http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.972411

RESEARCH ARTICLE

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Development of gel-forming lyophilized formulation with recombinant human thrombin Andrej Mura´nyi1,2, Peter Bartosˇ1, Eduard Tichy´1, Jana Lazova´1, Jana Psˇenkova´1, and Maria´n Zˇabka2 1

hameln rds a.s., Horna´, Modra, Slovak Republic and 2Department of Galenic Pharmacy, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic Abstract

Keywords

The objective of this work was development and evaluation of gel-forming lyophilized formulation with recombinant human thrombin for topical administration. The influence of pH, ionic strength and buffer type on protein stability was evaluated as part of the pre-formulation screening studies. Results indicated an optimal pH from 6.0 to 7.0 and increased stability with increasing content of sodium chloride. The tested buffer types had no significant effect on thrombin stability. For further development, thermosensitive PluronicÕ F-127 was employed as a bulking and gelling agent. Physical and mechanical characterization and viscosity measurement confirmed the gel-forming properties of the formulation at the application temperature of 32  C. Several techniques (addition of well-soluble polyols, different freezing protocols and reconstitution under vacuum) were tested to decrease the reconstitution time. The obtained results revealed that a vacuum in the vial headspace is crucial for acceptable reconstitution. The freeze drying process has no negative impact on recombinant thrombin stability, and this was confirmed by reverse-phase-HPLC, activity assay and optical density measurements.

Gel forming, Pluronic F-127, recombinant, reconstitution time, thrombin

Introduction Thrombin is a serine protease that catalyzes the conversion of fibrinogen to fibrin, induces cross-linking of the fibrin clot through factor XIII activation and activates platelets1. Therapeutically, it is commonly used as a haemostatic agent to control surgical bleeding. Until recently, most commercially available thrombin products were isolated from bovine or human plasma, but sourcing thrombin from plasma carries the potential risk of transmitting plasma-born pathogens such as HIV, hepatitis B and C, Creutzfeldt–Jakob disease or bovine spongiform encephalopathy2. The use of bovine thrombin can also lead to immunological reactions and related post-operative complications3. Advances in molecular biology in the past decade have led to investigation and approval of recombinant human thrombin (rhThrombin). Although rhThrombin has similar efficiency and safety profile to thrombin from bovine or human plasma, it has significantly lower risk of immunologic impact4 without potential risk of viral or prion transmission. rhThrombin is a disulfidelinked dimer composed of an A-chain (36 amino acids) and B-chain (259 amino acids). Under environmental conditions, it is subjected to specific autocatalytic cleavage5. Lyophilization is a well-established approach to stabilize therapeutic proteins and extend their shelf-life. Commonly Address for correspondence: Andrej Mura´nyi, hameln rds a.s., Horna´ 36, 900 01, Modra, Slovakia. Tel: +421/33/6904 192. E-mail: andrejmuranyi @gmail.com

History Received 16 January 2014 Revised 8 September 2014 Accepted 29 September 2014 Published online 27 October 2014

existing freeze-dried products are reconstituted before administration to form a clear solution with low viscosity; but this evaporates rapidly and flows out from application site and must therefore be frequently applied. The semisolid form with higher viscosity should be a reasonable improvement. Thrombin in water-based solution is unstable, so commercially available products are formulated as frozen solutions or lyophilizates6–8. Poor thrombin stability in the aqueous environment of hydrogel formulation is therefore highly assumed, and sterile filtration of such a viscous dispersion is impossible. The aim of this work is to develop a sterile gel-forming lyophilized product with rhThrombin. Poloxamer (PluronicÕ F-127) has been used as a bulking gel-forming agent. Pluronic F-127 is a commercially available polyoxyethylene–polyoxypropylene triblock copolymer that forms a viscous solution with Newtonian behavior at low temperature, but a gel at elevated temperature9. Furthermore, it is considered to have positive effects on skin burns and wound healing10. Its benefits are well documented in miscellaneous applications as a drug delivery vehicle for biomolecules and small molecules11–15. In our study, PluronicÕ F 127 (PF-127) was employed as the in situ gel-forming excipient. The presented formulation is suitable for preparation as an ordinary sterile lyo-product with sterile filtration and pump filling procedure. The liquid formulation state after reconstitution enables simple manipulation and measuring and direct application on bleeding areas or surgical gauzes and sponges. Its viscosity and mechanical structure are rapidly changed almost immediately after contact with the body surface,

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Table 1. Effect of pH and buffer type on stability of recombinant human thrombin.

Formulation

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Effect of pH 25 mM sodium phosphate buffer, 400 mM NaCl, pH 5.0 25 mM sodium phosphate buffer, 400 mM NaCl, pH 5.5 25 mM sodium phosphate buffer, 400 mM NaCl, pH 6.0 25 mM sodium phosphate buffer, 400 mM NaCl, pH 6.5 25 mM sodium phosphate buffer, 400 mM NaCl, pH 7.0 25 mM sodium phosphate buffer, 400 mM NaCl, pH 8.0 Effect of buffer type 25 mM citrate buffer, 400 mM NaCl, pH 6.2 25 mM potassium phosphate buffer, 400 mM NaCl, pH 6.2 25 mM histidine buffer, 400 mM NaCl, pH 6.2 25 mM sodium succinate buffer, 400 mM NaCl, pH 6.2

Residual content of rhThrombine (HPLC) (%)

Residual activity (%)

Optical density (mAU)

0.2 ± 0.1 45.2 ± 4.6 60.6 ± 3.7 57.0 ± 3.2 41.4 ± 2.1 21.2 ± 2.8

0.3 ± 0.2 40.5 ± 6.2 53.6 ± 2.0 50.3 ± 2.2 36.1 ± 2.3 17.1 ± 1.4

307 ± 13 38 ± 6 50 ± 20 14 ± 2 12 ± 3 49 ± 3

70.0 ± 0.2 67.4 ± 4.1 70.5 ± 1.4 68.3 ± 2.4

69.8 ± 3.3 64.7 ± 1.1 62.0 ± 1.9 69.3 ± 1.7

22 ± 2 20 ± 7 30 ± 2 23 ± 1

Table 2. Effect of ionic strength on stability of recombinant human thrombin.

Formulation 10 mM succinate buffer, pH 6.2, 50 mM NaCl, 5 mM CaCl2 10 mM succinate buffer, pH 6.2, 200 mM NaCl, 5 mM CaCl2 10 mM succinate buffer, pH 6.2, 500 mM NaCl, 5 mM CaCl2 100 mM succinate buffer, pH 6.2, 50 mM NaCl, 5 mM CaCl2 100 mM succinate buffer, pH 6.2, 200 mM NaCl, 5 mM CaCl2 100 mM succinate buffer, pH 6.2, 500 mM NaCl, 5 mM CaCl2

thus creating a film on the wound surface to enhance haemostatic effects. Moreover, after rapid manual warming, the semi-solid form should be spread over the bleeding site directly from a suitable container or by gloved surgical hands, so this procedure can be applied in areas inaccessible to commonly used application devices or surfaces where solution flow out is especially undesired.

Residual content of rhThrombine (HPLC) (%)

Residual activity (%)

Optical density (mAU)

30.2 ± 2.4 50.3 ± 2.1 57.6 ± 1.6 48.3 ± 2.7 51.6 ± 1.6 61.3 ± 1.4

23.4 ± 3.6 37.2 ± 2.5 48.1 ± 2.1 39.7 ± 1.7 44.9 ± 2.3 52.9 ± 3.2

160 ± 12 63 ± 6 43 ± 3 58 ± 4 36 ± 2 29 ± 2

buffer type and ionic strength on rhThrombin stability was evaluated. To accelerate degradation, the high temperature stress conditions of 37  C for two weeks and freeze/thaw stress conditions of seven cycles; 70  C/one hour following 30  C/ 15 min were applied. The remaining drug content, specific activity and optical density were then measured analytically as detailed below. The results and tested formulations are listed in Tables 1 and 2.

Materials and methods Materials The following materials were used: rh-Thrombin (Scil Proteins Gmbh, Halle, Germany), PluronicÕ F 127 (Anatrace, Maumee, OH), sodium chloride (Ph.Eur., Merck Chemicals, Bratislava, Slovak Republic), succinic acid (p.a, Mikrochem a.s., Pezinok, Slovak Republic), sodium hydroxide (Ph.Eur., Merck Chemicals), D-sorbitol (Ph.Eur., Parteck 400 SL, Merck Chemicals), Dmannitol (Ph.Eur., Merck Chemicals), water for injection (Ph.Eur., Imuna Pharm a.s., Sˇarisˇske´ Michalˇany, Slovak Republic), formic acid (p.a., Merck Chemicals), deionized water (Ph.Eur., Hameln rds a.s., Modra, Slovak Republic), acetonitrile (p.a., Merck Chemicals), thrombin from human plasma (Merck Chemicals), phosphoric acid (p.a. Sigma Aldrich, Bratislava, Slovak Republic), chromogenic substrate S-2238 (Chromogenix, Instrumentation Laboratory, Inc., Bedford, MA), potassium phosphate dibasic, potassium phosphate monobasic (Ph.Eur., Mikrochem a.s.), calcium chloride (Ph.Eur., Mikrochem a.s.), citric acid monohydrate, trisodium citrate dihydrate (Ph.Eur., Merck Chemicals), L-histidine (Ph.Eur., Merck Chemicals), sodium phosphate dibasic and sodium phosphate monobasic (Ph.Eur., Merck Chemicals). Preformulation screening studies Preformulation stability screening studies were performed to find suitable formulation for further development. The effect of pH,

Sample preparation Samples for preformulation screening were prepared by buffer exchange from a rhThrombin stock solution on HiTrapÕ desalting columns (GE Healthcare Life Sciences, Little Chalfont, UK). The protein concentration was determined by UV spectrophotometry at 280 nm with extinction coefficient 66 390 L mol1 cm1. The protein concentration was diluted to 0.3 mg/mL with suitable buffer. Samples were then filtrated through 0.2 mm low protein-binding filter (polyethersulfone (PES) membrane, Pall Port, Washington, NY) and aseptically transferred to sterile cryotubes. Each sample was prepared twice. Gel-forming lyophilized formulation Sample preparation Pluronic F-127 solutions (Table 3) were prepared by the ‘‘cold method’’ described by Schmolka10. Succinate buffer was prepared from succinic acid and sodium chloride and adjusted to pH 6.2 with 1 M sodium hydroxide. The well-soluble polyols, sorbitol and mannitol, were employed in some formulations as well. rhThrombin was added to a final concentration of 0.03% w/w (0.3 mg/g). The entire mixture was then hydrated and dispersed overnight at 2–8  C. The pH was checked after equilibration at 20  C, solutions were filtrated through 0.2 mm low protein-binding filter (PES membrane, Pall), and 2.17 g volumes were

Development of gel-forming thrombin formulation

DOI: 10.3109/03639045.2014.972411

3

Table 3. Tested formulations during development. Sample

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1 2 3 4 5 6 7 8 9 10

Formulation 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 25 mM succinate buffer, pH 0.03% w/w rh-Thrombin

6.2, 6.2, 6.2, 6.2, 6.2, 6.2, 6.2, 6.2, 6.2, 6.2,

150 mM 150 mM 150 mM 150 mM 150 mM 150 mM 150 mM 150 mM 150 mM 150 mM

NaCl, NaCl, NaCl, NaCl, NaCl, NaCl, NaCl, NaCl, NaCl, NaCl,

14% 15% 16% 17% 20% 16% 16% 16% 16% 16%

w/w w/w w/w w/w w/w w/w w/w w/w w/w w/w

PluronicÕ PluronicÕ PluronicÕ PluronicÕ PluronicÕ PluronicÕ PluronicÕ PluronicÕ PluronicÕ PluronicÕ

F F F F F F F F F F

127 127 127 127 127 127 127 127 127 127

0.5% 1.0% 0.5% 1.0% 0.5%

w/w w/w w/w w/w w/w

D-sorbitol D-sorbitol D-mannitol D-mannitol D-sorbitol,

Table 4. Freeze-drying protocols with different freezing procedures. Temperature ( C)

Time (h)

Pressure (mbar)

Moderate freezing Pre-cooled shelf Annealing

Process

Phase Freezing Freezing Freezing Freezing Freezing Freezing Freezing Primary drying Primary drying Primary drying Primary drying Secondary drying Secondary drying

Moderate freezing

Pre-cooled shelf

+5/50 – – – – 50

– – – – – 50 50 50/25 25 25/5 5 5/+30 +30

Annealing

Moderate freezing

Pre-cooled shelf

+5/50 50 50/10 10/10 10/50 50

2 – – – – 2.5

– – – – – 3 0.5 3 18 4 2 6 7

aseptically transferred into 6 mL tubing glass vials (Medical Glass, Bratislava, Slovak Republic). The vials were partially stoppered with lyo stoppers (West Pharma, Exton, PA). Prepared samples were freeze-dried in lyophilizer Christ 2-10 D (Martin Christ, Osterode am Harz, Germany) with three different lyophilization protocols varied in the freezing step. The moderate freezing protocol at approximately 0.5  C/min, thermal annealing for two hours at 10  C and loading onto pre-cooled shelves at 50  C. Stoppering was performed under vacuum. The freeze-drying programmes are listed in Table 4. Sample analysis Several analytical methods were used to evaluate the solutions’ rheological and physical–mechanical properties and also qualitative sample parameters before and after lyophilization. Rheological and mechanical properties and reconstitution time were evaluated with a gel base lacking thrombin. Reverse-phase HPLC The reverse-phase HPLC method tested the content and purity of the rhThrombin solution. The samples were diluted with water for injection 1:1 before analysis to decrease viscosity and to avoid potential gel formation during measurement. The employed chromatographic conditions were as follows: HPLC (Agilent 1200, Santa Clara, CA), stationary phase: Zorbax SB-C8 (150  3 mm, 3 mm), mobile phase: A – 0.1% formic acid in water and B – 0.085% formic acid in acetonitrile. Column

Annealing 2 1 1.5 2 1.5 1

Atmospheric Atmospheric Atmospheric Atmospheric Atmospheric Atmospheric Atmospheric/0.1 0.1 0.1 0.1 0.1 0.1/0.01 0.01

temperature: 25  C, flow rate: 0.5 mL/min, autosampler temperature: 20  C, injection volume: 10 mL, detection: DAD (280 nm). Gradient: 0 min – 5% B, 82 min – 60% B, 83 min – 5% B, 92 min – 5% B. Optical density To reveal the insoluble aggregates formed during lyophilization and storage, optical density measurement was performed at 350 nm in 95-well UV transparent microplates (BioTek Synergy II reader, Winooski, VT, filling volume 200 mL). Corresponding placebo samples were used as blank solution during measurement. Each measurement was performed in triplicate. Specific activity Thrombin-specific activity was measured after the required dilution with chromogenic substrate S-2238 using the enzymatic test method. Chemically, chromogenic substrate S-2238 is H-Dphenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochloride. The p-nitroaniline group promoting absorbance changes is released following reaction with specific enzymes. The kinetics of S-2238 substrate hydrolysis at 37  C was monitored at one-minute intervals over 10 min, with absorbance measurement performed at 405 nm (BioTek Synergy II reader). The increased absorbance at 405 nm is proportional to the enzymatic activity. The specific activity was calculated by average absorbance increase per minute with the standard calibration curve from human plasma thrombin. Each measurement was performed in triplicate.

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Reconstitution time

Results and discussion

For the determination of reconstitution times, the samples were subjected to the following protocols. The time until a solution without visible particles was obtained was recorded as the reconstitution time. If reconstitution was not finalized in 60 min, the time was no longer recorded because longer reconstitution time is generally unacceptable for clinical use. Each measurement was performed in triplicate. Setup 1: The desired amount of water for injection with temperature 2–8  C was added by Hamilton pipette to the lyophilized cake, and the sample was rapidly shaken and then left without agitation at that temperature. Setup 2: The desired amount of water for injection with temperature 2–8  C was added by Hamilton pipette to the lyophilized cake, and the sample was shaken manually for five minutes. The sample was then refrigerated for one minute, and this cycle was repeated until the sample completely dissolved. Setup 3: The desired amount of water for injection with temperature 2–8  C was added by Hamilton pipette into the lyophilized cake, and the sample was left without agitation at room temperature. Reconstitution time dependence on a vacuum in the vial headspace was also evaluated. Selected vials were opened, and following the addition of water, the reconstitution time was monitored by the above-mentioned procedures.

Preformulation screening studies

Residual moisture Karl Fischer titration (KF titrator DL 38, Mettler Toledo, Greifensee, Switzerland) method according to the Ph.Eur Article 2.5.12 Method A was used to determine residual moisture in the freeze-dried samples. This measurement was performed in triplicate. Viscosity measurement The developed formulation viscosities were evaluated with placebo solutions (samples 1–5, as in Table 3) at 20  C (preparation temperature) and 32  C (application temperature). Structural viscosity was measured at shear rate from 1 to 19.94 s1 with rotational viscometer Haake ViscotesterÕ VT 550 (Thermo Fisher Scientific, Inc., Waltham, MA) and SV II ST measuring system. Each measurement was performed in triplicate. Physical–mechanical characterization The physical–mechanical properties (hardness, adhesiveness, adhesive force and cohesiveness) were measured by CT3 – 1000 Texture analyzer (Brookfield Engineering Labortories, Inc., Middleboro, MA) with the TA-BEC test set. The sample was compressed twice during measurement at 0.5 mm/s1 to 5 mm depth with the texture profile analysis (TPA) test. The probe return speed was equal to the test speed, and this enabled mechanical property calculations by TexturePro CT V1.4 software (Brookfield Engineering Labortories, Inc.). Each measurement was performed at 20  C and 32  C in triplicate. Stability evaluation

Preformulation studies were performed to ensure suitable formulation for further lyophilization, because pH and ionic strength are fundamental parameters affecting therapeutic protein stability16. The influence of different buffer types on thrombin stability was also evaluated, because selective crystallization of buffer salts and buffer interactions during freezing can initiate protein aggregation and chemical degradation17. It is an accepted phenomenon that freezing can induce protein denaturation by ice crystals and ice– water interfaces, therefore high temperature and freeze/thaw cycles were employed during our studies as the most appropriate method to degrade therapeutic proteins. Data from our experimental runs indicated rhThrombin had maximum stability in the 6–7 pH range (Table 1). These observations are also in line with published data for thrombin isolated from human plasma18. Freeze/thaw stress conditions revealed a decrease in specific activity at pH 5 with activity maintained in the tested 6–8 pH range (Figure 1). The decreased activity at lower pH is linked with thrombin precipitation and this was confirmed by the huge increase in optical density and also by chromatographic evaluation. In contrast, the presence of high concentrations of degradation products was detected at higher pH. Both bovine and human thrombin undergo autolytic degradation particularly at higher pH18,19. The maintained activity at pH 8 during the freeze/thaw study indicated the necessity of longer time and higher temperatures for autocatalytic and chemical degradation reactions. All tested buffer types were compatible with recombinant thrombin and exhibited similar degradation rate during the accelerated temperature and freeze/thaw stress tests (Table 1, Figure 2). In this study, the succinate buffer was chosen for further development. According to Fenton et al., salt ions are necessary to maintain conformational integrity in human thrombin18. Moreover NaCl had a positive effect on thrombin activity20. A similar supposition is also mentioned in the literature in connection with recombinant thrombin, but unfortunately it is not supported by experimental results21. The results obtained during our preformulation studies confirmed salt content requirement in the formulation (Table 2). Higher residual activity and protein content was detected in formulations with higher ionic strength, while increased optical density in low salt content samples indicated insoluble precipitate formation. Gel-forming lyophilized formulation It is recognized that poloxamer solution viscosity is affected by sodium salts22. Moreover, the presence of sodium chloride in the formulation can reduce the collapse temperature of the freeze-dried formulation, and therefore high NaCl content is Residual acvity aer 7 freeze/thaw cycles 100 80 [%]

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4

60 40 20

Stability was established by measuring the following sample stability indication parameters; specific activity, purity, protein content and optical density before lyophilization and after lyophilization and reconstitution and also after one month storage in laboratory (25  C) and elevated temperature (40  C) storage conditions.

0 pH 5

pH 6

pH 7

pH 8

pH

Figure 1. Residual activity of recombinant thrombin in potassium phosphate buffer with different pH after seven freeze/thaw cycles.

Development of gel-forming thrombin formulation

generally undesirable23. However, based on our findings, the presence of salt in the system is necessary to maintain both activity and storage stability. Therefore, sodium succinate buffer solutions with 150 mM NaCl and appropriate poloxamer content were evaluated. The rheological and physical–mechanical characteristics of samples without thrombin are listed in Tables 5 and 6, respectively. The results unambiguously confirmed the gelforming properties of our tested formulations. Sol–gel transition took place at the 32  C application temperature, and this was manifested in increased viscosity and a significant change in texture properties. Although the mechanical properties of pluronic solutions with 14–17% w/w at 20  C were almost identical, the values at the 32  C application temperature were directly proportional to poloxamer concentration. The reason for this behavior is micelle formation at higher temperatures9. The flow curves of the resultant gel have non-Newtonian behavior and a hysteresis loop of thixotropic character (Figure 3). Texture profile analysis is currently, widely used in development and manufacture to characterize the various hydrogel formulations24,25. This simple and rapid technique provides information on gel mechanical parameters, including hardness, adhesiveness and cohesiveness. In our study at elevated temperature, solutions with concentrations of Pluronic F-127 15–20% exhibited high values of these parameters. Gel hardness expresses

Residual acvity aer 7 freeze/thaw cycles 100 80 [%]

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DOI: 10.3109/03639045.2014.972411

60 40 20 0 citrate

hisdine

succinate

potassium phosphate

Buffer type

Figure 2. Residual activity of recombinant thrombin in different buffers (pH 6.2) after seven freeze/thaw cycles.

gel applicability to the desired site, while adhesiveness indicates retention time at the wound site and cohesiveness signifies the strength of internal bonds participating in the gel’s structural recovery after application. The correlation of mechanical properties with therapeutic outcome has been previously discussed in research articles. In general, gels should have low hardness to be easily administered to the skin with minimum work. Moreover, high adhesiveness and cohesiveness ensures prolonged adhesion to the skin or mucosa and complete structural recovery following administration26,27. Our in situ gel formulation can be applied in a liquid form with high hardness values after sol–gel transition so that it gives beneficial film properties on the wound surface. Furthermore, the high adhesiveness of this film enhances the mechanical haemostatic effect. In summary, the 20% Pluronic F-127 solution was definitely unsuitable for fill-finish operations because of its high viscosity at 20  C, and the 14% solution had insufficient gel forming properties, so this was also excluded. Our study determined that the 16% w/w PF-127 concentration had suitable qualities for our purpose, so this preparation was chosen for further procedures and the evaluation of reconstitution time. Reconstitution time is an important issue in highly concentrated freeze-dried products, and in this study long lasting reconstitution time was assumed because of the specific excipient with high concentration in our formulation. Several strategies to decrease reconstitution time were suggested. These included the use of sufficiently soluble polyols in formulation (mannitol and sorbitol), different freezing procedures and closure of vials under vacuum. Our results of reconstitution times under different evaluation protocols are summarized in Figures 4 and 5. In this study, Setup 3 required more than 60 min reconstitution time in all tested samples, and it was therefore considered definitely unsuitable for use. The freezing phase is one of the most critical operations in the freeze-drying process. It defines the final structure and pore size and it can also affect reconstitution time28. Specifically, annealing step where the product is held at predetermined temperature for a specified time can induce both ice crystal growth and enlarge lyophilizate cake pore size29. The assumption that faster reconstitution is achieved this way because of easier penetration of the reconstitution medium into the cake pores was not confirmed. To

Table 5. Samples physical-mechanical properties and viscosity measured at 20 ± 1  C.

Sample 1 2 3 4 5

Pluronic concentration (% w/w)

Hardness (g)

Adhesive force (g)

Adhesiveness (mJ)

Cohesiveness

Viscosity (mPa.s) D ¼ 19.94 s1

14 15 16 17 20

6.8 ± 0.1 6.6 ± 0.0 6.7 ± 0.3 6.5 ± 0.0 134 ± 3.3

2.5 ± 0.0 2.6 ± 0.1 2.5 ± 0.1 2.7 ± 0.1 108.3 ± 1.9

0.07 ± 0.01 0.07 ± 0.01 0.09 ± 0.02 0.11 ± 0.02 6.06 ± 0.08

0.81 ± 0.06 0.78 ± 0.05 0.69 ± 0.12 0.52 ± 0.17 1.32 ± 0.08

58 ± 27 65 ± 38 85 ± 11 135 ± 27 10376 ± 331

Table 6. Samples physical–mechanical properties and viscosity measured at (32 ± 1)  C.

Sample 1 2 3 4 5

5

Pluronic concentration (% w/w)

Hardness (g)

Adhesive force (g)

Adhesiveness (mJ)

Cohesiveness

Viscosity (mPa.s) D ¼ 19.94 s1

14 15 16 17 20

8.6 ± 1.8 62.4 ± 0.9 70.5 ± 1.7 80.3 ± 4.1 151.6 ± 5.9

2.6 ± 0.1 50.1 ± 1.1 56.3 ± 1.8 64.9 ± 2.8 125.3 ± 4.7

0.16 ± 0.06 2.37 ± 0.15 2.85 ± 0.13 3.00 ± 0.49 4.25 ± 0.70

0.32 ± 0.03 1.24 ± 0.13 1.23 ± 0.04 1.22 ± 0.06 1.18 ± 0.08

221 ± 90 7398 ± 193 9746 ± 628 12 355 ± 156 18 297 ± 745

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60

Setup 1 (without agitaon) Setup 2 (manual agitaon)

Reconstuon me [min.]

50 40 30 20 10

Figure 3. Flow curve for formulation 3 at (32 ± 1)  C.

Reconstuon me [min.]

60 annealing

moderate freezing

pre-cooled shelf

Under Vacuum

Under Atmosphere

vial headspace

Figure 6. The reconstitution time of freeze-dried sample (16% PF-127) prepared by moderate freezing protocol reconstituted under vacuum and under atmospheric pressure.

50 40 30 20 10 0 16% PF-127

16% PF-127, 16% PF-127, 16% PF-127, 16% PF-127, 0.5% sorbitol 1% sorbitol 0.5% mannitol 1% mannitol Formulaon

Figure 4. The reconstitution time of freeze-dried products prepared by different freezing procedures evaluated by Setup 1 (refrigerated sample, without agitation).

60 Reconstuon me [min.]

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0

annealing

moderate freezing

pre-cooled shelf

50 40 30 20 10 0 16% PF-127

16% PF-127, 16% PF-127, 16% PF-127, 16% PF-127, 0.5% sorbitol 1% sorbitol 0.5% mannitol 1% mannitol Formulaon

Figure 5. The reconstitution time of freeze-dried products prepared by different freezing procedures evaluated by Setup 2 (manual agitation).

the contrary, samples prepared in the annealing step dissolved even slower than those in a moderate freezing or pre-cooled shelf procedure; presumably because the lower specific surface area leads to poor rehydration behavior30. Polyols in formulation had no or negligible effect on the reconstitution rate in the manual agitation protocol; as demonstrated in Figure 5. A more apparent influence was noted using different type of reconstitution protocol (Figure 4), but differences between the employed polyols were insignificant. The reason for such a marginal effect was probably due to their small amount in formulation, when their molecules were stuck in surrounding poloxamer matrix. Moreover, the mannitol was probably not allowed to crystallize in this high density structure during the freezing phase. Another important

issue we attended to in the reconstitution process was the effect of vacuum inside the vial. Although reconstitution under vacuum is a relatively new concept, it has been previously published in literature that this approach has potential in reducing reconstitution time31. Based on our observations in Figure 6, vacuum reconstitution has quite a significant impact on reconstitution rate. The addition of water for injection into vials with a vacuum headspace led to disintegrated freeze-dried cake, a higher surface area and improved dissolution rate. In contrast, the reconstitution medium did not mix with the cake in aired samples and they remained segregated (Figure 7). Following short-term manual vial inversions, larger agglomerates occurred, and these remained undissolved. The final reconstitution time for the developed formulation was approximately 20 min, depending on the reconstitution protocol employed. No obligatory guidelines are available for the reconstitution time. Maximal reconstitution time is set in drug product specification on a case by case basis. Nevertheless, the majority of therapeutic proteins in conventional formulation reconstitute within one minute, even for collapsed cakes30,32. In contrast, highly concentrated protein formulations very often have a long-lasting reconstitution time lasting up to 0.5–1 h33,34. Some of these are already marketed and successfully used in clinical practice, for example, XolairÕ 35 or HaemosolvateÕ 36. As previously mentioned, lyophilization is a common procedure for stabilizing sensitive biopharmaceutical products such as proteins and peptides. However, proteins are exposed to various stresses during this process, including low temperature stress, solute concentration, ice-crystal formation, pH changes and loss of hydration shell; all of which can lead to protein unfolding and loss of activity37. In order to maintain stability, suitable formulation must be developed and optimized. rhThrombin in inappropriate formulation is susceptible to precipitation/aggregation during the freeze-drying procedure21. To evaluate the stability of rhThrombin, the sample 10 solution formulation depicted in Table 1 was analyzed before lyophilization, after lyophilization (upon reconstitution) and after one month storage at 25  C and 40  C. Our results are summarized in Table 7. The freeze-dried cake appearance after lyophilization is acceptable from a commercial viewpoint (Figure 7). No significant changes were detected in evaluated parameter activity, protein content, optical density or purity either after lyophilization or after one month at 25  C. However, some increase in sample optical density was detected at 40  C, thus indicating formation of insoluble

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Figure 7. Evaluated product (from left to right): lyophilizate. After addition of reconstitution media under vaccum; after addition of reconstitution media into open vials; and after reconstitution.

Table 7. Quality parameters of drug product before and after lyophilization.

Before lyophilization After lyophilization Lyophilizate (after one month at 25  C) Lyophilizate (after one month at 40  C)

Reconstitution time (min.) (Setup 1)

Residual moisture (%)

Activity (IU/mL)

rhThrombin content (HPLC) (mg/mL)

Purity (HPLC) (%)

Optical density (mAU)

– 530 530 530

– 0.5 ± 0.03 Not analyzed Not analyzed

864 ± 73 930 ± 28 781 ± 60 779 ± 128

0.310 ± 0.03 0.298 ± 0.02 0.309 ± 0.01 0.311 ± 0.02

90.53 ± 0.8 91.35 ± 0.2 92.15 ± 0.3 90.76 ± 0.6

7±1 5±4 10 ± 5 28 ± 6

aggregates. Despite our promising results, a more comprehensive stability study has to be performed as further development work.

Conclusion We have developed a gel-forming lyophilized formulation with rhThrombin; with an emphasis on improving reconstitution rate. Our results confirmed that stoppering vials under vacuum is crucial to obtain the most acceptable reconstitution time. While formulation at the application temperature of 32  C has suitable mechanical properties for spreading and remaining on bleeding areas, the solution structure at 20  C is appropriate for wellestablished sterile production with sufficient filtration and filling. In addition, lyophilization of this formulation has no negative impact on the stability of the evaluated proteins, and this was successfully confirmed by our analytical measurements.

Acknowledgements The authors are grateful to Anasta´zia Pola´kova´ and Helena Katonova´ for technical assistance with viscosity and structure measurements.

Declaration of interest This work was supported by project ‘‘Industrial research for new drugs on the basis of recombinant proteins’’, code ITMS: 26240220034. We support research activities in Slovakia. This project is being co-financed by the European Union.

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