of influence significantly unfriendly effectively, especially radiolabeled material, composition or molecule.The example radio-protector is a serum albumin, as bovine serum albumin and human serum albumin, and xitix.

In the operation of this quadrat method, at least the first kind of radio-protector of significant quantity is positioned in selected collection container or the susceptor, and by radiolabeled material is put into selected collection container or receptor simply will be basically purifying or basically purifying and filtering radioactive marker substance add radio-protector.

Can use a series of or a plurality of containers or receptor, as a series of or a plurality of bottles, and working method make equal portions purifying basically or basically purifying and filtering radiolabeled dispensed materials to each container, receptor or bottle.Any such one or a series of container, receptor or bottle can be equipped with one or more required compositions of significant quantity in advance, as radio-protector, pharmaceutical diluents or carrier or the like.

Therefore, basically purifying or basically purifying and filtering radiolabeled material can be dispensed to or be collected on the pharmacology in the acceptable preparation, randomly also contain a kind of of radio-protector, or be dispensed in a plurality of such preparations.Therefore, this method can be used to prepare purifying basically or purifying basically and filtering radiolabeled material, as radiopharmaceuticals, is scattered in a plurality of receiving flasks, preferably makes each bottle contain acceptable unitary dose on the pharmacology.

As mentioned above, method of the present invention can be used for any material of radio-labeling or molecule basically, as any biotic component or molecule, for example protein, polypeptide, peptide, antibody and antibody molecule, lipid, carbohydrate, glycoprotein, protein-polysaccharide and small molecules.The isotopic labeling method that can stand the molecule of electrophilic substitution that can oxidized formation mixing halogen species with one or more is preferred aspect of the present invention.In the method for radiolabeled protein, polypeptide or peptide such as antibody and antibody molecule, radio-labeling can be operably connected to one or more tyrosine, Histidine or tryptophan residue in protein, polypeptide or the peptide.

In the specific preferred embodiment, the invention provides the method with iodine isotope radio-labeling target molecule, target molecule is preferably protein, polypeptide, peptide, antibody or antibody molecule.Such method can be included under the existence of oxygenant, mode with continuous basically effectively hybrid target molecule, iodine isotope and oxygenant in passing through the process of reaction tubes, the target molecule and the iodine isotope of significant quantity are applied to reaction chamber or reaction tubes, use iodine isotope radio-labeling target molecule thus.

Ad hoc approach with iodine radiolabeled protein, polypeptide, antibody or antibody molecule comprises:

In the presence of the oxygenant of significant quantity, to contain first kind of solution of target protein, polypeptide, antibody or antibody molecule and contain the iodine radio-labeling, second kind of solution pumping of preferred iodine-131 or iodine-125 passed preferably uninterrupted pumping basically and passed reaction chamber or reaction tubes;

Making agent, iodine radio-labeling and target protein, polypeptide, antibody or antibody molecule pass in reaction chamber or the reaction tubes process one in pumping and react, and therefore produce radioiodinated protein, polypeptide, antibody or antibody molecule; With

Pumping is collected radioiodinated protein, polypeptide, antibody or antibody molecule after passing reaction chamber; And it is preferred

Basically collected radioactive thyroprotein matter, polypeptide, antibody or the antibody molecule of purifying.

In radioactive thyroprotein matter, especially in antibody and the antibody molecule, preferably move one or more pumps oxygenant, target protein or antibody and radio-labeling are moved forward in reaction chamber or the reaction tubes with predetermined flow velocity.Chien shih oxidizer oxygen radio-labeling formed reactive mixing halogen species when predetermined flow velocity provided enough or competent." reactive mixing halogen species " is preferably can react the material that produces radiolabeled protein or antibody with the tyrosine and the histidine residues of target protein or antibody.Preferably, the substance that do not have of described situation is destroyed, and does not for example have substantive radiolysis.

Preferably radiolabeled proteins or antibody are pumped reaction chamber or reaction tubes.More preferably, then radiolabeled proteins or antibody are pumped into purification column, pass purification column by pumping and come purifying, collect then.

In another serial embodiment, the invention provides the method for addressing relative unit or equipment in the manufacturing.The example manufacture method of radio-labelled molecule device comprises:

At least first and second reagent container are provided;

Reaction chamber or reaction tubes with first end and second end are provided;

The peristaltic pump that has at least first and second passage is provided;

At least first purification unit, allocation units or collection container are provided;

Use first reagent pipe first reagent container fluid to be connected in first end of reaction chamber or reaction tubes;

Use second reagent pipe second reagent solvent fluid to be connected in first end of reaction chamber or reaction tubes;

First reagent pipe is passed first passage of peristaltic pump;

Second reagent pipe passed second passage of peristaltic pump; With

The second end fluid of reaction tubes is connected in purification unit, allocation units or collection container.

These methods may further include the reaction tubes second end fluid is connected in the import of purification unit and the outlet fluid of purification unit is connected in first collection container at least.

If desired, can be with the applying with oxygenant to the small part internal surface of reaction chamber or reaction tubes, as 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril.Similarly, can put into reaction chamber or reaction tubes with being attached to the material such as the particulate of oxygenant such as N-toluene(mono)chloride sulphonamide.

Can make device or equipment that peristaltic pump wherein further comprises the 3rd passage.These methods may further include provides the 3rd reagent container; Use the 3rd reagent pipe the 3rd reagent container fluid to be connected in first end of reaction tubes; And the 3rd reagent pipe passed the 3rd passage of peristaltic pump.

Any such device or equipment have been made among the present invention, then in the one or more method of the present invention, can advantageously use and do not need over-drastic experiment, as disclosed in this, comprise the Equivalent that those of ordinary skills are disclosed according to the present invention and this area knowledge can be learnt.

The accompanying drawing summary

Further aim of the present invention with it being played used other feature and consequent advantage, will become clear from the description of following particular preferred embodiment of the present invention, it is shown in the accompanying drawing kind, the whole parts of related numeric representation, wherein:

Fig. 1 has described the iodide ion experience balanced reaction in the aqueous solution and has formed reactive H ₂OI ⁺Material;

Fig. 2 has shown and has passed through H ₂OI ⁺The iodate of tyrosine and histidine residues in the protein.

Fig. 3 is the explanation of N-toluene(mono)chloride sulphonamide (chloramine-T) molecular structure;

Fig. 4 is 1,3,4,6-tetrachloro-3 α, the explanation of 6 α-hexichol glycoluril (Iodogen) molecular structure.

Fig. 5 has illustrated by chloramine-T catalysis and has formed highly reactive mixing halogen species and modify Histidine and tyrosine by the reactive halogen material;

Fig. 6 has represented to adhere to the particulate (iodo-pearl) of N-ammonia tolylsulfonyl amine groups;

Fig. 7 has described by on the pearl or the catalytic labeled reactant of the sulfuryl amine group that connects on the particulate illustrated in fig. 6, and it forms the iodine sulphonamide intermediate product of tyrosine and histidine residues in highly reactive iodination of protein;

Fig. 8 has illustrated by Iodogen catalysis and has formed highly reactive mixing halogen species and modify Histidine and tyrosine by the reactive halogen material.

Fig. 9 uses I ¹³¹The synoptic diagram of radiolabeled protein's apparatus of the present invention and method embodiment.

Figure 10 is the diagram of example device of the present invention and method.

Figure 11 is the diagram of another embodiment of radio-labeling apparatus and method of the present invention.

Figure 12 is the diagram of different embodiments of the present invention.

Figure 13 has illustrated and has used the reaction vessel radiolabeled protein's who applies Iodogen the apparatus of the present invention and the further embodiment of method.

Figure 14 has illustrated and has used apparatus of the present invention of the reaction vessel radiolabeled protein who applies Iodogen and another embodiment of method; With

Figure 15 has illustrated and has used apparatus of the present invention of the reaction column radiolabeled protein who fills the iodine pearl and another embodiment of method.

The description of illustrative embodiment

The present invention relates to new " a row formula " marking method and device, it allows fast, the high-quality radio-labeling of scale operation is treated protein and antibody dosage.Below will discuss each in these aspects of the present invention in more detail.

A. used catalyzer in the protein iodate

Radioiodination is that the chemically modified molecule makes it contain the process of one or more radioiodine atoms.The early stage research of protein modification is assert that iodine forms reactive ion H in the aqueous solution ₂OI ⁺, as shown in Figure 1.Reactive iodide ion (I ⁺) can modify the imidazole group of tyrosine side chain and Histidine, they are oxidized to disulphide perhaps to modify side group or catalysis, as shown in Figure 2.Except tyrosine (it has given the most stable iodate product) and Histidine, indolyl radical that also can the mark tryptophane.Radioiodinated more effective ways use chemical reagent to generate reactive iodine material.With radioiodine as ¹³¹I and ¹²⁵I link coupled to protein main method is that oxygenant comprises but is not limited to N-salt sulfonamide derivatives such as N-toluene(mono)chloride sulphonamide (chloramine-T) and 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril (Iodogen) by the use oxygenant.The chemical structure of chloramine-T (C-T) and Iodogen is shown among Fig. 3 and Fig. 4 separately.

Height reactive halogen material and the compound or the material rapid reaction that have aromatic ring structure, wherein aromatic ring structure has the carbon atom that electron donating group enough activates on encircling and stands electrophilic substitution.Therefore, contain OH, NH separately ₂Or phenol, anils or the alkyl benzene amine of NHR part are highly susceptible to by iodate.Therefore, in the protein, tyrosine side chain phenolic group and Histidine side chain imidazolyl are easy to iodate, as shown in Figure 5.Tryptophan residue in the protein also can obtain mark by its indyl.

Known in the art almost do not have influence to protein active usually with radioiodine atom adding protein molecule, unless its avtive spot has obtained modification in processing.In addition, iodine is relatively little molecule and can not causes the steric hindrance problem.As mentioned above, the site of modification is tyrosine and Histidine side chain, and tryptophane.With each phenol foundation group altogether two iodine atoms modify tyrosine, and Histidine can mix an iodine, as shown in Figure 5.

The reaction of iodine causes oxidation to form reactive, mixing halogen species, IC1 (Fig. 5) subsequently in chloramine-T and the solution.Then as mentioned above, IC1 fast and any site that can stand electrophilic substitution in the target molecule react.Because in the chloramine-T water soluble solution, react completely and carry out in mutually at solution, its cause introducing reactive iodine than use insoluble or the immobilization oxygenant as respectively with Iodogen or iodine pearl height, as described below.

Referring now to Fig. 6, it has shown the embodiment of immobilization chloramine-T analogue.As directed, derived ps particle or pearl contain N-toluene(mono)chloride sulfuryl amine group.Different with water-soluble oxidizers, the oxidation capacity of immobilization oxygenant is subjected to its fixedly restriction of support surface, and it has significantly reduced iodine and has mixed macromolecular speed. and slow if desired speed and low mixing, this is favourable.Still limit the tyrosine iodate is become single iodine form, therefore avoided solubleness or active disadvantageous effect causing excessive grooming.

Then, another mechanism of using derive particulate iodotyrosine and the Histidine of Fig. 6 has been described with reference to Fig. 7.Opposite with the mechanism of the use chloramine-T shown in Fig. 5, do not form the mixing halogen species in this reaction.Alternate is that sulfuryl amine group forms highly reactive iodine sulphonamide intermediate product, as directed iodotyrosine of this intermediate product and histidine residues.

Next width of cloth figure, Fig. 8 has shown and has used insoluble oxygenant Iodogen to form a method again of mixing halogen species.Because in the water insoluble solution of Iodogen, therefore as the agent of solid type radioiodination.The iodine pearl difference of oxide group Covalent Immobilization on solid support wherein, Iodogen is coating before the iodate on solid support or the reaction vessel.Chloramine-T is similar with using, and the reaction of iodine causes forming the reactive halogen species IC1 that mixes in Iodogen and the solution, and is as directed.IC1 and the reaction target molecule rapid reaction that can stand the electrophilic replacement are as discussed above then.

B. successive one row formula radio-labeling

As mentioned above, the present invention relates to use control mobile reactant in reaction tubes or container continuously, a row formula, mobile phase radiolabeled protein, form efficient, large-scale Radiolabelling method.Although surprising progress, the present invention is applicable in a plurality of embodiments, for example, and as described below in conjunction with Fig. 9 to 15.

With reference to Fig. 9, the apparatus of the present invention of radiolabelled antibody and the embodiment of method have been shown especially.This device can comprise 100, the second reagent containers 102 of first reagent container and the 3rd reagent container 104, by 106, the second reagent pipes 108 of first reagent pipe and the 3rd reagent pipe 110 the reagent container fluid is connected in mixing section 114 separately.Before incorporating mixing section 114 into, each reagent pipe is passed pump 112.The preferably controllable multi-channel peristaltic pump of pump 112, as be used for the pump of liquid chromatography.Mixing section 114 fluids are connected in first end 116 of reaction tubes or reaction vessel 118.Second end, 120 fluids of reaction tubes 118 are connected in purifying chamber or purification column 122.

Purification column 122 contains the trapping agent that is incorporated into not connection or free radioactivity active element and reaction reagent, but allow the protein of mark to pass through. purification column 122 can contain resin anion(R.A) and or the polystyrene of chloromethylation, or material of catching iodine oxide and free-iodine well known in the art.Protein with mark is collected in the collection container 124 then.System can be had no particular limits from being adjusted to very large scale very on a small scale by diameter and the suitable peristaltic pump of use that changes pipe.Can also control speed of reaction and labelled amount by flow velocity that changes solution and length and/or the diameter that changes reaction tubes 118.Therefore, those ordinarily skilled in the art are disclosed according to the present invention, will understand to change combination effectively.

For example, usually according to the size of batch to be produced, can use the reagent pipe of any possibility diameter.If the amount of cryoproteins matter or antibody is big, preferred antibody reagent pipe diameter is bigger, and oxygenant and radiolabeled reagent pipe diameter are less.Can also come adjusting reaction time with the speed (flow velocity) of pump and the length of reaction tubes.Therefore, the present invention does not have significant restriction.Disclosed according to the present invention, can use reaction tubes narrow or that approach to produce single dose and use larger-diameter reaction tubes to produce hundreds of dosage now.

The present invention is better than in batches that the important difference and the advantage of the art methods of mark comprise, only has the active and short production time internal labeling of small portion almost unrestrictedly to measure the ability of protein or antibody under reaction conditions.Before the present invention, can not the many dosage of mark in single batch, because the high radioactivity in the large volume batch has destroyed antibody, making can not the occurrence flag reaction.The present invention has overcome all such defectives.

Fig. 9 has also shown the reagent container of filling all ingredients solution.Container 100 has been filled water-soluble oxidizers such as chloramine-T (C-T) 101, and container 102 has been filled antibody to be marked 103, and container 104 has been filled ¹³¹I 105.When ejector priming 112, enter in the mixing section 114 so reagent pumped into from reagent container in their reagent pipes separately, every kind of reagent obtains mixing therein.Mixing solutions is entered in the reaction tubes, therein C-T 101 Hes ¹³¹I105 reaction forms the reactive halogen species IC1 115 that mixes, and therefore the tyrosine and the reaction of Histidine side chain of itself and antibody 103 are used ¹³¹The I traget antibody, as above described in conjunction with Fig. 5.Make mixing solutions by the most of antibody in the next enough mark mixing solutionss of reaction tubes 118 preset times, if not whole antibody.As those skilled in the art will recognize that, this labeling process can be infinitely continuous, and therefore the marked product of high volume is provided.After the mixing solutions that contains marked product 117 now passes reaction tubes 118, enter purification column 122, wherein from described solution, remove any residual free ¹³¹I 105, halogen species 115 and C-T101.Purification column allows antibody product 117 and any unlabelled antibody 103 of mark to pass, and it is collected in the collection container 124, as shown in Figure 9.

Then, shown the diagram of Fig. 9 device, had whole described parts, and comprised that further air outlet 128 prevents to form pressure and or vacuum in container that installs and pipeline with reference to Figure 10.Also shown around container 104 that contains radioiodine and the lead shielding 130 that contains the collection container 124 of radiolabeled antibody 117.Can also adding arbitrarily in the shown device, a row formula filter 126 filters purification column 122 effusive solution before.One row formula filter is sterile filters preferably.

Referring now to Figure 11, another embodiment in conjunction with Fig. 9 and the described device of Figure 10 has been described.In this embodiment, reagent pipe 106,108 and 110 passes single peristaltic pump from each reagent container 100,102 and 104 separately.Wherein, pipe 106 passes first pump 132, and pipe 108 passes second pump 134 and manages 110 and pass the 3rd pump 136.Then the reagent manifold is gone in the mixing section 114, its fluid is connected in first end 116 of reaction tubes 118.The second end fluid of reaction tubes 118 is connected in purification column 122.The purification column fluid connects a row formula filter 126 then.The effluent of filter 126 is collected in the collection container 124.

It is separately to control the flow velocity of each pump that each reagent container uses the specific advantages of branch turn on pump.This can optimizing and control solution in the labelled amount of every kind of target protein or antibody.As non-limiting example, by the flow velocity of

pump

132 and 136 flow velocity with respect to pump 134 is set at slowly, obtain single iodate of tyrosine, in resulting solution, contain more antibody and less oxygenant and reactant thus, therefore reduced tyrosine residues two iodinating chances in the protein.Therefore can control ratio between every kind of reactant by the flow velocity that changes each pump.Therefore those skilled in the art will recognize that the versatility of this setting.

Referring now to Figure 12, shown the different synoptic diagram of selecting embodiment fully of explanation apparatus of the present invention.In one embodiment, still as above described in conjunction with Fig. 9 and Figure 10, comprise three reagent containers 100,102 and 104, multi-channel peristaltic pump 112, reaction tubes 118, purification column 122 and collection container 124.In another embodiment, particulate or sterile filters 126 are positioned between purification column 122 and the receiving flask 126, as directed.Still in another embodiment, use dialysis unit 138 to substitute purification column 122 and come the radiolabeled product of purifying.Preferably radio-protector 140 is added in the final radiolabeled product 117 and prevent the radiation destruction product.Radio-protector can include but not limited to, serum albumin for example is as human serum albumin, bovine serum albumin, horse serum albumin with from the serum albumin of other Mammals kind; Sodium ascorbate; With other absorbing radiation and not with the material of radio-labeling product reaction.

Remain another embodiment of the present invention kind, only used two

reagent containers

102 and 104, rather than three.In this embodiment, oxygenant and molecule to be marked, protein or antibody 103 mix in container 102, and with labelled with radioisotope ¹³¹I 105 puts into container 104.Use pump 112 reaction tubes 118 to be passed in these two kinds of solution pumpings then and come traget antibody 103 with enough acquisition grace times with predetermined flow velocity.As mentioned above, the solution that uses purification column 122 purifying pipes 118 wash-outs to come out is then removed free radioiodine 105, oxygenant 101 and any reactive intermediate product 115.Preferred post 122 is anion-exchange columns.Perhaps, can use dialysis unit 138 to come purified product, or use post 122 and dialysis unit 138 to come purified product simultaneously.

Then describe Figure 13, still described of the present invention another and selected embodiment fully.In this embodiment, use two reagent containers 102 and 104.Each container is connected mixing section 114 by reagent pipe 108 separately with 110

fluids.Pipe

108 and 110

peristaltic pumps

134 and 136 that pass separately.Mixing section 114 fluids are connected in first end of reaction chamber 118.Reaction chamber 118 applies water-insoluble oxygenant 142.Oxygenant 142 is Iodogen preferably, and is as directed.Second end, 120 fluids of reaction chamber 118 are connected in purification column 122, and its fluid is connected in filter 126.The effluent of filter 126 is collected in the receiving flask 124.In conjunction with Fig. 8 as mentioned above, radio-labeling ¹³¹I forms the reactive halogen species IC1 115 that mixes by the Iodogen oxidation.The flow velocity of

pump

134 and 136 is set in antibody 103 and the radioiodine 105 that predetermined speed provides mark aequum or required ratio.Make the mixture of antibody 103 and radioiodine 105 maximize the mark of antibody 103 by reaction chamber 118 preset times.Therefore this obtains by setting flow velocity.

See Figure 14 again, represented another embodiment of Figure 13 apparatus and method.This embodiment comprises by managing 109 fluids and is connected in the single container 107 of reaction chamber 118 first ends 116.Pipe 109 passes peristaltic pump 111.Reaction chamber 118 is connected in purifying filter 122 at second end, 120 fluids, and its fluid is connected in filter 126.The also filtering product 117 of resulting purifying is collected in the collection container 124.As directed, antibody 103 and radio-labeling 105 are positioned in the container 107.Use pump 111 pipe 109 to be passed in this mixture pumping then and make that oxygenant 142 and mark 105 reactions on 118 walls of chien shih chamber when enough are arranged, produce halogen species 115 with set rate.Tyrosine and histidine residues covalent attachment on halogen species and the antibody 117 produces radio-labeling product 117. as mentioned above then

Then with reference to Figure 15, what apparatus of the present invention and method be described one selects embodiment more fully.With above-mentioned opposite with the described embodiment of Figure 14 in conjunction with Figure 13, the particulate that adheres to oxygenant 144 shown in reaction chamber 118 is filled.Reagent pipe 118 and 110 fluids of reagent container 102 and 104 by separately are connected in mixing section 114.Mixing section 114 fluids are connected in first end 116 of chamber 118.The second end fluid of chamber 118 is connected in purifying chamber 122, and its fluid is connected in filter 126.Purified product 117 is collected in the container 124, and is as directed.In the use, container 102 is filled for example cold antibody, and container 104 is filled radioiodine 105.Process pump 134 and 136 pumps into the solution in container 102 and 104 antibody 103 and the radio-labeling 105 that obtains required ratio in the mixing section 114 with predetermined flow velocity then.Solution mixed in mixing section 114 before entering reaction chamber 116 then.In case in chamber 116, be attached to the oxygenant of particulate 144 and the radioiodine in the solution and react and form highly reactive toluene iodide sulphonamide intermediate product, with tyrosine in the iodination of protein and histidine residues, as above described in conjunction with Fig. 7.From purifying chamber 122, filter any residual radioiodine 105 then in the effusive solution.Then purification solution is passed filter 126 and remove macrobead in the solution.The filtering solution of purifying that will contain radiolabelled antibody 117 then is collected in the container 124.

C. continuous large-scale protein labeling

The target that the open middle demonstration development and Design of the present invention is used for the extensive Radiolabelling method of pharmaceutical industries has realized.As mentioned above with following examples in the inventive method, can be counting GBq radiolabeled protein in the short production time, and therefore extensively apply to produce various diagnosis and radiotherapy medicine.

Before the present invention, several parameter limit extensive labelled protein, comprise antibody.Important limiting factor is a gross activity, the volume of mark batch and the radiolysis of product.For example, in the art methods, in single container, hatch protein to be marked and radio-labeling, make all radio-labelings in entire reaction course, contact with protein.In addition, radio-labeling and protein keep in touch the extra time that needs purifying.Along with common reaction and purifying time, this has the defective that causes protein denaturation and cohesion.In addition, the individual need shielding of the such batch processing mode labeling technique of operation needs the lead shielding of 4-6 inch usually in whole process.Except these difficulties, the given activity of the product that prior art and existing method are produced is restricted; Expensive and the not raising in proportion of process.

Novel method of the present invention has been avoided these difficulties by mark batch materials in a continuous manner, only has small portion protein to contact with radio-labeling in any given time, as above in conjunction with Fig. 9,10,11,12,13 and 15 described.Although be suitable for using any radio-labeling, the present invention uses the iodine-131 particularly advantageous.For example, with opposite with the yttrium mark, use the method for prior art problem more to be arranged, in particular for the embodiment beyond the small-scale mark with the iodine-131 mark with reagent such as technetium.When attempting with the extensive mark of iodine-131, radiolysis is very significant problem, the practicality that it has destroyed protein or antibody and has limited these technology.

The present invention has overcome the various defectives in the existing radio-labeling technology, especially uses those of iodine-131 mark.Be better than hatching separately in " batch processing mode ", biotic component to be marked, protein or antibody contact with " continuous mode " with iodine-131 activity and oxygenant, making only has protein, iodine-131 activity and the oxygenant of small portion to mix in any given time, as described above.Oxygenant, as chloramine-T or Iodogen oxidation iodine-131, the radical reaction on itself and the biotic component to be marked is as the tyrosine on protein and the antibody, as above described in conjunction with Fig. 1 to 8 then.

The size of labelling apparatus of the present invention or equipment can be different, but generally include three different treating parts.These are shown in the synoptic diagram of Fig. 9 and in the graph of Figure 10.Referring again to Fig. 9 and Figure 10, the first part of process utilizes that three in local lead shielding 130 separate contains reagent, educt or reactant: the reacted constituent holder 100,102 of protein 103, chloramine-T 101 and iodine-131 105 and 104 (Figure 10).Second section comprises from each reagent holder 100,102 and 104 separately and enters single mixing tube 114 by pump 112 (preferred triple channel peristaltic pump), enters the reaction chamber that carries out labeled reactant or the pipe 106,108 and 110 of reaction tubes 118 then.The third part of process relates to a row formula purifying, comprises a row formula purification unit 122 and a collection device 124.Various purifier apparatus are suitable, and are as volume-exclusion post, anion-exchange column, ion exchange column and dialysis equipment, as above described in conjunction with Figure 11.Sterile filters 126 and receiving flask 124 are followed in purification unit or equipment 122 preferred back.Receiving flask preferably contains additive and stablizer such as the radio-protector that is useful on finished product.

Although the embodiment of the present invention that is described among Fig. 9 and Figure 10 is used three reacted constituent holders that separate, protein and chloramine-T can be incorporated in first composition holder and with iodine-131 solution and remain in second the composition holder that has local lead shielding, as shown in figure 12.Therefore, the invention provides the system of two holders, it can use two multi-channel peristaltic pumps.In another embodiment, can use water insoluble ingredients, as Iodogen, rather than chloramine-T, as above described in conjunction with Figure 13 and Figure 14.Can be dissolved in Iodogen in the chloroform and be used to apply reaction tubes 118.Iodogen also can be used in the pearl form, and as the iodine pearl, it comes down to chloramine-T in conjunction with pearl.

In any pattern, can make complete marking arrangement or the device that has aseptic processing element arbitrarily in the mark cover of shielding under aseptic condition, and finally minimize personnel's radiation burden, the bottle that contains iodine-131 solution can linking device.

In the operation of labelling apparatus of the present invention and method,, the educt or the reactant of reagent holder are introduced in reaction tubes or the reaction vessel, preferably used peristaltic pump no matter be two or three storeies.Therefore, reacted constituent only mixed before entering reaction tubes.Use the present invention also directly to regulate the reaction times, for example pass through to change the length and/or the diameter of reaction tubes, and by changing the flow velocity of pump.

By removing the just stop flag reaction simply of free-iodine and oxygenant, do not need to add any reaction reagent.From handle, remove these reagent by a row formula purifying, as be placed in a row formula purification column 122 of reaction tubes 118 ends by use.The protein or the antibody-solutions of mark are collected in the bottle, have preferably contained stablizer, radio-protector, for example xitix or inert protein such as BSA, and/or the final required any additives of preparation.This makes marked product be diluted to selected content in gathering system or bottle, for example, radiolabeled protein or antibody is distributed into single dose and quality control sample.This method can be moved the time period of prolongation, for example, and effectively labelled protein is distributed into the mode of 10,000 or the more bottles that are used for patient treatment.

Comprise that following embodiment proves the preferred embodiment that the present invention is specific.Those skilled in the art will recognize that disclosed representative method, device and method for compositions, device and the composition of finding according to the inventor plays a good role among the embodiment in the invention process, and therefore think the preference pattern that constitutes its enforcement.Yet, those skilled in the art are with disclosed according to the present invention, recognize and in disclosed particular, can carry out many changes and still obtain result similar, similar or of equal value and do not deviate from the spirit and scope of the present invention, as claims clear and definite.

Embodiment 1

An extensive row formula radiolabeled protein continuously,

This embodiment and embodiment have subsequently described successful development extensive radiolabeled method and apparatus has the advantage that can be used in the pharmaceutical industry.

The present inventor has used apparatus and method of the present invention, especially abovely comes radio-labeling in conjunction with Fig. 9 and the described apparatus and method of Figure 10, in batches up to the 487GBq in conjunction with monoclonal antibody ¹³¹I (table 1).About 40 minutes of whole process need comprises the reaction times, purifying and final preparation.Free-iodine is 0.1 to 2.1%.The overall yield minimum is 85%, and maximum is 96%.Antibody to every batch of mark carries out the Size Exclusion Chromatograph SEC analysis, and it shows insignificant antibody cracking and cohesion and is higher than 95% complete radiolabeled antibody monomer.The exemplary results of scale operation is listed in the table 1.Carry out cell and test the high quality that proves product in conjunction with potential, with respect to the unmarked antibody of criticizing together in producing, it demonstrates and surpasses 80% potential.

Table 1

Batch	Active	Chloramine-T	Antibody	Cumulative volume	Reaction times	Productive rate	Productive rate	Free-iodine
Batch	Active	Chloramine-T	Antibody	Cumulative volume	Reaction times	Productive rate	Productive rate	Free-iodine		GBq	mg	mg	mL	Min.	GBq	％	％
A	47.9	1.8	60	3×3.3	10	42.9	90	1.4		GBq	mg	mg	mL	Min.	GBq	％	％
A	47.9	1.8	60	3×3.3	10	42.9	90	1.4	B	25.2	0.9	30	3×3.3	10	21.4	85	0.1
C	288.2	25	450	3×20.0	10	256.5	89	1.8	B	25.2	0.9	30	3×3.3	10	21.4	85	0.1
C	288.2	25	450	3×20.0	10	256.5	89	1.8	D	487.3	30	1000	2×10+50	10	466.8	96	2.1

Embodiment 2

A row formula radio-labeling and pass through dialysis purification continuously,

This embodiment is particularly related to the radio-labeling product that uses dialysis to come purifying large scale production method of the present invention.

Among this embodiment, protein to be marked is Lym-1 antibody.The further details that relates to Lym-1 antibody and use thereof is provided in U.S. Patent No. 4,724,213, and denomination of invention is " mouse hybridoma Lym-1 and the diagnosis antibody that therefore produces "; U.S. Patent No. 4,861,579, denomination of invention is " suppressing B-lymphocyte in the Mammals by the anti-B-lymphocyte antibody of administration "; U.S. Patent No. 5,194,594, denomination of invention are " antibody of modification "; With U.S. Patent No. 6,007,817, denomination of invention is " conjugate that promotes vascular permeability "; Wherein every piece is incorporated herein by reference with its integral body at this.

Use has the unitary configuration of two 4ml reagent containers and dialysis purification with I-131 label L ym-1 antibody, as above in conjunction with Figure 12 summarized.First reagent bottle is filled the 5mg Lym-1 that contains among 80mCiI-131 (sodium salt), the 250ml and the mixture of 3ml 0.1M potassiumphosphate.In second reagent bottle, with chloramine-T and the mixing of 3ml potassiumphosphate of 100 μ l 0.5mg.The flow velocity of peristaltic pump is set at 0.6ml/ minute, and the reverse flow of dialysis solution (0.9%NaCl) is set at 8ml/ minute.Reaction times in this batch reaction tubes is 12 minutes.The efficient of calculating dialysis purification is 99.9%.Only come stopped reaction, and do not need to add Sodium Pyrosulfite by dialysis.Output of this batch or labeling effciency are 92%.

Use in second mark batch of identical configuration, first 4ml reagent bottle is filled the mixture that contains 5mg Lym-1 and 3ml 0.1M potassiumphosphate among 71mCi I-131 (sodium salt), the 250ml.Second 4ml reagent bottle filled the chloramine-T that is dissolved in 100 μ l 0.5mg in the 3ml Repone K.The flow velocity of peristaltic pump is set at 0.6ml/ minute, and similarly the reverse flow of dialysis solution (0.9%NaCl) is set at 8ml/ minute.Reaction times in this batch reaction tubes is 19 minutes.Output of this batch or labeling effciency are 88%.The potential of resulting radio-labeling Lym-1 antibody or binding affinity (with respect to unlabelled Lym-1 antibody) are determined as 94%.

Embodiment 3

A row formula radio-labeling and pass through ion-exchange purification continuously,

This embodiment has described the radio-labeling Lym-1 antibody that uses ion exchange column to come purifying to make by the inventive method.

Use as above and dispose antibody with I-131 (sodium salt) radio-labeling Lym-1 in conjunction with Fig. 9 and the described the present invention of Figure 10.With reference to Figure 10, in this mark batch, first reagent bottle 100 is filled the 30mg chloramine-T in the 10ml DI water, fills the solution that 50ml contains 1g Lym-1 antibody in second reagent bottle 102, fill 13170mCi I-131 among the 10ml 0.1M PBS, pH7.4 with the 3rd reagent bottle 104.23ml pasc reaction pipe 118 is used for this radio-labeling batch.Use ion exchange column 122 to come the resulting radio-labeling product of purifying.The flow velocity of this configuration is 2.3ml/ minute, and it uses the 23ml reaction tubes to give 10 minute reaction times.The labeling effciency of this batch is determined as 95.6%.

Embodiment 4

Continuous, a row formula radio-labeling and a purifying of ch-TNT-1/b antibody

This embodiment has put down in writing second kind of radiolabeled antibody using ion exchange column to come purifying to be made by the inventive method.

In this research, protein to be marked is Ch-TNT-1/b antibody.More details about TNT antibody are provided in U.S. Patent No. 6,071,491, and denomination of invention is " detection of downright bad malignant tissue and an associated treatment ", and it specially is incorporated herein by reference with its integral body at this.

Use the as above similar configuration described in the embodiment 3 with I-131 (sodium salt) mark Ch-TNT-1/b antibody, with 17ml pasc reaction pipe and 3ml Dowex ion exchange column.The volume that every kind of reagent place uses is as follows: 4ml I-131,4ml antibody and 22ml chloramine-T, with and separately concentration be shown in the following table 2.Receiving flask is filled radio-protector, comprises the serum albumin of 1.4 to 1.5g sodium ascorbates and 37 to 39ml 25%.Then resulting radio-labeling product is diluted in 0.9% salt solution and obtain final concentration.Be shown in result in the table 2 and show 88% or higher output or labeling effciency, use 10 minutes wash-outs or reaction times, use configuration as shown in figure 10.

Table 2

Batch	Active	Chloramine-T	Antibody	Cumulative volume	Reaction times	Residual activity	Productive rate	Productive rate
Batch	Active	Chloramine-T	Antibody	Cumulative volume	Reaction times	Residual activity	Productive rate	Productive rate		mCi	mg	mg	mL	min	mCi	mCi	％
A	2960	9.6	216	30	10	54	2564	88		mCi	mg	mg	mL	min	mCi	mCi	％
A	2960	9.6	216	30	10	54	2564	88	B	3216	9.6	233	30	10	62	2910	92
C	3120	9.2	233	30	10	54	2759	89	B	3216	9.6	233	30	10	62	2910	92
C	3120	9.2	233	30	10	54	2759	89	D	2820	10.1	231	30	10	154	2543	90

Generally speaking, as demonstrating by above-mentioned work embodiment, so the new row formula marking method of the present invention allows rapid large-scale to produce high-quality treatment protein and antibody dosage.These methods can be used in a plurality of embodiments.System can be adjusted to very large scale and not have specific limited from very little reaction volume, for example by selecting suitable pipe.Continuous flow makes in principle production lot without limits.This is being important from preclinical study to the small-scale clinical trial to extensive patient treatment; because the mark therapeutical agent that can use identical method to prepare to be used for single patient or hundreds and thousands of patients, it is important for FDA approval treatment production technology.

All parts that further advantage of the present invention is a device can asepticly make and can dispose arbitrarily; Pipe can take out from pump and abandon, and making does not need conventional the cleaning.After using very for a long time, radiolysis will show can abandon and replace pipe and purifier apparatus.However, the time that the operation of continuous mode of the present invention can prolong, this is to use batch mark of prior art and existing method not reach.The cost that the present invention reduces also is a significant benefits, studies show that based on this and can save 10 to 30 times of costs.This method also is easy to automatization, and the present invention can use in hospital.

Therefore the invention provides effective mark novel method, especially labelled protein and antibody.According to instruction of the present invention, now can adjustable pipe, the capacity of flow velocity, receiving flask or the like, however, obtained commercial and the especially scale operation of antibody of biochemical quality status stamp product.

***

All patents of mentioning in this specification sheets, patent application, temporary patent application and other publication specially are incorporated herein by reference with its integral body at this.

Disclosed according to the present inventionly can implement, make and use, and not need undue experimentation at these disclosed all methods, device and composition.Though describe method of the present invention, device and composition in detail with reference to specific preferred embodiment, those of ordinary skills it is evident that the present invention is not subject to those accurate embodiments.On the contrary, in view of disclosed in this invention, will understand can with many improvement and change utilization to device and composition and utilization to the step or series of steps of said method, and do not deviate from notion of the present invention, spirit and scope.Be clear that also chemistry can replace reagent described herein with biological relevant particular agent, and obtain same or analogous result.In addition, those of ordinary skills only use normal experiment will recognize the scheme that maybe can determine with particular equivalence described herein.Think that all such are included in the scope of claim with change, improvement, variation, similar replacement and equivalents in the scope in the claim equivalence meaning.

Claims

1. be used for coming with radio-labeling the radio-labeling device of the continuous mode of tagged molecule, described device comprises:

The reaction chamber or the reaction tubes that comprise first end and second end;

First reagent container, it is connected with the described first end fluid of described reaction chamber or reaction tubes by first reagent pipe, and described first reagent pipe coupling is in first pump at least;

Second reagent container, it is connected with the described first end fluid of described reaction chamber or reaction tubes by second reagent pipe, and described second reagent pipe coupling is in pump;

Radiation shielding, wherein at least one described reagent container is contained in the described radiation shielding; With

First purification unit at least that comprises import and outlet, the described import of described purification unit is connected with the described second end fluid of described reaction chamber or reaction tubes.

2. the device of claim 1, wherein said device comprises reaction tubes.

3. the device of claim 2, wherein said reaction tubes is predetermined length and predetermined diameter.

4. arbitrary device in preceding claim, wherein said first reagent pipe and described second reagent pipe are connected in described first pump separately.

5. the device of claim 4, wherein said first pump is a multi-channel peristaltic pump.

6. the device of claim 5, wherein said multi-channel peristaltic pump is by computer-controllable system.

7. the device of claim 1, wherein said reaction chamber or reaction tubes contain oxygenant.

8. the device of claim 7, wherein said reaction chamber or reaction tubes apply described oxygenant to the small part internal surface.

9. the device of claim 8, wherein said oxygenant is 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril.

10. the device of claim 7, wherein said reaction chamber or reaction tubes contain the particulate group who is attached to described oxygenant.

11. the device of claim 10, wherein said oxygenant are N-toluene(mono)chloride sulphonamide.

12. the device of claim 1, wherein said device further comprise the 3rd reagent container that is connected with the described first end fluid of described reaction chamber or reaction tubes by the 3rd reagent pipe, described the 3rd reagent pipe coupling is in pump.

13. the device of claim 12, wherein said first, second and the 3rd reagent pipe be connected in described first pump separately.

14. the device of claim 12, wherein said first, second and the 3rd reagent pipe be connected to separately first, second and the 3rd pump.

15. the device of claim 14, wherein said first, in second and the 3rd pump each be peristaltic pump.

16. the device of claim 14, wherein said first, in second and the 3rd pump each be by computer-controllable system.

17. the device of claim 1, wherein said purification unit contain at least the first kind of material in conjunction with iodine, mixing halogen species and iodine oxide material.

18. the device of claim 1, wherein said purification unit comprise at least the first kind of ion exchange column, anion-exchange column or volume-exclusion post.

19. the device of claim 1, wherein said purification unit comprise first dialysis unit at least.

20. the device of claim 1, wherein said device further comprise first allocation units or the collection container at least that is connected with the described outlet fluid of described purification unit.

21. the device of claim 20, wherein said device further comprises the filter with entrance end and exit end, and the described entrance end of described filter is connected with the described outlet fluid of described purification unit; And the described exit end of described filter is connected in described collection container.

22. the device of claim 1, wherein said molecule are protein, polypeptide or peptide.

23. the device of claim 1, wherein said molecule is an antibody molecule.

24. the device of claim 1, wherein said radio-labeling are iodine-131 or iodine-125.

25. the continuation method of preparation radio-labelled molecule, be included under the condition of the described radiolabeled molecule of effective generation and in the time length, with significant quantity treat radiolabeled molecule and radio-labeling together with the aid mark agent of significant quantity radio-labeling device by continuous mode, described device comprises:

Second reagent container, it is connected with the described first end fluid of described reaction chamber or reaction tubes by second reagent pipe, and described second reagent pipe coupling is in pump; With

First purification unit at least that comprises import and outlet, the described import of described purification unit is connected with the described second end fluid of described reaction chamber or reaction tubes;

And from described device, collect described radiolabeled molecule.

26. the method for claim 25, wherein said device comprises radiation shielding, and wherein at least one described reagent container is contained in the described radiation shielding.

27. the method for claim 25 or 26, wherein said radio-labeling are oxidable radio-labelings, and wherein said aid mark agent is an oxygenant.

28. the method for claim 27, wherein said method comprises:

First kind of solution that will contain target molecule and oxygenant is put into described first reagent container;

To contain oxidable radiolabeled second kind of solution and put into described second reagent container;

Described first kind of solution and described second kind of solution pumping are passed described reaction chamber or reaction tubes, wherein said target molecule, described oxygenant and described oxidable radio-labeling react in the process of passing described reaction chamber or reaction tubes, therefore produce the mixture that contains radiolabeled molecule;

Described purification unit is passed in described mixture pumping; With

Collect the radiolabeled molecule of purifying basically from described purification unit.

29. the method for claim 27, when reaction chamber or reaction tubes comprise the oxygenant described in the claim 7, wherein said method comprises:

First kind of solution that will contain target molecule is put into described first reagent container;

Described first kind of solution and described second kind of solution pumping are passed described reaction chamber or reaction tubes, the oxygenant that contains in wherein said target molecule and described oxidable radio-labeling and described reaction chamber or the reaction tubes reacts in the process of passing described reaction chamber or reaction tubes, therefore produces the mixture that contains radiolabeled molecule;

Described purification unit is passed in described mixture pumping; With

30. the method for claim 27, when described device further comprises the 3rd reagent container described in claim 12, wherein said method comprises:

Oxygenant is put into described first reagent container;

Target molecule is put into described second reagent container;

Oxidable radio-labeling is put into described the 3rd reagent container;

Described reaction chamber or reaction tubes are passed in described oxygenant, described target molecule and described oxidable radio-labeling pumping, wherein said target molecule, described oxygenant and described oxidable radio-labeling react in the process of passing described reaction chamber or reaction tubes, therefore produce the mixture that contains radiolabeled molecule;

Described purification unit is passed in described mixture pumping; With

31. the method for claim 27 is wherein passed described reaction chamber or reaction tubes with described molecule and described oxidable radio-labeling pumping.

32. the method for claim 31, wherein said molecule and described oxidable radio-labeling quilt uninterrupted pumping basically pass described reaction chamber or reaction tubes.

33. the method for claim 31 or 32 is wherein passed reaction chamber with described molecule and described oxidable radio-labeling pumping.

34. the method for claim 31 or 32 is wherein passed reaction tubes with described molecule and described oxidable radio-labeling pumping.

35. the method for claim 34, wherein said reaction tubes is predetermined length and predetermined diameter, is enough to make described molecule, described oxidable radio-labeling to contact with described oxygenant to produce the described radiolabeled molecule of aequum at time per unit.

36. the method for claim 31 or 32 is wherein passed described reaction chamber or reaction tubes by multi-channel peristaltic pump with described molecule and described oxidable radio-labeling pumping.

37. the method for claim 36 is wherein passed described reaction chamber or reaction tubes by computer-controlled multi-channel peristaltic pump with described molecule and described oxidable radio-labeling pumping.

38. the method for claim 27, wherein said oxygenant be present in described reaction chamber or reaction tubes to the small part internal surface, and wherein said molecule and described oxidable radio-labeling and described oxygenant react in the process of passing described reaction chamber or reaction tubes.

39. the method for claim 38, wherein said oxygenant is 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril.

40. the method for claim 27, wherein said oxygenant is present on the particulate that contains in described reaction chamber or the reaction tubes, and wherein said molecule and described oxidable radio-labeling and described oxygenant react in the process of passing described reaction chamber or reaction tubes.

41. the method for claim 40, wherein said oxygenant are N-toluene(mono)chloride sulphonamide.

42. the method for claim 27 is wherein passed described reaction chamber or reaction tubes with every kind of pumping in described molecule, described oxidable radio-labeling and the described oxygenant.

43. the method for claim 42 wherein pumps into described reaction chamber or the reaction tubes from the reagent container that separates every kind in described molecule, described oxidable radio-labeling and the described oxygenant.

44. the method for claim 42, wherein the mixture with described molecule and described oxygenant pumps into described reaction chamber or the reaction tubes from first reagent container, and wherein described oxidable radio-labeling is pumped into described reaction chamber or the reaction tubes from second reagent container.

45. the method for claim 42, wherein said oxygenant are N-toluene(mono)chloride sulphonamide.

46. the method for claim 27 further comprises described radiolabeled molecular application in first purification unit at least and from wherein separating the radiolabeled molecule of purifying basically.

47. the method for claim 46, wherein said purification unit comprise first ion exchange column, anion-exchange column or volume-exclusion post at least.

48. the method for claim 46, wherein said purification unit comprise first dialysis unit at least.

49. the method for claim 46 comprises that further the radiolabeled molecule with described purifying basically filters by the sterile filters that is connected in described purification unit.

50. the method for claim 27 further comprises at least the first kind of radio-protector added in the radiolabeled molecule of described purifying basically.

51. the method for claim 50, wherein said radio-protector are bovine serum albumin, human serum albumin or xitix.

52. the method for claim 27, wherein said molecule are protein, polypeptide or peptide.

53. the method for claim 52 is wherein used the described protein of described radio-labeling mark on Histidine, polypeptide or peptide.

54. the method for claim 52 is wherein used the described protein of described radio-labeling mark on tryptophane, polypeptide or peptide.

55. the method for claim 52 is wherein used the described protein of described radio-labeling mark on tyrosine, polypeptide or peptide.

56. the method for claim 52, wherein said molecule is an antibody molecule.

57. the method for claim 27, wherein said oxidable radio-labeling is iodine-131 or iodine-125.

58. be used for the manufacture method of the continuous mode radio-labeling device of radio-labelled molecule, described method comprises:

At least first and second reagent container are provided;

The reaction chamber or the reaction tubes that comprise first end and second end are provided;

At least first pump is provided;

Radiation shielding is provided, and wherein at least one described reagent container is contained in the described radiation shielding;

At least first purification unit is provided, and it comprises import and outlet;

Use first reagent pipe that described first reagent container is connected with the described first end fluid of described reaction chamber or reaction tubes, described first reagent pipe coupling is in described first pump at least;

Use second reagent pipe that described second reagent container is connected with the described first end fluid of described reaction chamber or reaction tubes, described second reagent pipe coupling is in pump; With

Described second end of described reaction tubes is connected with the described inlet fluid of described purification unit.

59. the method for claim 58, wherein said first pump at least are to have the peristaltic pump of first passage and second passage at least.

60. the method for claim 59, wherein said first reagent pipe and described second reagent pipe coupling are to described first pump.

61. the method for claim 58 comprises first allocation units or collection container at least are provided; And directly or indirectly the described outlet of described purification unit is connected with described first allocation units at least or collection container.

62. the method for claim 58, further comprise with oxygenant apply described reaction chamber or reaction tubes to the small part internal surface.

63. the method for claim 62, wherein said oxygenant is 1,3,4,6-tetrachloro-3 α, 6 α-hexichol glycoluril.

64. the method for claim 58 further comprises the particulate that is attached to oxygenant is put into described reaction chamber or reaction tubes.

65. the method for claim 64, wherein said oxygenant are N-toluene(mono)chloride sulphonamide.

66. the method for claim 59, wherein said peristaltic pump further comprise the 3rd passage.

67. the method for claim 66 comprises:

The 3rd reagent container is provided;

Use the 3rd reagent pipe that described the 3rd reagent container is connected with the described first end fluid of described reaction tubes; With

Described the 3rd reagent pipe passed described the 3rd passage of described peristaltic pump.