CZ306818B6 - A method of suppressing freeze-induced protein denaturation when freezing and/or lyophilizing biologically active substances - Google Patents

Abstract

The invention relates to to the method of suppressing freeze-induced protein denaturation when freezing and/or lyophilizing biologically active substances in the form of a solution. The essence of the invention lies in the fact that the pH change is determined before and after the freezing test, then a cryoprotectant from the group of salts with a different affinity to the resulting ice is added to the solution before the next freezing, which causes the opposite changes in the local proton concentrations in the solution being frozen during the freezing compared to the solution frozen without the addition of this salt.

Description

Technical field

The invention relates to a method of suppressing the frost denaturation of proteins upon freezing and / or lyophilization of biologically active substances in solution form with the addition of at least one neutral salt to stabilize the pH of the buffer in solution.

BACKGROUND OF THE INVENTION

One of the most effective modern ways of storing biologically active substances is to freeze or freeze-dry (vacuum freeze), which involves freezing the stored substance and subsequently subliming the water at low pressure and temperature. This method is suitable for long-term storage of proteins in research laboratories as well as in the biotechnology and pharmaceutical industries. During freezing, the active substances are partially or completely degraded. For this reason, the entire freezing process is optimized, which is time-consuming and costly. In practice, optimization is often based on empirical experience rather than scientific knowledge that is incomplete or contradictory. This experience cannot be well used in determining industrial manufacturing processes. Therefore, there is an effort to analyze and minimize the effects of freezing on the degradation of biologically active substances.

A large group of substances stored by lyophilization are solutions or substances with initially high water content. The solution before freezing or lyophilization contains many components, in addition to the active substance (e.g. protein) also various excipients such as buffer, cryoprotectant or lyoprotectant, which lower the temperature of homogeneous nucleation, increase the recrystallization temperature and reduce the amount of free water in the system. surface tension, etc. When freezing such a solution, processes may occur that result in protein denaturation. One of these processes is the gradual crystallization of the individual buffer components, which can significantly change the pH of the frozen solution. An example is the sodium phosphate buffer (Na-P), where freezing leads to significant acidification of the solution due to gradual crystallization. The literature reports that upon freezing the sodium phosphate buffer at a buffer concentration of 50 mM or 100 mM and an initial pH of 7.4, the pH drops by more than 3 units to a final pH of 4.2. When the potassium phosphate buffer (K-P) is frozen, there is only a slight change in acidity, because the difference in solubility of monohydrogen phosphate and dihydrogen phosphate is not so pronounced.

Neutral salts often affect the stability of proteins in solution and also during freezing and thawing. Most of the authors suggest that the stabilizing effect of salt is related to the position of ions in the Hofmeister series (Carpenter, JF and JH Crowe (1988). THE MECHANISM OF CRYOPROTECT1ON OF PROTEINS BY SOLUTES. Cryobiology 25 (3): 244-255 Anchordoquy, T.

J., K. Izutsu, et al. (2001). Maintenance of quatemary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying. Archives of Biochemistry and Biophysics 390 (1): 35-41., Chen, Y.H. and Z. Cui (2006). Effect of salts on the freezing denaturation of lactate dehydrogenase. Food and Bioproducts Processing 84 (C1): 44-50). If the salt is preferentially excluded from the surface of the protein (the so-called salting salt), it tends to stabilize the protein. More recently, attempts have been made to stabilize LDH in freezing with salting out salts. However, the difference in restored LDH activity in the presence of salts and LDH in buffer alone was minimal or even none, or high salt concentrations (> 0.5 M) were needed to stabilize LDH, with lower salt concentrations leading to destabilization of LDH (Carpenter, JF and JH Crowe) (1988). THE MECHANISM OF CRYOPROTECTION OF PROTEINS BY SOLUTES Cryobiology 25 (3): 244-255., Chen, YH and Z. Cui (2006), Effect of salts on the freezing denaturation of lactate dehydrogenase, Food and Bioproducts Processing 84 (C1): 44-50.). These result

They question the stabilizing effect of salts in freezing by means of the priority elimination mechanism, which thus cannot be used to rationalize and optimize the freezing process.

The addition of salts to the solution prior to freezing may also affect the pH of the frozen solution. The added salt may affect the solubility of the buffer components and the amount of residual water in the frozen solution. The pH change in freezing may also be associated with neutralization of the freezing potential, which is caused by the uneven distribution of positive and negative ions between the ice and the non-frozen solution, and the charge imbalance is then neutralized by diffusion of H ⁺ or OH from solution to ice. The results of the above experiments did not result in a method of suppressing the effects of frost degradation that would allow the freezing and thawing process to be carried out in industrial practice with minimal losses due to the denaturation of the protein portion of the frozen substance.

The pharmaceutical freezing agents used so far in the pharmaceutical practice are dimethylsufoxide (DMSO) or glycerol, but these are toxic and have undesirable side effects.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide an industrially applicable method of suppressing the frost denaturation of proteins which removes the above drawbacks and is easily applicable to a wide variety of substances.

SUMMARY OF THE INVENTION

This object is solved by providing a method of suppressing the frost denaturation of proteins upon freezing and / or freeze-drying of biologically active substances in solution. It is an object of the present invention to first perform a test freezing of the solution, determining the pH change of the solution before and after the test freezing. Subsequently, cryoprotectant from the group of salts containing anions and cations with different affinities to the ice formed upon freezing of the solution is added to the solution before further freezing. A cryoprotectant is a salt whose anions and cations have different affinities to the ice formed upon freezing the solution. For example, if the solution is acidified during freezing due to the partial crystallization of the buffer present, such a salt is added to the solution that the anions are more incorporated into the ice than the cations. There will be an excess of negative ions in the ice and an excess of positive ions in the remaining solution. This charge imbalance will then be neutralized by the flow of protons from the remaining non-frozen solution to the ice and the remaining solution will become more basic. Thus, the addition of an inert salt prevents the acidity of the frozen solution from being changed and thus the denaturation of the active substance.

If the pH of the solution drops as a cryoprotectant in the test freeze, a salt whose anions have a higher affinity for ice than cations is added. The salt is at least one of tetramethylammonium chloride (TMAC), sodium chloride (NaCl), sodium iodide (Nal), sodium fluoride (NaF), sodium bromide (NaBr), potassium chloride (KCl), calcium chloride (CaCl ₂ ) , magnesium chloride (MgCl ₂ ), barium chloride (BaCl ₂ ). Otherwise, when the pH of the remaining non-frozen solution increases due to the change in proton concentration in the test freeze solution, a salt whose cations have a higher affinity for ice than anions is added to the solution. Such a salt is preferably at least one of sodium sulfate (Na ₂ SO ₄ ), sodium nitrate (NaNO ₃ ), ammonium chloride (NH ₄ Cl) and magnesium sulfate (MgSO ₄ ). As described above, the use of neutral salts can replace most commonly used substances such as DMSO or glycerol as cryoprotectants, which often have undesirable side effects.

The change in pH of the test freezing solution is determined by adding an acid-base indicator to the solution, e.g., cresol red. The solution is then frozen and the change in protonation is determined from the change in the acid-base indicator spectrum. Other methods, such as a low temperature electrochemical electrode, can be used to determine the change in protonation. Cresol red can occur in three different forms depending on the surrounding pH. Each of these forms absorbs in a different area of visible light so that the individual forms can be spectroscopically differentiated from the area ratio

The acidity of the solution (both liquid and frozen) can be approximately determined from the individual peaks. The positions of the absorption maxima of the individual forms and the corresponding pK _and solution values are given below.

Form A Π Form B [| form C (Z _max = 518 nm) pX l 10.10 (λ _max = 434 nm) pK _and ² = 8.15 (λ, λ = 573 nm)

In the frozen state, the absorption maxima of all three forms show a slight bathochromic shift. The pH of the frozen solution can also be determined by spectroscopic evaluation.

The advantages of the method of suppressing the frost denaturation of the proteins according to the invention reside in its easy applicability on a wide variety of substances and in the removal of toxic cryoprotectants from solutions containing the substance to be frozen.

Clarification of drawings

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be explained in more detail by means of the drawings in which:

Fig. 1 UV-Vis spectra of cresol red (2.5 ^χ ΙΟ ' ⁵ M) in liquid (----- / solid curve) and frozen solutions of 50 mM Na-P (--- / dashed curve) and 50 mM KP ( ----- / dashed line). The initial pH of the solutions was 7.50.

Fig. 2 UV-Vis spectra of cresol red (2.5 χ 10 ^-5 M) in liquid (solid curve) and frozen solutions of 50 mM Na-P (dashed line) and 50 mM Na-P with 0.1 M TMAC (dashed line). The initial pH of the solutions was 7.50.

FIG. 3 Restored DbjA activity in 50 mM Na-P or 50 mM K-P without addition of TMAC (?) and with addition of TMAC at a final concentration of 0.1 M (?). The samples were frozen and thawed 3 times, the freezing temperature was 77 K. Error bars represent standard deviations.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the specific embodiments of the invention described below are presented by way of illustration and not by way of limitation of the examples to the examples. Those skilled in the art will find, or will be able to detect, using routine experimentation, more or less equivalents to specific embodiments of the invention specifically described herein.

The inhibition of the frost denaturation of proteins in the freezing of biologically active substances was verified using a model enzyme. DbjA haloalkane dehalogenase isolated from Bradyrhizobium japonicum USDA110 of the haloalkane dehalogenase class (EC 3.8.1.5), which catalyzes the hydrolytic cleavage of the carbon-halogen bond in halogenated aliphatic hydrocarbons, was used as a model enzyme. The pH optimum of DbjA is about 9.7, but more than 90% of the activity is maintained in the pH range of 7.7 to 10.4. DbjA is stable over a wide pH range, retaining its secondary and tertiary structure in the pH range of 6.2 to 10.1. The specific activity of DbjA was determined as the rate of catalyzed conversion of 1,2-dibromoethane to 2-bromoethanol in glycine buffer at pH 8.6 at 37 ° C. Iwasaki method (Iwasaki, I., S. Utsumi, et al. (1952) was used to monitor the decrease in reactant concentration / increase in product concentration. Bulletin of the Chemical Society of Japan 25 (3): 226-226) or by gas chromatography with a flame ionization detector (Chaloupkova, R., Z. Prokop, et al. (2011). Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature, Febs Joumal 278 (15): 2728-2738.

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The enzyme was frozen by immersing the polypropylene tube in 77 K liquid nitrogen or in a 233 K ethanol bath. The freezing time did not affect the loss of enzyme activity. Then, the sample was thawed by immersion in a 22 ° C water bath for 15 minutes. This freeze-thaw cycle was repeated several times to make the effect of freezing on the loss of activity more pronounced. Specific activity was measured immediately after thawing and also after 24 hours and there was no significant difference - loss of activity was irreversible and was a permanent denaturation of the enzyme due to freezing. Various batches of DbjA were used during the experiments and sufficient measurements were made to statistically process the results.

First, the DbjA enzyme solution (c = 0.1 to 0.2 mg / ml) was repeatedly frozen (1x - 5x) in buffer alone without the addition of other substances. 50 mM Na-P and 50 mM KP buffer were used, both pH 7.5. Na-P buffer tends to be strongly acidified during freezing due to the crystallization of Na ₂ HPO ₄ from solution. In contrast, the pH changes only slightly when freezing the KP buffer solution. To verify this trend, the acidity of frozen buffer solutions was determined spectrophotometrically (Figure 1). The solid curve in the graph is a liquid solution of cresol red in buffer (Na-P or KP) at pH 7.5. Two forms of cresol red are present in the solution - monoprotonated form B and deprotonated form C. After freezing of the KP solution, form C almost disappeared - the solution became slightly acidified (dashed line). Form C is no longer present in the spectrum of the frozen Na-P solution, except for Form B, there is also a significant peak for the diprotonated form A - the solution is significantly acidified (dashed curve). The loss of enzyme activity after freezing and thawing also correlates with the acidity of the buffer solutions. When a solution of DbjA in 50 mM Na-P or KP buffers was subjected to 3 freeze-thaw cycles at 77 K and thawing, its mean thawing activity (referred to as recovered activity) was (65 ± 11)% for the enzyme in KP buffer and only (19). ± 11)% for enzyme in Na-P buffer.

In the next step, the acidity change of the solution was suppressed by the addition of neutral tetramethylammonium chloride (TMAC) salt. Addition of TMAC to the solution results in a marked change in the pH of the frozen solution towards more basic values than freezing without its addition, probably due to neutralization of the Workman-Reynolds freezing potential. The tetramethylammonium cation is bulky, so its incorporation into the ice structure is minimal compared to chloride anions. Thus, there is an excess of negative charge in the ice, which is then neutralized by the proton flow from the residual non-frozen solution to the ice. This makes the residual solution more basic. Thus, the addition of TMAC to the phosphate buffer results in only a slight change in the freezing acidity of the non-frozen solution.

Figure 2 shows a comparison of the acidity of frozen solutions of 50 mM Na-P buffer (dashed line) and 50 mM Na-P buffer with 0.1 M TMAC (dashed line). It can be seen from the figure that significant acidification of the buffer solution during freezing was suppressed by the addition of 0.1 M TMAC (Form C is again present, the peak for Form A disappeared).

The recovered enzyme activity after freezing and thawing in buffer in the presence of TMAC was determined under the same conditions as in the previous measurements, except that TMAC at the desired concentration was added to the enzyme solution in the buffer prior to freezing. The most suitable concentration of TMAC was 0.1 M experimentally. The average recovered DbjA activity at 3 K freezing cycles at 77 K and thawing in 50 mM KP buffer with 0.1 M TMAC was (106 ± 14)% and in 50 mM Na-P buffer with 0.1 M TMAC was (105 ± 23)%. A comparison of the recovered DbjA activity values in the buffer without TMAC addition and with 0.1 M TMAC addition is shown in Table 1 and Figure 3. The Welch two-sample / -test was applied to the results, confirming that between the average recovered DbjA activity values without and with 0.1 M TMAC, there is a statistically significant difference.

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Table 1: Average DbjA activities after 3 freeze-thaw cycles. The freezing temperature was 77 K.

Recovered DbjA activity after 3 freeze / thaw cycles /% Buffer type without TMAC with 0.1 MTMAC 50 mM K-P 65 ± 11 106 ± 14 50 mM Na-P 19 ± 11 105 ± 23

At higher concentrations of TMAC (0.5 M and 1 M), a decrease in the specific activity of the non-frozen solution was observed, possibly due to inhibition of the dehalogenation reaction by the chloride. Despite this decrease, the activity of the enzyme solution in 50 mM Na-P buffer with 0.5 M and 1 M TMAC was recovered after 5 cycles of freezing (77 K) and thawing of approximately 90%.

Industrial applicability

The method of suppressing the frost denaturation of proteins in the freezing and / or lyophilization of biologically active substances according to the invention can be used in the field of molecular biotechnology, pharmacy, food industry, where it is necessary to store proteins in frozen or lyophilized state.

Claims (4)

A method for suppressing the frost denaturation of proteins in the freezing and / or lyophilization of biologically active substances in the form of a solution, characterized in that: - a test freeze of the solution is first carried out, determining the change in pH of the solution before and after the test freeze, - subsequently, cryoprotectant from the group of salts containing anions and cations with different affinity to the resulting ice when freezing the solution is added to the solution before further freezing, - when the pH of the solution drops during test freezing, a salt whose anions have a higher affinity for ice than cations is added to the solution as a cryoprotectant, where the salt is at least one of tetramethylammonium chloride (TMAC), sodium chloride (NaCl), sodium iodide (NaI), sodium fluoride (NaF), sodium bromide (NaBr), potassium chloride (KCl), calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ) and barium chloride (BaCl ₂ ), - while increasing the pH of the solution during the test freeze, a salt whose cations have a higher affinity for ice than the anions where the salt is at least one of sodium sulphate (Na ₂ SO ₄ ), sodium nitrate, is added to the solution as a cryoprotectant (NaNO ₃ ), ammonium chloride (NH ₄ Cl), magnesium sulfate (MgSO ₄ ). 2. The method of claim 1, wherein the change in pH of the test freeze is determined by adding an acid-base indicator to the solution. Method according to claim 2, characterized in that the acid-base indicator is cresol red. Method according to claim 1, characterized in that the pH change of the solution in the test freeze is determined by spectroscopic evaluation of the solution. 2 drawings