The Introducing of the Human Fibrinogen and Thrombin Virus Inactivation Directly in the Affinity Chromatographic Process before Manufacturing Fibrin Sealant
- 1. Neutromics Ukraina TOV, 12, Melnikova str, Kyiv, Ukraine
- 2. Shijir International LCC, Raining Valley, Ulaanbaatar, Mongolia
hFg: human fibrinogen; hProThr/hThr: human prothrombin/ human thrombin ; PAI-1: Plasminogen Activator Inhibitor-1; NIH – units of thrombin activity is equivalent to 1.1 to 1.3 IU of thrombin FVIII/vWF – complex of Factor VIII and von Willebrand factor; MW: Molecular Weight ; S/D: Solvent/Detergent; LEV/ LEVs – virus (es) enveloped by lipid membrane NLEV/NLEVs: Non-Enveloped Virus(es); IFA method: method of immunefluorescent analysis; FITC: fluorescein isothiocyanate; RTPCR method: Real-Time PCR Method; DBC: Dynamic Binding Capacity; TDC: temperature-depending binding capacity ; EC: ion exchange chromatography; AC: Affinity Chromatography; GC: Gas Chromatography; FID detector : Flame Ionization Detector ; UVdetector: ultra violet detector; human prothrombin; thrombin; fibrinogen; affinity peptides; virus inactivation
Fibrin glue/sealant results from the mixture of a fibrinogen source (from plasma or heterologous/autologous cryoprecipitate) with a thrombin source (bovine, human, or recombinant). Fibrin glue is a non-FDA-approved thrombin/preparation, and it has been widely used in Europe for many years. Fibrin sealants are FDA-approved alternatives to fibrin glue and have some advantages, such as standardization of production, over locally made fibrin glues, but are more expensive. Fibrin glues/sealants can be used for multiple surgical purposes, including as topical hemostat, sealant, or adhesive. Multiple fibrin or thrombin containing products are FDA approved for use .
Fibrin sealant is a two-component material consisting of the human fibrinogen (hFg) and thrombin (hThr). In the presence of small amounts of calcium and factor XIII, the hThr converts hFg into insoluble fibrin, the final stable form of the agent. Fibrin sealant now has over a century of development and use. Bergel used fibrin first as a hemostat in 1909 , Young and Medawar used it as an adhesive in 1940 , Matras used concentrated fibrinogen for nerve attachment in 1972 , and the FDA approved liquid fibrin sealant in 1998 [5,6] as well as a fibrin sealant patch in 2010 [7,8]. The fibrin sealant category of hemostats  is divided into four classes as noted in : human pooled plasma fibrinogen and thrombin, individually obtained units of human plasma (possibly platelet-enriched) mixed with bovine thrombin and collagen, and dry human pooled plasma fibrinogen and thrombin fixed on either an equine collagen or oxidized regenerated cellulose patch.
The safety of sealant’s composed liquid agents is most dependent on the fact that multiple donor plasma pools are used for their preparation [11,12]. Both contain highly concentrated forms of hFg (70-85 mg×mL-1) and hThr (500-1000 IU×mL-1), respectively, derived from large pools of human plasma. Thus, these forms of human pooled plasma fibrin sealant may be associated with viral disease (Parvovirus B19, hepatitis, HIV) or prion disease (CJD - Creutzfeldt Jakob) transmission [11,12].
A variety of methods for obtaining the hFg and hThr required for fibrin sealant are available although not FDA approved for any specific indications including ammonium sulfate  or cold  precipitation. The most frequently employed alternatives to the FDA approved commercial products at present involve plasma fractionation devices . In either case, the components of the fibrin sealant should be previously highly purified and the virus inactivated.
Fibrinogen purification has traditionally been obtaibed from side-fraction of the purification of FVIII from cryoprecipitate using heparin affinity chromatography , or directly from plasma through a series of ethanol precipitation steps . The more complicated method can include several chromatographic steps on cation exchanger, hydrophobic gel and/or dye gel [15,16] or mixed mode gel (for example, Capto MMC) . Recently, the tetra- or hexapeptide ligands (GPRP [18,19], FLLVPL ), identified from a peptide library, was claimed to allow, at experimental scale, isolation of fibrinogen under a highly pure and functional state. Such affinity-purified fibrinogen retained a FXIII crosslinking activity claimed to be required to prepare elastic and high tensile- strength fibrin sealants [18-20].
A simplified procedure was described for the purification of prothrombin (hProThr) from pooled human plasma. The initial steps, which are common to prior purification techniques, include adsorption onto and elution from barium citrate, ammonium sulfate fractionation, and DEAE- Sephadex chromatography. These chromatographic conditions permit the separation of proThr, Factor X and IX from each other, yielding fractions with apparent homogeneity in several electrophoretic systems .
Crude hProThr enriched from human plasma was directly activated to generate alpha-thrombin without prior exhaustive purification of the. Activated Thr was separated from other components in a single/double step(s) by taking advantage of its highly specific affinity to heparin immobilized on a matrix support of Sepharose CL-6B  or by SP Sepharose ion-exchange chromatography and subsequently over Phenyl Sepharose column . It was demonstrated the ease with which at least 22-25 mg of a highly purified enzyme (>97% homogeneous) with specific activity of at least 4000- 6000 NIH×mg-1 can be obtained per 1 L of plasma.
Baumbach and other  discovered ? group of peptides characterized by their ability to bind to prothrombin and thrombin. This group included the following peptide affinity ligands: QLWGSH, RQLWGSH, HQLWGSH, and YFPGPYL. The preferred ligands were QLWGSH and YFPGPYL. Authors also described ? method of purifying prothrombin and thrombin which comprises passing ? protein containing solution over ? substrate which has bound upon it ? peptide affinity ligand of the defined sequence and then eluting the prothrombin or thrombin. Nogami et al. , was observed covalent cross-linking between the FVIII 484-509 peptide and thrombin and demonstrate that clustered basic residues within the 484-509 region of the A2 domain play a part of key role in thrombin-binding. Minami et al. , demonstrated that the activated factor VIII (VIIIa) forms the tenase complex and markedly amplifies the activation of factor X as a cofactor of factor IX and The A2 residues 484-509 partially contribute to cleavage at Arg372 by thrombin. They showed that FVIII A1 residues 340-350 (NEEAED (sY) DDDL) involving sulfated Tyr346 contained the thrombin binding-site.
After this work, dozens of articles were published, where the ProThr/Thr interaction sites with various proteins and peptides were identified by the method of molecular docking. From the point of view of the development of affinity ligands for the chromatographic isolation of ProThr from the donor plasma the dates of Nogami et al. , and Minami et al. , Taneda et al. , and Ahmed et al. , in which a number of amino acids were identified in the anion-binding exosites I and II of thrombin, which are involved in the binding of FXa during the activation of prothrombin into thrombin have a great value.
In addition, it should be taken into account that for the production of fibrin glue, it is necessary to efficiently activate prothrombin into thrombin, and then quickly isolate thrombin with high purity and activity. In this case, the isolation and additional purification of thrombin without its self- fragmentation is preferable on an affinity sorbent for the following reasons: 1) the use of an activator “attached” to the chromatographic adsorbent, 2) the rapid binding of thrombin on a chromatographic gel, 3) the use of bound thrombin to activate free-flowing prothrombin, 4) possibility of integration the activation and purification processes into a single chromatographic system. This approach allows implementing the method of prothrombin activation by thrombin immobilized on a chromatographic gel, proposed by Strukova . Given the rather high cost of thrombin from donor blood, it was more economical to use as an activator the α-specific thrombin-like enzyme (Ancistron-B) previously isolated from the venom of Agkistrodon blomhoffii ussuriensis , immobilized on a chromatographic gel.
Naturally, the isolated protein components of fibrin glue must be processed to remove possible viral contaminants.
Treatment with solvent/detergent (S/D) is a widely used method for ensuring the virus safety of the manufactured proteins from human plasma. It is important to note two significant weaknesses of the S/D treatment. First, it did not inactivate non-enveloped viruses (NLEVs) and it was necessary for further NLEVs reduction to use wet/dry heat treatment at 60-100°? [31- 33], or nanofiltration [34,35], or UV-/gamma irradiation . Second, the exposure at 25-37°? to S/D a relevant labile highmolecular weight protein  can cause its denaturation which is summarized with additional losses of the denatured target product caused by high turbulence filtration process using for R/D and NLEVs removing .
Recently we demonstrated a simple and useful procedure for proteins virus inactivation directly in chromatographic column . This virus elimination was possible to introduce into purification process and obtain the factor of enveloped [LEVs] and non-enveloped (NLEVs) viruses concentration decrease values that were higher than provided by known technologies of virus inactivation in solution [36-40]. These results were based on the two well-known principals : 1). The chromatographic gel with high dynamic binding capacity should retain the target protein by binding a large number of sites to deprive the protein of degrees of freedom and thus save it from denaturing by S/D at elevating temperature of 30-45 °C, that treatment guarantees high degree LEVs elimination; and 2). The S/D treatment during 3-6 hours mechanically washes out NLEVs and LEVs traces/ fragments from the column.
The development of such affinity gels virus reduction treatment introduced in affinity chromatographic purification process with new peptide for human fibrinogen and thrombin is presented in this paper.
MATERIALS AND METHODS
All reagents were purchased from Sigma-Aldrich (Green Chemistry LLC, distributor in Mongolia) unless otherwise indicated. Reagents and equipment from other manufacturers are indicated below. Human (donor’s) plasma, obtained by plasmapheresis, was bought from Ulaanbaatar blood collection center (Mongolia, Ulaanbaatar).
Peptide array library
Peptide array library was calculated according to 7 developed principles described before  using nucleotide sequence of the human genes and amino acid (aa) sequences of the hFg and hProThr from Protein Knowledgebase - UPKB (UniProtKB/ Swiss-Prot).
Array and FMOC solid phase peptide synthesis, SPOT peptide array experiments
The synthesis and SPOT peptide array experiments were carried on as described before [41-43].
Peptide-affinity chromatographic gel synthesis
All developed by early publish method [41-43] 7- and 8-mer peptides were coupled with WorkBeads 40/10000 ACT matrix according to manufacturer’s recommendations (Bio-Works Technologies AB, Uppsala, Sweden).
A peptide 1% solution in Na-carbonate-bicarbonate buffer, pH 9.4, was pumped with flow 10 Vc (column volume) per hour through ECOPLUS glass columns Ø15×120 mm (YMC Europe GmbH, Germany, Dinslaken), packed with 3 mL WorkBeads 40/10000 ACT matrix (Bio-Works Technologies AB, Uppsala, Sweden), bed Ø:height 15×15 mm (packing density factor 1,15-1,20) during 48 hours at 4 °C. All other manipulations were carried on according to recommendation of activated gel manufacturer. The peptide density on the WorkBeads gel 40/10000 was determined by quantifying the difference between total peptide quantity in start solution and unbound peptide by fluorescence of tryptophan in the spacer measured by Typhoon Trio (GE Healthcare AB, Sweden, Uppsala).
Finally, the column was rinsed with 60 mL of deionized water to remove the blocking agent and, before using, – with working buffer for equilibration or, before storage, – with 20% ethanol.
Target Protein determination
The protein’s quantity was measured by the standard methods with Human Fibrinogen ELISA Quantitation kit (GenWay Biotech. Inc., San Diego, USA), Human Prothrombin (Factor II) and Human Thrombin ELISA kits (both kits from ASSAYPRO LLC, St. Charles, USA), Rox Prothrombin (Rossix AB, Molndal, Sweden) and Human Thrombin (ASSAYPRO LLC) chromogenic activity assay kits using analytical equipment Typhoon Trio Imager, Biotrak II visible plate reader and UV/VIS-spectrophotometer Ultrospec 3300pro (all from GE Healthcare AB, Sweden, Uppsala). The total protein in the collected samples was determined by Bradford method  with Stoscheck modification .
Determination of the peptide-affinity chromatographic gel dynamic (DBC) and temperaturedependent binding (TDC) capacities
The DBC and TDC of the peptide-affinity gels were determined on ECOPLUS glass columns Ø15×120 mm (YMC Europe GmbH, Germany, Dinslaken) packed with 3 mL of IEC gel, bed Ø:height 15×15 mm (packing density factor 1,15-1,20).
DBCs were determined according to standard procedure . TDCs were determined by the following way. Target proteins was applied on the affinity gel. The quantity of protein samples was calculated 10% lower than maximum level of adsorbent DBC at 20°C. After sampling and washing the column/buffer temperature were slowly raised on 5 degrees each step and determined the target protein quantity in eluates. 5 or more independent experiments were performed according to chromatographic conditions stipulated above.
The apparent Kd
The apparent Kd of each protein-peptide interaction was determined by standard procedure described by Baja et al. , for intact hFg, hProThr and hThr interactions with development affinity peptides.
Partially target proteins purification
All chromatographic experiments were performed using ÄKTAexplorer system (GE Healthcare AB, Uppsala, Sweden) and different size ECOPLUS glass columns (YMC Europe GmbH, Germany, Dinslaken).
The target proteins – hFg and hProThr – were manufactured by early described cascade technology from human blood plasma  with our following modifications. The batch of donor’s plasma was divided on high (H) and low-middle molecular weight (L/M) protein fractions eluted from Sepharose 6 Fast Flow (GE Healthcare AB, Sweden, Uppsala) according to our developments . On the next steps the H fraction was used for hFg and the L/M fraction for hProThr/hThr manufacturing.
Human prothrombin purification
L/M fraction  contained hProThr was concentrated to protein content 10 mg×mL-1 and applied on ECOPLUS column Ø50×120 mm packed with 120 mL of ANX Sepharose FF (GE Healthcare AB, Uppsala, Sweden) with flow rate 6 mL×min-1 of the 2.5 mM Na3-citrate buffer, containing 25 mM NaCl and CaCl2. Then column was washing with 2 Vc 10 mM Na3-citrate buffer, containing 25 mM NaCl and CaCl2, with the flow rate 10 Vc×h-1 and prothrombin fraction was collected in 2 Vc 25 mM Na3-citrate buffer, containing 85 mM NaCl and CaCl2, with the flow rate 5 Vc×h-1. This fraction was buffer exchanged to citric acid-Na2HPO4, pH 6.0, and concentrated to protein content 10 mg×mL-1 and applied on ECOPLUS column Ø25×120 mm (with cooling jacket) packed with 15 mL VVIVPADR (*VVI)-WorkBeads gel for creating a configuration of the gel bed to achieve the best flow direction of the buffer (the ratio Ø to height 1:0.5÷1:1), which was previously showed . Then column was twice washing by 2 Vc of application buffer and 2 Vc of the 20 mM citric acid-Na2HPO4, pH 4.2. The application and washing flow rate - 2 Vc×h-1. The prothrombin was eluted by 2 Vc of 20 mM citric acidNa2HPO4 buffer, pH 2.6. The eluate was automatically titrated by 0.05 M NaOH to pH 6.2-6.0.
Human prothrombin activation by immobilized α-specific thrombin-like enzyme
Collected pure prothrombin was applied on a column with immobilized α-specific thrombin-like enzyme (Ancistron-B) previously isolated from the venom of Agkistrodon blomhoffii ussuriensis  to hydrolyze prothrombin and obtain active thrombin. This method of thrombin activation, however, with immobilized thrombin, was proposed by Strukova . The eluate from the column was directly applied on the ECOPLUS column Ø25×120 mm (with cooling jacket) packed with 15 mL PFLRAWAI(*PFL)-WorkBeads 40/10000 gel. The process was cycled for 6 hours at a temperature of 10–12°C, i.e., the eluate from the column with immobilized Ancistron-B was applied on the column with thrombin-affinity gel, thrombin was captured by the gel, and non-activated prothrombin was again applied on the column with immobilized Ancistron-B. And thus, within 6 hours, more than 90% of the thrombin, that was bound on the affinity column, was activated. After 6 hours, the PFLRAWAI (*PFL)-column was transferred to the direct in-column virus inactivation/elimination by S/D treatment process.
In-column S/D treatment of target proteins and its final purification by chromatography method
hFg was partially purified from blood plasma by H-fraction separation . H-fraction, buffer changed on application buffer citric acid-Na2HPO4, pH 6.0, and concentrated to protein content 10 mg×mL-1, was applied on ECOPLUS column Ø50×120 mm (into cooling jacket), packed with 110 mL FFFFRIF(*RIF)-WorkBeads 40/10000 gel, with flow rate 2 Vc×h-1. 2 Vc of the application buffer was used for column washing with same flow rate. After these manipulations the column with captured hFg was ready for virus inactivation/elimination process.
The peptide-affinity column ECOPLUS column Ø25×120 mm (with cooling jacket) packed with 15 mL PFLRAWAI(*PFL)- WorkBeads 40/10000 gel, and with coupled activated thrombin was included in thermoregulated chromatographic system, described several early published papers in details [39, 42,43].
In the same works, the scheme of the process itself is also presented in detail and the stages of manipulations with the temperatures of the column and buffers in the process of virus inactivation/elimination are described. It should be noted that the removal of model viruses [39, 42, 43] from hFg and hThr was carried out according to the scheme used for upper IgG , Table 3, max T°=35 °C] due to dates of TDC that the critical zone for proteins retentions by gels is beginning after 38-39 °C [Table 4].
The S/D treatment was beginning after target proteins capturing on the affinity column. The experimental scheme and stages of the virus-inactivation/elimination process, developed and presented earlier, did not change when studying the behavior of chromatographically bounded target proteins under the temperature-dependent treatment. The exceptions were temperature levels and the using one R/D blend only: TnBP/ Triton X-100.
- The common conditions of S/D treatment were the following:First washing: 30 Vc of equilibration buffer (0,5 Vc×min-1) to complete absence of virus material in eluate.
- The raising of S/D inactivation buffer gradient: 30 Vc inactivation buffer with flow rate 0.5 Vc×min-1 was automatically created by programming linear increasing concentration from 0 to 100% and applied on the column. The final inactivation buffer included 1.0%/2.5% TnBP/Triton X-100.
- The raising inactivation temperature gradient: on the beginning of inactivation process for inactivation buffer and column the termostabilized gradient was started from 20 to 35°? during 60 min, therefore a temperature gradient grew in parallel to S/D gradient in all cases. Stages 2 and 3 combined in one process named in the finale table Gr.
- The second washing after raising gradients – actual treatment process: column was washed by 90 Vc under 35°? with same flow rate 0.5 Vc×min-1; the column temperature was kept at corresponding buffer temperature (Gs).
- The falling down S/D inactivation buffer and temperature gradients: after the washing the reverse S/D and T° gradients were used to reach the initial conditions for 60 minutes - 30 Vc with same flow rate 0.5 Vc×min-1 (Gf).
- The third column washing: when the start T° 20°? was reached simultaneously with falling down gradient finishing, the column was washed by 10 Vc equilibration buffer, flow rate 0.5 Vc×min-1.
The elution of target proteins was carried out with 2 Vc of 20 mM citric acid-Na2HPO4 buffer, pH 2.6, 0.04 Vc×min-1. The eluate was automatically titrated by 0.05 M NaOH to pH 6.2-6.0.
The all eluates were collected for concentration/yield and purity of target proteins determination and their impurities and ligands determination by ELISA, and electrophoresis, and HPLC analysis.
Column regeneration and sanitization were performed with 2 Vc of 0.1 M glycine-HCl, pH 2.5, and 2 Vc of 0.05 M NaOH with flow rate 0.5 Vc×min-1.
Virus titer and nuclear acid content
Virus titers were calculated using the method of Kaerber  and Spearman  and were expressed as log 50% tissue culture infectious dose (TCID50) [39, 42,43].
For extraction and purification viral RNA/DNA from the protein samples “QIAamp Viral RNA/DNA Mini Kit” from QIAGEN China Co., Shanghai, China was used. Quantitative Real-time PCR was performed on the Rotor-Gene Q 2plex HRM System instrument (QIAGEN China Co., Ltd., China, and Shanghai). The data was expressed as log10 of ratio between virus DNA/RNA detected in infected protein sample before and after purification [39, 42,43].
In general, in all virus inactivation studies the virus titer was a primary parameter and virus titer determination was always followed by infectivity studies. This choice, when it is necessary to determine whether the residual amount of infectious virus, remains reasonable until no suggestion that the new, accurate and adequate methods for this purpose there are more reliable. In fact, due to the destruction of the lipid membrane by the S/D LEV viruses lose the infectious properties and cannot be determined by the Kaerber  and Spearman . Methods. Furthermore, we lost a possibility to calculate the process mass balance  using the virus titer.
We received full repetition of previously detected pattern in the previous investigations: the method of the virus titer determining gave an adequate result according to FVIII/vWF infectivity in the case of LEV and NLEV models . The LEV titer determination didn’t leave the hope to calculate process kinetics since the loss in virus infectivity due to the particles destruction and virus proteins denaturation. The virus titration was more acceptable for NLEV infectivity mass balance determinationmaximum 24% of the total infectivity was lost against 4-6% determined by RT-PCR. Unfortunately, the titration accuracy was very poor again (variants deviation more than 18-22% in the best cases) and didn’t allow definite conclusions.
Quantification of viral nucleic acids by RT-PCR in proteins complex and buffer fractions obtained during inactivation allowed to calculate the process mass balance within 97.0 ± 2.9%. It means that instability of the virus nucleic acid (in most experiments not more than 3-4% of losses, rarely up to 4.1%) allows to calculate the process kinetic to assess what happens to model virus in the process of S/D treatment with small variant’s diversity not higher than 8-15%. In contrast, diversity of variants of virus titer of target proteins was more than 16-33% .
Compared with the results of previous publication , where we could not accurately detect the ratio of virus particles “leaching” and destruction in the process of inactivation, it was introduced a separate definition of viruses in the steps up to the temperature of inactivation, most inactivation and washing after inactivation.
Therefore, the residual infectivity of treated hFg and hThr was determined by virus titer and virus nucleic acid concentration, but kinetic of treatment process by virus nucleic acid only.
S/D determination in the target proteins after virus inactivation
The solvent level in the final proteins concentrate after S/D treatment was measured by GC method with FID , the detergent level - by HPLC with UV-detector (both instruments from E-Chrom Tech, Taiwan) .
The statistical processing of results was carried out by the standard methods . A value of p<0.05 was considered statistically significant. Data was presented as a mean ± standard errors (SEM) of at least 5 independent experiments unless otherwise indicated.
Proteins sequences numeration
Proteins and peptides sequences numeration was done according to the whole molecule represented in UniProtKB (Swiss-Prot): human Fibrinogen alpha chain - P02671, beta chain - P02675, gamma chain - P02679; human Prothrombin - P00734.
RESULTS AND DISCUSSION
Using the approach of calculating affinity peptides for target proteins, developed in previous investigations [41-43], we identified several potentially affinity peptides for hFg, hProThr/ Thr and separately for hThr.
High affinity peptides calculation to the human Fg specific coupling
Bennet et al. , Smolarchuk et al. , Mosesson , Lancellotti et al. , Vu et al. , Zafar et al.  and others, established that peptides 85-98, 112-125, 196-209 of α-chain, 432-445 of the γ-chain, 439-452 of the γ-peptide can be sites of interaction with glycoprotein IIb-IIIa complex (GPIIb-IIIa) , PAI-1 , anion-binding exosite (ABE)-II of thrombin [58-60] or histidine-rich glycoprotein , platelet integrin receptor aIIbβ3 .
Our analysis showed three important facts: 1) at the hFg sequence there are several similar peptides which were added into the Figure 1: 48-61 from the β-chain and 407-420 from the γ-chain, 2) antisense amino acid sequences of these peptides are very similar and include between two wide anionic zones a powerful charged cationic amino acid (sometimes two amino acids), represented by arginine or lysin, less often glutamic acid, 3) the both anionic zones represented mostly by leucine, valine and phenylalanine; less often by alanine, glycine and tryptophan (Figure 1A).
Besides Zamolodchikov et al , Giau and An  discovered that Aβ5-25 peptide was the most critical region for the interaction between β-Amyloid (Aβ) and hFg as which can be inhibited by specific monoclonal and polyclonal antibodies against the central region of Aβ (Figure 1B).
Kaufman et al. , in the process of the development of the affinity peptides to hFg purification through the creation of a peptide library came to the conclusion that the most promising peptide is FLLVPL, which is very similar in structure and ionic properties to those considered by us (Figure 1C).
Based on the above dates and “summarized” antisense peptide obtained after recognition of the most stable/matching structure (shown by black arrows) with additional arbitrarily chosen amino acid (shown inside a black dotted frame) an initial library of potential affinity peptides was created [Table 1]. We decided to lengthen it to 8-mer, because previous work has shown a preference for the octapeptide on a short spacer. These peptides were tested on the hFg binding in 0.05 M Na3-citrate buffer, pH 7.2, – a future coupling buffer in the chromatographic process of hFg purification.
The “summarized” peptide (2 FLLARLA8 amino acids) and its daughter peptide with additional leucine (1 LFLLARLA8 ) showed approximately equal and high affinity to hFg (Table 1, no statistically significant changes of affinity - p>0.1) enough for strong coupling protein and for its desorption at nondenaturation level pH=2,5÷3,2.
However, the presence of the eighth amino acid in the peptide showed a tendency to decrease its affinity to hFg, moreover, leucine at this position was the poor and best of the tested anionic amino acids isoleucine and tryptophan (not represented in Table 1), respectively. It became clear that the reason is not the size or magnitude of negative charge of the eighth amino acid, but rather its hydrophobicity (HPIL=+3.8, pIL=5.98, MWL=166.7; HPII=+1.3, pII=6.02, MWI=166,7; HPIW=−0.9,
pIW=5.89, MWW=227,8). In the case of the affinity peptide interaction with a linear sequence of hFg, this implies another statement: the interaction pair for leucine can be a hydrophilic, weakly positively charged amino acid, and according to Biro  it should be E, D, N, or, in extreme cases, Q or K. The analysis hFg sequences shown in literature [48-53] and offering sequences by our search leads us to the understanding that L1 cannot be a component of the affinity peptide, since it discovered a high fluctuation of the putative pair for the interaction (L, G, D, K, M, P and T) from hydrophilic to hydrophobic amino acids, and only in one case does desired pair (D). Besides the isoleucine and tryptophan mentioned above as well as the indicated in the Table 1 leucine only worsened the affinity of the peptide to hFg (for all cases the statistical difference were significant, p<0.05). Thus, L1 - residue should be excluded from the affinity peptide construct as insignificant and, moreover, subversive.
As for the central positively charged core of the peptide, arginine provides the highest affinity for hFg compared to lysine and glutamic acid. Effective interaction with the protein is facilitated by the electrostatic properties of the amino acid, since the hydropathy of all three amino acids is practically the same (HPIR=−4.5, pIR=10.76; HPIK=−3.5, pIK=9.74; HPIE=−3.5, pIE=−3.22).
The R6 /K replacement definitely shows a tendency to decrease the peptide affinity (no statistically significant changes, p>0.1), while the R6 /E substitution, although it does not abolish the interaction of the peptide with the protein, practically brings it closer to the border of nonspecific adsorption (statistically significant changes, p<0.001).
Appearance of practically neutral, weakly hydrophilic G (HPIG=−0.4, pIG=5.97) or strongly hydrophobic L (HPIL=+3.8, pIL=5.98) and V (HPIV=+4.2, pIV=5.96) significant changes in the affinity of the peptide does not lead (p>0.1 in all cases) after the changing of F2 (HPIF=+3.8, pIL=5.98) in the peptide. However, the trend of affinity change clearly demonstrates a certain effect of the amino acid charge, but not its hydropathy in the following rank F>V=L>G. It is possible that G falls out from the equation between L and I due to its weakly hydrophilicity.
Mutations L3 /(F, Y, V, A), L4 /(F, Y, I, A) and L7 /(F, I, G) have the same effect on the affinity of the peptide as in the case of the F2 mutation: the higher negative charge of the hydrophobic amino acid - higher affinity and the presence of low hydrophilicity reduces affinity (F>Y>L=I≥V=A>G).
It should be noted that than mutations will locate closer to the central positively charged core its effect on the affinity of the peptide will be lower. However, it is also true that mathematical statistics are not ready to confirm this claim, which is based probability only on the trends of peptide affinity increasing/ decreasing.
An attempt to increase the affinity of the peptide by mutations L3 /K and L5 /E, the possibility of which was indicated by the antisense sequence 112-125 of the alpha chain hFg and the sequence 17- 25 of β-amyloid, had negative result only. Strong positively charged hydrophilic lysine (HPIK=−3.9, pIK=9.74) in position 3 and strong negatively charged hydrophilic glutamic acid (HPIE=−3.5, pIE=−3.22) catastrophically reduced the affinity of the peptide to hFg (statistically significant changes in both cases, pK0.5), then according to the effect on increasing the affinity of the substitutions A8 amino acids were arranged in the following order: I>F=V>A> G>W. We did not find statistically significant changes (in cases F, I and V p>0.5, in cases G and W p>0.2), but the trend of the results shows that in the case of position 8 of the peptide, the hydropathy of the amino acid is more important than its charge.
Thus, for the next affinity test, the FLLARLA peptide becomes 1 F2 F3 F(Y)4 F(I)5 R6 F(I)7 F(V/I). All these sequences are given in Table 2.
The results of determining the peptides 31-46 affinity to hFg [Table 2] indicate that all peptides have a sufficiently high affinity to be used as chromatographic ligands.
It should be noted that the affinity to hFg previously proposed by Kaufman et al. , the FLLPL peptide was tested in our system [Table 2]. It is obvious that, both in terms of construction and affinity, no special differences were found with the peptides proposed by us. But for further work, three peptides FFFFRIF, FFYIRIV and FFFIRIV which have the better affinity properties compared with peptide FLLPL (statistically significant changes in all cases, p<0.05), were selected for synthesis of chromatographic gels.
High affinity peptides calculation to the human Thr specific coupling
A technique similar to the one above was used to develop affinity ligands for the chromatographic purification of thrombin. Thrombin uses three principal sites - active site, exosite I, exosite II - for recognition of its cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II .
On Fig. 2 presents an analysis of the results of experimental studies from 13 mainly, from our point of view, literature sources [28, 65-77] on the identification of sequences or separate amino acids of thrombin, which can be sites of interaction with a number of human proteins or chemicals or pharmaceuticals, namely: FVa, FVIII, FIX, FXa, FXIII, fibrinogen and its peptides, platelet glycoprotein GPIbα, thrombomodulin, PAR-1, PAR-3, PAR-4, IgA, heparin, hirullin, hirugen, triabin, modamin, rhodniin, boophilin, gallic acid, flavonoids, etc.
The researchers identified two exosites I and II and several amino acid sequences that take part in the thrombin interactions, and these sites do not have enzymatic activity, that is, they contribute to the substrate fixation on the surface of thrombin, and/or, on the contrary, are the zones of prothrombin fixation during it activation into thrombin, and/or are the sites of thrombin interaction with inhibitors. It is possible that certain sites perform the generally described triple function. Unfortunately, we did not have enough dates to bring these speculations to a conclusion, however, there are more than enough dates to identify possible affinity peptides for thrombin.
First of all, we focused our attention on the 425RIGKHSRTRYERNIEK440 peptide (Figure 2, exosite I – red letters into the blue field), especially since Ahmed et al. , showed that this peptide reliably inhibited the activation of prothrombin to thrombin by blocking the binding of prothrombin by the hydrolytic complex at the same site. The development an affinity peptide against this site would be very acceptable in view of the fact that in non-activated prothrombin exosite I is not formed and exposed  and thus, using an affinity peptide as a ligand it would be possible to separate prothrombin and thrombin.
The other our interest was incited by Derkach et al. , who revealed the affinity of the peptide 501AGYKPDEGCRD ACEGDSGGPFV523 (Figure 2, amino acid sequence – yellow letters into the pink field) to αvβ3 integrin. Authors showed the peptide 501-523 interaction with integrin αvβ3, the member of the integrin family of transmembrane receptors, leading to nitric oxide synthesis.
The antisense 8-mer peptides with overlapping 2 (for short) or 3-4 (for long) amino acids were calculated according our development [41-43] against 425-440 and 501-523 sequences are representing in the Table 3.
It should be pointed out that based on our understanding of the protein interaction in the linear form of sense and anti-sense amino acid sequences and taking into account the formation of the 564C- C594 bridge in the thrombin heavy chain, we assumed that there could be three such linear thrombin sequences (Figure 3), against which antisense peptides was calculated.
The affinity determination of the antisense peptides listed in Table 3 revealed the following. Peptide PFLRAWAI (No. 50) had the highest affinity (pH 50% 3.47±0.12) to human thrombin in the series of peptides 48 > 52 > 47. Other peptides 41, 51, 53 had a low affinity, approaching nonspecific adsorption (?? 50% > 4.75). The affinity of peptides 55-66 decreased sharply with a decrease in the content of amino acids, which are antisense to sense belong to the basic fragment (AGYKPDEGKRGDA) of the peptide proposed by Derkach et al. . It should be noted that the activity of affinity peptide No. 55 (RPIFGLLP, pH 50% 3.44±0.10) was the highest and equaled with the affinity of peptide No. 56 (GLLPFAPL, pH 50% 3.8±0.09; no statistically significant changes, p>0.5). If antisense peptides 57, 60 and 64 still interacted with thrombin (pH 50% 4.58±0.16, 4.63±0.11 and 4.49±0.14, respectively), then other antisense peptides did not recognize thrombin at all.
Two statements are possible that would not contradict the obtained results, namely: a) only the following sequence AGYKPDEGKRGDA (± 2-3 more amino acids) is the interaction site in human thrombin or/and 2) only the AGYKPDEGKRGDA sequence is remaining linear and available for interaction with the affinity peptides, calculated by our method.
Another result was shown that peptides PFLRAWAI (No. 50) and RPIFGLLP (No. 55) and GLLPFAPL (No. 56) did not interact with prothrombin. It suggested previously advanced assumption that as minimum two sites of thrombin remain masked, hidden until the activation hydrolysis of prothrombin . Thus, these peptides can be used for affinity chromatographic separation of thrombin from prothrombin.
High affinity peptides calculation for the human ProThr specific coupling
On the way of the affinity peptides development to human prothrombin, we decide to shifted our attention as far as possible from the thrombin sequence in order to avoid the conformational opening of its exosites I and II during the interaction of prothrombin with any affinity peptides.
Friedmann et al. , was used nuclear magnetic resonance spectroscopy of recombinant prothrombin F2 domain to identify seven residues within kringle K2 that are significantly responsive to FVa binding: 220Q, 222Q, 224R, 225L, 226A, 227V, 228T (our numeration described in “Materials and methods”). The whole sequence 215PDRGQQYQGRLAVTTH230 includes around 80% hydrophil amino acids that allows to hope for a surface, i.e. accessible, orientation of this peptide. Against this sequence the antisense 8-mer peptides with overlapping 2 amino acids were calculated (No. 67 GLAPVVIV, No. 68 APVVIVPA, No. 69 VVIVPADR, No. 70 IVPADRHW and No. 71 PADRHWWL) and examined on affinity to hProThr in described early conditions. All peptides shown satisfactory affinity to hProThr, but the affinity of peptide No. 67 was extremely high (pH 50% 3.19±0.12) and was not dissociated from hProThr even at pH=2.0. The most acceptable affinity was observed for peptides No. 69 and 70 (pH 50% 3.64±0.09 and 3.71±0.15, respectively). Peptide 71 had too low affinity for use as a chromatographic ligand (pH 50% 4.57±0.13).
Note: The red color letters A and R shown that sense amphoteric amino acid serine is able to determine two antisense amino acids alanine and arginine. By the blue color letters shown the antisense sequences of the basic fragment of sense peptide AGYKPDEGKRGDA.
Synthesis peptide-affinity chromatographic gels and their properties determination
From the proposed modified peptides, the best results of hFg binding showed peptides No. 34 FFFFRIF (pH-50% = 3.41, Kd = 61 nM) and No. 39 FFYIRIV (pH-50% = 3.41, Kd = 73 nM) and No. 43 FFFIRIV (pH-50% = 3.41, Kd = 70 nM) [Table 2], hThr binding - No. 50 PFLRAWAI (pH-50% = 3.47, Kd = 94 nM), No. 55 RPIFGLLP (pH-50% = 3.44, Kd = 87 nM) [Table 3], hProThr binding - No. 69 VVIVPADR (pH-50% = 3.64, Kd = 118 nM).
The most effective peptides were affinity peptides to hFg (these were specially selected, taking into account the high molecular weight protein virus inactivation planned in-column), but each of the six listed can be used for chromatographic purification of Fg, Thr and ProThr as an affinity ligand. Their chromatographic properties were determined in a separate study. So far, we have obtained seven high affinity peptides that interact with the protein molecules at the different sites.
Considering the fact that binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix and ligand to facilitate efficient binding and create a more effective and better binding environment. The length of spacer arms is critical. Too short or too long arms may lead to failure of binding or even non-specific binding. In general, the spacer arms are used when coupling molecules less than 1000 Da. Due to these stipulations for coupling affinity peptides to the gel matrics we used tested before short spacer with 2-3 tryptophan including as for easy amount coupled peptide calculation as for the ligand leaching from the chromatographic matrics.
After peptides “sealing” the following chromatographic gels were obtained into the 3 mL ECOPLUS glass columns: *RIFWorkBeads 40/10000 with a peptide density 24.1 mg×mL-1 (~23.6 μmol×mL- 1 ), *RIV-WorkBeads 40/10000 (a peptide density 21.3 mg×mL-1, ~22.3 μmol×mL-1), *FIR- WorkBeads 40/10000 (a peptide density 22.8 mg×mL-1, ~24.2 μmol×mL-1), *PFL-WorkBeads 40/10000 (a peptide density 21.7 mg×mL-1, ~18.4 μmol×mL-1), *RPI-WorkBeads 40/10000 (a peptide density 19.5 mg×mL-1, ~21.4 μmol×mL-1) and *VVI-WorkBeads 40/1000 (a peptide density 22.8 mg×mL-1, ~21.7 μmol×mL-1).
The dynamic binding capacity (DBC) with enough residence time 14-20 min for all hFg and hProThr affinity gels was between 32-58 mg×mL-1. The enough residence time for coupling hFg for
*FIR-WorkBeads was 20 min while for *RIV-WorkBeads 18 min and for *RIF-WorkBeads 14 min only. The DBC for these gels was different too: *FIR-WorkBeads – 32 mg×mL-1, *RIVWorkBeads – 51 mg×mL-1, *RIF-WorkBeads – 58 mg×mL-1. *PFL- , *RPI- and VVI-WorkBeads DBCs were 44, 40 mg×mL-1 of hThr and 39 mg×mL-1 hProThr, respectively.
During the process determination of DBC with pure hFg or hThr or hProThr it was shown that target chromatographic fraction eluted with 20 mM citric acid-Na2HPO4 buffer, pH 2.6, contained all these proteins in native state. The hProThr was applicated on columns packed with *RIF/*RIV/*FIR– WorkBeads 40/10000 gel. Then the target proteins were washed with equilibration buffer from *RIF- and *RIV-columns, i.e. they were didn’t capture by these two gels affine to hThr. The *FIR– WorkBeads 40/10000 gel bound around 3-5% of applied hProThr which was eluted by citric acid- Na2HPO4 buffer, pH 2.6. So, it was decided to refuse from using this column in the further investigations (the reason was following: column assignment was hThr purification).
The testing *RIF- and *RIV-columns with H-fraction  and *PFL-, *RPI-, and *VVI-columns with L/M  fraction of the blood plasma we determined the following. The residence time 14 min
*RIF – FFFFRIF, *RIV – FFYIRIV, *FIR – FFFIRIV [for hFg]; *PFL – PFLRAWAI, *RPI – RPIFGLLP [for hThr], *VVI – VVIVPADR [for hProThr].
for *RIF- and 18 min *RIV-column was not enough – it was obtained less than 57 and 50%, respectively, from the applied total hFg with H-fraction. The residence time prolongation to 25 min increased the hFg binding by gels to the maximum of 92 and 80%, respectively. Given that 80% coupling is an average result for affinity *RIV-WorkBeads 40/10000 gel, it was decided to continue with *RIF-column only. The purity of the hFg eluted from the *RIF- WorkBeads 40/1000 gel was around 83%. No hydrolytic activity in this eluate was not found.
The residence time 15 min for coupling target proteins to *PFL-, *RPI- and *VVI- WorkBeads 40/10000 gels was completely enough and maximum: 89% and 83% from the total hThr and 84% from the total hProThr at the L/M blood plasma fraction. The purity of the hProThr eluted from the
*VVI- WorkBeads 40/10000 gel was around 88%. The hThr purity (it was added to this fraction extra) was 82% and 87% after elution from *PFL-, *RPI- WorkBeads 40/10000 gels, respectively. In this case it was receiving two discussion results: first chromatographic gel coupled higher quantity of the hThr (89%) but with amazing purity (82%) or smaller quantity (83%) with better purity (87%). During the discussion what variant is better the higher quantity receive an advantage with the idea that if greater purity of thrombin is required, one more chromatographic step can be applied.
So, in the next investigations *RIF-WorkBeads 40/10000 gel was used to obtain fibrinogen, *VVI- WorkBeads 40/10000 gel - to obtain prothrombin, and *PFL-WorkBeads 40/1000 gel - to obtain thrombin.
The DBC and temperature depended dynamic capacity (TDC) of the peptide-affinity gels, as expected, dropped sharply with increasing speed (>250 cm×h-1) or buffer temperature (>38- 40°C) (Figure 4). Any way the other conclusion from the results of the peptide-affinity gels properties determination was following: the direct process of hFg and hThr virus inactivation/elimination was possible to carry on under the temperature 35°C and buffer flow rate maximum 250 cm×h-1. The temperature around 40°C is already in the zone of the critical retention of the target proteins by chromatographic gel.
Unfortunately, *VVI-WorkBeads 40/10000 gel did not pass the test of both high speed and, accordingly, increased buffer temperature. Therefore, virus inactivation is carried over to the last steps of prothrombin hydrolytic activation and thrombin purification.
Human prothrombin purification and hydrolysis by immobilized a-specific thrombin-like enzyme
hProThr was enriched from blood plasma as L/M fraction  and partially purified by ion exchange chromatography on ANX by IEC. Last fraction after peptide affinity chromatography on VVIVPADR (*VVI)-WorkBeads gel acquired a purity of 82%. Its yield after last step of chromatography was 89%. The activation process with immobilized α-specific thrombin-like enzyme (Ancistron-B) previously isolated from the venom of Agkistrodon blomhoffii ussuriensis  shown the activation more than 90% prothrombin to active thrombin. Such method of thrombin activation, however, with immobilized thrombin, was proposed by Strukova . It is important that whole activated thrombin very fast was coupled by PFLRAWAI (*PFL) - WorkBeads gel on the column which was completely ready to be involved in the direct in-column virus inactivation/elimination process.
Direct in-column virus inactivation/elimination by S/D treatment during human fibrinogen and thrombin purification by peptide-affinity chromatography
hFg was partially purified from blood plasma by H-fraction separation  and finally purification on ECOPLUS column Ø50×120 mm (with cooling jacket) packed with 110 mL FFFFRIF(*RIF)- WorkBeads 40/10000 gel.
Of cause, just for model investigations of virus inactivation/ elimination the target proteins at first were eluted from purification column, infected by viruses and then applied on columns again.
Solvent/detergent (S/D) treatment of hFg and hThr was performed according to scheme shown earlier [39, 42,43] and described in the “Methods…” Our published dates and previous studies on model virus inactivation of streptokinase peptide (SK1–61), lysozyme, fibrino (geno) lytic enzyme , complex FVIII/vWF, ceruloplasmin  and immunoglobulins [42,43] dates have shown that proper virus removal during chromatographic purification depends on the nature of the S/D and their concentration, time and temperature of action on the protein adsorbed by the chromatographic gel. The most effective virus inactivation/elimination conditions were selected for the presented work, namely: TnBP/Triton X100 concentrations 1.0% and 2.5%, respectively; temperature 35-45°C and exposure to high T° for 5 hours (linear buffer gradient, which T° increases from 20 to 35-45°C during 1 hour; exposure to constant buffer T° 35-45°C during 3 hours; linear buffer gradient, which T° decreases from 35-45 to 20°C during 1 hour). In the case of hThr and hFg virus-inactivation the max temperature of the process was allowed 35 °C by critical zones defined TDC (Figure 4).
Results of the S/D virus inactivation of thrombin and fibrinogen directly in a chromatographic column depending from process temperature are presented in [Tables 4,5].
For further comparison we continue to evaluate the inactivation by three or two mentioned methods in the present study. In fact, we received full repetition of previously detected pattern: the method of the virus titer determining gave an adequate result according to target protein infectivity in the case of LEV and NLEV models [39, 42,43]. The LEV titer determination didn’t leave the hope to calculate process kinetics since the loss in virus infectivity due to the virus particles destruction and protein denaturation. The virus titration was more acceptable for NLEV infectivity mass balance determination - maximum 18% of the total infectivity was lost against 4-6% determined by RT-PCR. Unfortunately, the titration accuracy was very poor again (variants deviation more than 15-20% in the best cases) and didn’t allow definite conclusions.
Determination of viral proteins by IFA approximately simulated the virus titer and nucleic acids measurement but could in no way to confirm the presence or absence of infectivity. Quantification of viral nucleic acids by RT-PCR in target proteins and buffer fractions obtained during inactivation allowed to calculate the process mass balance within 95.4±4.9%. It means that stability of the virus nucleic acid (in most experiments not more than 3-4% of losses, rarely up to 6.5%) allows to calculate the process kinetic to assess what happens to model virus in the process of S/D treatment with small variant’s diversity not higher than 8-10%. In contrast, diversity of variants of virus titer of target proteins was more than 25-30%.
Note: 1: Gr, Gs, Gf – process stages, more explanation at the section “Materials and Methods - Total IgG peptides-affinity purification…”
Note 2: NA – nucleic acid
Note 3: bsl – value was below the method’s sensitivity level.
On the other hand, the virus titer determination compared with the determination of its nucleic acid in the eluate samples is very indicative in terms of resistance of the virus to the S/D mixture. For example, if the titer of most LEVs at the eluate (phase Gr) was almost on the limit of detection, and nucleic acids amount was 2-4 Log, it can mean only one thing: S/D mixture destroyed the virus envelope with loss of virulence and nucleic acids were released into solution. If we found equal amount of virus in the eluate by titer and nucleic acid, it means that LEVs almost were not damaged by S/D, but S/D caused dissociation of virus and target proteins and virus was washed out of the column. Thus, the process of virus elimination occurs with CPVNLEV and BEVNLEV (Table 4,5, stage Gr, Gf). Thus, it’s well known that LEVs destruction by S/D is 3-4 times more effective than that for NLEVs  that also supported by our data shown before [39, 42,43] and in the [Table 4]. BVDVLEV conduct itself is a bit asymmetric, namely: partially destroyed and partially undamaged washed out, which confirms its previously determined medium resistance to S/D treatment .
Note: 1: Gr, Gs, Gf – process stages, more explanation at the section “Materials and Methods - Total IgG peptides-affinity purification…”
Note 2: NA – nucleic acid
Note 3: bsl – value was below the method’s sensitivity level.
In the present investigation we demonstrated again that virus inactivation by S/D treatment of peptide/protein preparation directly in the chromatographic column cooperates two processes, namely: 1) destruction of a virus particles by solvent and/or detergent (mainly for LEVs with minor effect for NLEVs) and 2) dissociation and washing away of virus material which was associated with protein where the outcome that was successful both for LEVs and NLEVs. The fibrinogen and thrombin yield and purity were not measurably changed when the virusinactivation/elimination was introduced in the chromatographic purification process.
Target protein yield and activity after in-column treatment
Our results suggested that proteins “nailed” to chromatographic gel didn’t lose any activity and can be eluted from column with satisfied yield. Small (lower than 7%) fluctuations in the target protein yield and activity we explained by ordinary deviations in chromatographic process. No solvents and detergents were detected in target protein preparations.
With each new study, we are increasingly finding that biochemical process of protein-protein interaction occurs due to hydropathic and electrostatic contact between linear sequences built from amino acid partner couples. This emphasizes once again the non-randomness and the logical genetic program (sense and antisense sequences on each of the interaction partner proteins) of recognition, such as receptor recognition by the effector. Conversely, the interaction due to conformational convergence of partner amino acids seems random and very complicated for genetic programming. From the same point of view, it no longer seems speculative and fantastic that the interaction amino acids partner couples (or by J. Biro sense-antisense) are the same genetic code for proteins as the nucleotide pairs for nucleic acids.
Based on the above the definition at the MOG receptor RV the linear peptides of effective interaction with virus antisense peptides and more than 66-81% extraction of aRIgG from a mixture of IgG1-4 plasma donors suggests that MOG peptides 130RDHSYQEE137 or 232HRRLAGQF239 may be a key receptor sequence for RV. On the other hand, we hope that peptides QHGPLTDV/RDHHGTHE, which we developed in this study, could work no less effectively in an organism as blockers of the virus landing site on the cellular receptor. If the assumptions made in the following experiments are proven, it will pave the way for the development of reliable natural peptide safeguards pharmaceuticals not only against viruses and bacteria.
Authors express profound gratitude to the staff of pilot plant of scientific and manufacturing firm Shijir International Co. Ltd, Sukhbaatar sq., Bodi Tower building, Ulaanbaatar, Mongolia, for the opportunity to intervene in the real manufacturing processes and to test the represented method on the pilot plant in the Raining (Boroo) Valley of Mongolia.
This study was funded by Neutromics Ukraine TOV (Ukraine) and Shijir International LCC (Mongolia) in equal shares. The scientific data and practical results obtained by authors belong to Neutromics Ukraine TOV (Ukraine) and Shijir International LCC (Mongolia) and fixed by the agreement and the priority certificate for the granted patent of Ukraine. Both companies have allowed publication in the open press with the transfer of rights on the article to the appropriate publisher.
COMPLIANCE WITH ETHICAL STANDARDS CONFLICT OF INTEREST STATEMENT
Authors declare no conflict of interest. The study was performed at a time when authors were employees of Neutromics Ukraine TOV and working together at the biotechnology pilot plant in Raining (Boroo) Valley, Mongolia.
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by all authors too. The first draft of the manuscript was written by Serhiy
P. Havryliuk and Heorgii L. Volkov and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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