1111111111111111 IIIIII IIIII 11111 1111111111 1111111111 111111111111111 11111 1111111111 11111111
`US 20070238860Al
`
`c19) United States
`c12) Patent Application Publication
`SCHLEGL
`
`c10) Pub. No.: US 2007 /0238860 Al
`Oct. 11, 2007
`(43) Pub. Date:
`
`(54) METHOD FOR REFOLDING A PROTEIN
`
`(30)
`
`Foreign Application Priority Data
`
`(76)
`
`Inventor:
`
`Robert SCHLEGL, Wien (AT)
`
`Correspondence Address:
`MICHAEL P. MORRIS
`BOEHRINGER INGELHEIM CORPORATION
`900 RIDGEBURY RD, P. 0. BOX 368
`RIDGEFIELD, CT 06877-0368
`
`(21) Appl. No.:
`
`11/695,950
`
`(22) Filed:
`
`Apr. 3, 2007
`
`(EP) .............................. EP 06112 443
`Apr. 10, 2006
`Publication Classification
`
`(51)
`
`Int. Cl.
`C07K 14147
`(2006.01)
`0~ vs.a ....................................................... ~oo~
`ABSTRACT
`(57)
`
`A method for refolding a protein by mixing a protein
`solution with a refolding buffer at mixing conditions that
`approximate ideal mixing. The method can be carried out
`batch wise, in a fed-batch mode or continuously with on-line
`solubilization of inclusion bodies.
`
`1 of 13
`
`Fresenius Kabi
`Exhibit 1022
`
`

`

`Patent Application Publication Oct. 11, 2007 Sheet 1 of 6
`
`US 2007 /0238860 Al
`
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`2 of 13
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`Fresenius Kabi
`Exhibit 1022
`
`

`

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`additives (optional)
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`3 of 13
`
`Fresenius Kabi
`Exhibit 1022
`
`

`

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`4 of 13
`
`Fresenius Kabi
`Exhibit 1022
`
`

`

`Patent Application Publication Oct. 11, 2007 Sheet 4 of 6
`
`US 2007 /0238860 Al
`
`Figure 4
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`
`5 of 13
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`Fresenius Kabi
`Exhibit 1022
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`Patent Application Publication Oct. 11, 2007 Sheet 5 of 6
`
`US 2007 /0238860 Al
`
`Figure 5
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`
`6 of 13
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`Patent Application Publication Oct. 11, 2007 Sheet 6 of 6
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`US 2007 /0238860 Al
`
`Figure 6
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`
`7 of 13
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`Fresenius Kabi
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`US 2007 /0238860 Al
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`Oct. 11, 2007
`
`1
`
`METHOD FOR REFOLDING A PROTEIN
`
`[0001] This application claims priority benefit to EP 06
`112 443, dated Apr. 10, 2006, the entirety of which is
`incorporated herein.
`[0002] The invention relates to the field of recombinant
`protein production
`[0003] Proteins for industrial applications, e.g. for use as
`biopharmaceuticals or fine chemicals, are either obtained by
`extraction and purification from a natural source, such as a
`plant or animal tissue or microorganisms, or by means of
`recombinant DNA technology.
`[0004] To produce a recombinant protein, the cDNA
`encoding the protein of interest is inserted into an expression
`vector and the recombinant vector is transformed into host
`cells, which are grown to express the protein. The host cells
`may be selected from microorganisms such as bacteria, yeast
`or fungi, or from animal or plant cells.
`[0005] Expression of a recombinant protein is a complex
`event. To obtain the correct conformation, the protein is
`associated with so-called "folding helper proteins" and
`enzymes. The folding helper proteins, also termed "chaper(cid:173)
`ones" or "minichaperones", interact in a complex way so
`that the protein regains its native conformation after passing
`through various intermediate states. Some of the intermedi(cid:173)
`ate states may be quite stable. Enzymes involved in protein
`maturation either catalyze the rapid formation of disulfide
`bridges (1; 2), the isomerization of prolyl-peptide linkages
`(3-6) or more complex modifications, such as the truncation
`of the protein, side chain modifications or modifications of
`the N- and C-terminus. When a protein is efficiently over(cid:173)
`expressed, the production of the nascent peptide chain
`occurs faster than the folding of the protein. For some
`proteins, an intermediate state may also form aggregates (in
`the following, the term "intermediate" forms also encom(cid:173)
`passes aggregate forms). Overall, aggregate formation
`occurs much faster than the complete folding of a protein (7;
`8).
`In expression systems, in which such conditions
`[0006]
`are present, the protein is deposited in the cells in a paracrys(cid:173)
`talline form, so-called "inclusion bodies", also termed
`"refractile bodies".
`[0007] Since the protein, when present in the form of
`insoluble inclusion bodies, is shielded from enzymatic
`attack like proteolysis, it cannot interfere with the physiol(cid:173)
`ogy of the cells. Therefore, recombinant DNA technology
`has taken advantage of this aberrant way of protein secre(cid:173)
`tion, e.g. for the production of the proteins that are toxic for
`the cells (9).
`[0008] Various steps have to be taken to obtain a protein
`from host cells, in which it is accumulated in a denatured
`form, i.e. a conformational state without biological activity,
`in its correctly refolded form. For example, bacterial cells
`carrying inclusion bodies are disintegrated, the inclusion
`bodies harvested by centrifugation and then dissolved in a
`buffer containing a chaotropic agent. The denatured protein
`is then transferred into an environment that favors the
`recovery of its native conformation. Before adopting its
`native state, the protein undergoes a transition through
`various semi-stable intermediates. Since intermediates in the
`early stages of the folding pathway have highly exposed
`hydrophobic domains, which are prone to associate, they
`tend to form aggregates. Obviously, intramolecular interac-
`
`tions are concentration-independent, whereas intermolecular
`interactions are concentration-dependent. The higher the
`protein concentration, the higher the risk of intermolecular
`misfolding, and vice versa. In principle, refolding, also
`termed "renaturation", may be considered as a race against
`aggregate formation, which usually follows second or higher
`order reaction kinetics, while refolding of the protein fol(cid:173)
`lows first order reaction kinetics (10).
`[0009] A protein can be refolded from its denatured con(cid:173)
`formation to the correctly folded conformation by transfer(cid:173)
`ring it into an environment that favors the change to the
`native conformation. During this rearrangement, the protein
`passes through several intermediate conformational states,
`which are prone to form aggregates. Depending on the
`individual protein and on the environmental conditions, the
`aggregates may precipitate. Independent of whether the
`aggregates remain soluble or whether they precipitate, this
`process leads to dramatic losses in the yield of correctly
`folded protein.
`[0010] During a folding reaction, several characteristic
`conformations are formed. Although the transition from one
`conformation to another is smooth and a characterization of
`the distinct conformations is not available yet, similar states
`have been reported for different proteins. Immediately after
`initiation of the folding reaction, the unfolded protein col(cid:173)
`lapses and a partly structured intermediate state is formed.
`This change in conformation is called burst phase and
`appears in the sub millisecond time scale. Rapid changes in
`spectroscopic properties, such as fluorescence and far UV(cid:173)
`CD are due to the molecular collapse of the protein. For
`lysozyme, molecular compaction and formation of globular
`shape was detected with small angle X-ray scattering and
`tryptophan fluorescence (11). Other examples of proteins
`where a burst phase was detected are ovalbumin (12), barstar
`(13), cytochrom C (14), dihydrofolat reductase (15) and
`a-lactalbumin (16). After the burst phase, a more compact
`structure is formed, the 'molten globule' intermediate. The
`molten globule is defined as state with native-like secondary
`structure but fluctuating tertiary structure (17). It was pro(cid:173)
`posed as a common intermediate in folding pathways and a
`number of proteins pass through a molten globule structure
`during folding. Intermediates in early folding steps cannot
`be detected, either due to very rapid or very little structural
`changes. In later folding events, reorganization of tertiary
`contacts takes place. These reactions are slow compared to
`formation of secondary and tertiary structure. They com(cid:173)
`prise generation and reshuffling of disulfide bonds, praline
`isomerization and domain pairing. Disulfide bond interme(cid:173)
`diates can be detected for example with reversed phase
`chromatography. Association of native monomers to bio(cid:173)
`logically active oligomers is the final step in the case of
`larger proteins.
`[0011] With some currently available methods, refolding
`of proteins is achieved either by diluting the protein in a
`refolding buffer in a batch or continuous mode (18-20). In
`these methods, batch wise dilution results in highly diluted
`protein solutions and therefore large process volume, which
`often is the bottleneck in industrial processes.
`In another approach the naturally occurring folding
`[0012]
`pathway is simulated by adding chaperons and/or minichap(cid:173)
`erons, and/or enzymes that catalyze certain steps in the in
`vivo folding pathway (2; 21-25). Complex refolding reactor
`systems comprising series of tanks have been designed to
`improve the refolding reaction (26).
`
`8 of 13
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`Fresenius Kabi
`Exhibit 1022
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`

`

`US 2007 /0238860 Al
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`Oct. 11, 2007
`
`2
`
`[0013]
`In another approach, the helper proteins and
`enzymes are immobilized to a solid phase. Then the protein
`solution is passed over a so-called "Packed Bed" that
`contains the immobilized helper proteins and/or helper
`enzymes, whereby the protein is folded into its native
`conformation (27-30). Since the folding helper proteins and
`enzymes must be present in a stoichiometric ratio, this
`process requires almost the same amount of helper proteins,
`which in tum have to be produced by recombinant DNA
`technology, as the finally obtained protein of interest. In
`addition, to improve folding, the helper proteins are usually
`fused to the protein of interest, which requires further
`processing of the fusion protein. For these reasons, this
`strategy is very cost intensive and not applicable on an
`industrial scale.
`[0014] WO 02/057296 discloses an on-line method for
`refolding a protein by dilution and subsequent separation.
`The solution containing unfolded protein is diluted with
`refolding buffer by mixing in a mixing chamber and the
`output of this dilution step is directly loaded onto the
`separation device, e.g. a chromatographic colunm. By
`optionally varying the length of the tubing between the
`mixing chamber and the colunm, the time for refolding the
`protein in solution-before it is bound to the colunm----can
`be adjusted. This system is limited to proteins with fast
`refolding kinetics and to proteins with low requirements as
`regards adjusting the conditions of the separation step to
`those of the antecedent refolding step.
`[0015] Dilution of the unfolded protein with the refolding
`buffer using a flow-type reactor was described by Terashima
`et al (31): Denatured lysozyme is continuously diluted in a
`small mixing unit and directed to a packed colunm with a
`flow that closely approaches a plug flow. The achieved
`refolding efficiencies in the flow type reactor are hardly
`superior to those of a batch system.
`[0016] Among the known refolding strategies, dilution is
`still the simplest methodology. In industrial scale applica(cid:173)
`tions, dilution is commonly used for refolding of recombi(cid:173)
`nant proteins, expressed as inclusion bodies. Typically,
`dilution is carried out in one step by mixing/diluting the
`solution containing solubilized protein with a diluent con(cid:173)
`taining a solubilizing agent in an amount necessary to reach
`the optimal level of dilution. When the concentration of
`solubilizing agent is below a certain threshold level, the
`protein start to regain its biologically active three-dimen(cid:173)
`sional conformation. Depending on the specific protein and
`the chosen folding conditions, refolding begins within mil(cid:173)
`liseconds to seconds. In this initial burst phase, the protein
`is highly susceptible to aggregation. To minimize aggrega(cid:173)
`tion, the protein concentration has to be kept low. After this
`initial refolding phase, the protein forms into a more com(cid:173)
`pact structure. This intermediate structure, which is some(cid:173)
`times termed 'molten globule', is defined as a state with a
`secondary structure that resembles that of the native protein
`and that is less susceptible to aggregation. Complete refold(cid:173)
`ing, including formation of disulfide bonds, praline isomer(cid:173)
`ization and domain pairing may take hours and up to several
`days.
`[0017] Usually, such dilution is carried out as a so-called
`"batch" dilution, in which the diluent is added in a defined
`volume, the "batch", to the unfolded protein solution. Batch
`dilution has many disadvantages when carried out at large
`scale. In commercial protein purification methods, depend(cid:173)
`ing on the dilution rate, the total volumes being handled at
`
`the same time can be very large, usually between several
`hundreds or thousand liters. In such processes, variations in
`refolding efficiency are caused by ill-defined operating vari(cid:173)
`ables with regard to feed rate and mixing, which result in
`non-robust processes during scale-up with (32).
`[0018] During batch refolding, all of the protein in the
`reactor is transiently present in the form of reactive inter(cid:173)
`mediates, resulting in a brief period of aggregation. There(cid:173)
`fore, optimum operation occurs at extremely low overall
`protein concentration. Additionally, refolding a protein in
`large volumes by batch dilution may cause some re-aggre(cid:173)
`gation of the protein, probably because the solution, at least
`as initially present in batch dilutions, is not homogeneous.
`This may result in a lower net yield of refolded protein. The
`non-homogeneity of the solution in batch dilutions results
`from the difficulty in timely achieving "ideal" mixing con(cid:173)
`ditions, which are required for obtaining homogeneity, in
`large volumes.
`[0019]
`Ideal mixing conditions in a refolding mixture are
`given when the composition of the mixture with respect to
`its physical-chemical properties is identical at each time
`interval for each infinite small volume element in the
`refolding tank. In theory, "ideal" mixing conditions result in
`a homogenous solution without concentration gradients of
`unfolded or partially refolded protein during dilution. Ideal
`mixing conditions are a function of a solution's "mixing
`time". Mixing time is the time needed for the molecules in
`a droplet between addition of the droplet to the solution and
`their even dispersion in the total volume of the solution.
`Variables affecting mixing time include the total volume of
`the solution, the size of the added volume, the size and
`configuration of the mixing chamber (vessel, tank), and
`other characteristics of the mixing device, e.g. whether
`stirring occurs and which type of stirrer is used, and the
`location of the inlets in the mixing chamber. The larger the
`volume of the solution and the larger the size of the reaction
`vessel, the longer is the mixing time and thus the longer it
`takes until the mixture, e.g. the solubilized protein solution
`and the diluent; will not be homogenous. As reported by
`Ram et al. (33), mixing time in process vessels used in
`biopharmaceutical manufacturing can last up to several
`minutes.
`[0020] Due to the concentration gradient present in a
`non-homogenous solution, there are variations of the pH
`value and ionic strength, which results in variations of the
`charges of the unfolded or partially folded protein causing
`the protein to refold incorrectly or interact improperly with
`nearby protein molecules. A high local concentration of
`unfolded protein in the regions of the mixing chamber where
`the unfolded protein is fed into the reactor, may lead to
`higher aggregation compared to an "ideal" mixing chamber.
`[0021]
`In so-called "fed-batch" processes, the unfolded
`protein is added to the refolding tank in a semi-continuous
`or pulse wise manner, which results in a lower actual
`concentration of folding intermediates and therefore less
`aggregation (34). Such methods have the advantage that the
`actual concentration of unfolded protein is kept low, while
`the final concentration of refolded protein can be increased.
`The composition in terms of the protein's state in the
`refolding mixture changes from the first molecule (virtual
`isolation, best chance of successful folding into native
`conformation) to the last molecule, which is added to a
`volume containing the correctly folded or misfolded proteins
`(worst chance of successful refolding). Like in batch meth-
`
`9 of 13
`
`Fresenius Kabi
`Exhibit 1022
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`

`

`US 2007 /0238860 Al
`
`Oct. 11, 2007
`
`3
`
`ods, renaturation that is conducted pulse-wise (fed-batch
`mode) can be only operated in a discontinuous way. In a
`fed-batch reactor, the amount of denaturing and reducing
`agents from the feed solution start to accumulate during
`addition of the unfolded protein until they reach a critical
`level at which the protein starts to unfold. Design equations
`for fed-batch refolding with regard to the folding and
`aggregation kinetics have been described by Dong et al. (35)
`and Kotlarsky et al. (36).
`It was an object of the invention to provide an
`[0022]
`improved method for obtained a protein in its refolded,
`biologically active form.
`[0023] The solution of the problem underlying the inven(cid:173)
`tion is based on refolding the protein under defined mixing
`conditions.
`[0024] The present invention relates to a method for
`obtaining a biologically active recombinant protein by
`reconstituting the protein from a denatured state to its active
`form, said method containing a steps of mixing a feed
`solution containing the protein in its denatured form and/or
`its biologically inactive intermediate forms with a refolding
`buffer under conditions that approximate ideal mixing,
`wherein
`i. the mixing time is ca. 1 msec to ca. 10 sec; and
`[0025]
`ii. the dilution rate F p:F B
`is ca. 1: 1 to ca.
`[0026]
`1: 100000, wherein
`[0027] F Pis the flow rate of said protein feed solution
`and
`[0028] F B is the flow rate of said refolding buffer.
`"Denatured form", in the meaning of the present
`[0029]
`invention, designates the biologically inactive, unfolded or
`predominantly misfolded form of the expressed protein of
`interest, as obtained as a product of the recombinant pro(cid:173)
`duction process, e.g. as obtained after dissolving the inclu(cid:173)
`sion bodies.
`"Intermediate forms" or "intermediates" in the
`[0030]
`meaning of the present invention, designates the forms that
`the protein passes through between its denatured form and
`its reconstituted (refolded) native and biologically active
`state. The intermediates, which are biologically inactive or
`have a lower biological activity than the native protein, may
`form aggregates.
`[0031] A "protein" in the meaning of the present invention
`is any protein, protein fragment or peptide that requires
`refolding upon recombinant expression in order to obtain
`such protein in its biologically active form.
`[0032] Preferred mixing times range from ca. 10 msec and
`ca. 5 sec, preferably from ca. 100 msec to ca. 1 sec.
`[0033] By maintaining a very high flow rate of the refold(cid:173)
`ing buffer and a low flow rate of the feed stream containing
`the unfolded protein, the method of the invention provides
`very high local dilution rates; preferred dilution rates range
`from 1:5 to 1:50000 and from 1:10 to 1:10000.
`[0034] Depending on the dimensions of the system, the
`flow rates may vary within a wide range, e.g. from µUmin
`in the case of laboratory scale to Liters/min in the case of
`industrial scale manufacturing.
`[0035] The concentration of the protein after dilution with
`refolding buffer is in the range of ca. 1 ng/ml to 10 mg/ml,
`for example ca. 100 ng/ml to ca. 5 mg/ml or ca. 1 µg/ml to
`ca. 1 mg/ml.
`[0036] The refolding buffer used for a given protein of
`interest is customized to the refolding requirements/kinetics
`of that protein. Refolding buffers are known in the art and
`
`commercially available; typical buffer components are gua(cid:173)
`dinium chloride, dithiothreitol (DTT) and optionally a redox
`system ( e.g. reduced glutathione GSH/oxidized glutathione
`GSSG), EDTA, detergents, salts, and refolding additives like
`L-arginine.
`[0037] As mentioned above, "ideal mixing" refers to con(cid:173)
`ditions that result in a homogenous solution without sub(cid:173)
`stantial concentration gradients in solution. By ideal mixing,
`infinitive short mixing times are achieved.
`[0038] Since the mixing conditions according to the
`method of the invention are close to ideal mixing, mixing of
`the protein feed stream with the refolding buffer occurs with
`similar or faster kinetics than the unfolding/aggregation
`kinetics of the protein, thereby reducing or completely
`preventing aggregation of the protein
`In the process of the invention, the actual protein
`[0039]
`concentration immediately after mixing is much lower as
`compared to conventional refolding methods.
`In its simplest embodiment, the method of the
`[0040]
`invention is a batch process that comprises, as its essential
`step, the above-defined mixing operation, in which a feed
`stream having a high concentration of unfolded protein and
`a low flow rate is combined with a refolding buffer solution
`having a high flow rate.
`[0041] This embodiment of the invention, which is sche(cid:173)
`matically shown in FIG. 1, is particularly useful for proteins
`that have very fast refolding kinetics, e.g. peptides and
`smaller protein fragments. The refolding buffer and the
`protein feed solution are independently fed from reservoirs
`to the mixing device. Having passed the mixing device, the
`highly diluted solution containing the refolded protein is
`collected in a tank. Optionally, before entering the tank,
`refolding additives may be added in the case of proteins that
`have not yet completely refolded during mixing to suppress
`or completely prevent unfolding/aggregation. Compounds
`useful as refolding additives are known in the art, examples
`are L-arginine, Tris, detergents, redox systems like GSH/
`GSSG, ionic liquids like N'-alkyl and N'-(omega-hydroxy(cid:173)
`alkyl)-N-methylimidazolium chlorides etc. The end of the
`process is reached when the reservoir of refolding buffer
`and/or protein solution is exhausted. At this point, the feed
`of unfolded protein (or the feed of buffer, respectively) is
`interrupted and the solution containing the protein in its
`refolded, biologically active form is withdrawn from the
`tank. In this embodiment of the invention, it is advantageous
`to have the mixing device equipped with means that control
`the temperature to exclude any, even minimal aggregation,
`e.g. cooling means.
`[0042] Mixing devices suitable for use in the method of
`the invention are any mixers that ensure fast mixing and
`short mixing times, e.g. tubular jet mixers or static mixers,
`e.g. commercially available mixers from Fluitec, CH, or
`Sulzer Chemtech, CH. In the simplest form of the method of
`the invention, the two streams can be combined into one
`stream by a branch connection without any additional spe(cid:173)
`cific mixing devices. Such a simple device can be used to
`achieve the desired mixing efficiency, albeit without precise
`control of mixing efficiency. In the case that the mixer is a
`high-throughput continuous flow device, accurate control of
`the flows is of particular importance. With such mixers,
`mixing times as low as a few milliseconds on the small scale
`or a few seconds on the large scale can be achieved. The
`mixing characteristics of such mixers most closely approxi(cid:173)
`mate "ideal mixing". The mixing ratio of the two streams is
`
`10 of 13
`
`Fresenius Kabi
`Exhibit 1022
`
`

`

`US 2007 /0238860 Al
`
`Oct. 11, 2007
`
`4
`
`adjusted such that a low protein concentration is maintained
`to minimize aggregation. After mixing the two streams, the
`protein starts to refold.
`[0043] The method of the invention is also referred to as
`"fast mix refolding".
`[0044] Except for proteins with fast refolding kinetics,
`which may already be completely refolded during mixing,
`the initial refolding steps take place in the mixing device and
`refolding is completed in the refolding tank or in the
`optionally present adjustment zone, e.g. in the plug flow
`reactor (PFR), as described below.
`In a further embodiment, the method of the inven(cid:173)
`[0045]
`tion comprises in addition, subsequent to the mixing step
`defined above and before the solution enters the refolding
`tank, a step in which the highly diluted mixture is transferred
`to a zone in which the protein is allowed to form more stable
`folding intermediates under precisely controlled conditions
`such that unfolding and formation of aggregates is sup(cid:173)
`pressed or completely prevented. This step is also referred to
`as "adjustment step", and the zone or the reactor in which
`adjustment occurs is referred to as "adjustment zone" or
`"adjustment reactor", respectively.
`In a preferred embodiment, the adjustment reactor
`[0046]
`is a plug flow reactor, i.e. a chemical reactor where the fluid
`passes through in a coherent manner, so that the residence
`time is the same for all elements. An ideal plug flow reactor
`has a fixed residence time:
`[0047] Any fluid that enters the reactor at time twill exit
`the reactor at time t+i:, where i: is the residence time of the
`reactor. In its simplest form, the plug flow reactor is a tube,
`optionally packed with solid material.
`[0048] The adjustment step provides the possibility of
`generating, for a defined volume and period of time, con(cid:173)
`ditions that favor stabilization of the partially refolded
`protein. This may be achieved by a short-term change of the
`pH value (increase or decrease) and/or change of the tem(cid:173)
`perature (heating or cooling) and/or addition of refolding
`additives, as defined above, in the adjustment zone. The
`adjustment step provides the advantage that optimal refold(cid:173)
`ing conditions, e.g. heating or cooling or addition of addi(cid:173)
`tives, need to applied only to a small volume as compared
`to the refolding tank, thus saving energy, reagents and costs.
`[0049] The mean residence time, i.e. the time that it takes
`for the solution to pass through the adjustment reactor, i.e.
`the tube in the case of a plug flow reactor, depends on the
`flow rate and the tube volume. The residence time should be
`long enough to allow the protein to fold into a more compact
`and stable structure, e.g. into a so-called 'molten globule'
`intermediate.
`[0050] By varying the design of the adjustment reactor,
`e.g. length and/or diameter of the tube, the residence time of
`a specific protein in the adjustment zone and thus its
`exposure to the selected adjustment conditions is adapted to
`the requirements of the protein, i.e. its specific refolding
`kinetics. In the case of fast refolding kinetics, refolding is
`usually completed already in the adjustment zone.
`[0051] After leaving the adjustment zone, the protein
`solution containing the refolded protein and/or the partially
`refolded stabilized intermediates is collected in the refolding
`tank, where, if still necessary, refolding is completed.
`In the embodiment that provides an adjustment
`[0052]
`step, the method of the invention may be conducted batch(cid:173)
`wise or preferably, by recycling the protein solution from the
`refolding tank, in the fed-batch mode.
`
`In the batch mode, the end of the process is reached
`[0053]
`when the reservoir of refolding buffer and/or protein solu(cid:173)
`tion is exhausted. At this point, the feed of unfolded protein,
`or of the refolding buffer, respectively, is interrupted and the
`protein solution is withdrawn from the tank. FIG. 2 shows
`the schematic drawing of the embodiment of the invention
`that is a fed-batch process comprising an adjustment step in
`combination with recycling of the protein solution. In this
`embodiment, the protein solution circulates at high flow
`rates from the refolding tank back to the feed inlet, where
`unfolded protein is freshly introduced into the system. In
`such embodiment, the recycled protein solution forms the
`refolding buffer solution.
`In a preferred embodiment, the method of the
`[0054]
`invention is performed on-line and, even more preferred, in
`a continuous mode. "On-line" means that refolding is con(cid:173)
`nected to one or more other steps, e.g. antecedent steps, of
`the overall process, e.g. solubilization of inclusion bodies.
`[0055] By running refolding continuously and on-line
`with solubilization of inclusion bodies, as depicted in FIG.
`3, time consumption and costs can be reduced and the yield
`of refolded protein increased as compared to known meth(cid:173)
`ods. The method of the invention ensures, in particular in its
`continuous on-line embodiment, fast and efficient process(cid:173)
`ing of inclusion body proteins, thereby reducing inadvertent
`variations, such as variations in refolding efficiency or
`product homogeneity. On-line solubilization of suspended
`inclusion bodies is preferred to their batch-wise solubiliza(cid:173)
`tion in a stirred tank, where the contact time between the
`molecules and the solubilizing agent has to be minimized or
`precisely controlled to avoid irreversible modification of the
`proteins. This is often the case when solubilization of the
`inclusion bodies is carried out at extreme pH values. Such
`irreversible modification of amino acid side chains could
`lead to reduced activity of the molecule.
`[0056] Exemplified by the embodiments in which the
`protein solution is recycled (FIGS. 2 and 3), very high local
`dilution rates (1: 1000, 1: 10000 or more) can be easily
`achieved depending on the ratio of the flow rate of the
`solution of unfolded protein F B (designated F3 in the Figure)
`and the flow rate of the circulating refolding buffer F B
`( designated F 5 in the figure). The protein concentration C4
`after
`dilution can be calculated by a simple mass balance as
`
`[0057] C3 is the concentration of the unfolded protein in
`the feed stream and C4 is the concentration of unfolded
`protein immediately after mixing. The flow rate of the
`circulating stream necessary to achieve the desired concen(cid:173)
`tration of unfolded protein after dilution (C4 ) can be simply
`calculated by neglecting C5 (refolded protein present in the
`reaction system, which is less susceptible to aggregation) as
`
`F 5~((C3xF3 )/C4 )-F3
`[0058] The tota