DECISION OF COUNCIL OF THE EURASIAN ECONOMIC COMMISSION

of July 4, 2023 No. 77

About modification of Rules of carrying out researches of biological medicines of the Eurasian Economic Union

According to article 6 of the Agreement on the single principles and rules of drug circulation within the Eurasian Economic Union of December 23, 2014, Item 88 of appendix No. 1 to the Regulations of work of the Eurasian economic commission approved by the Decision of the Supreme Eurasian economic council of December 23, 2014 No. 98, Council of the Eurasian economic commission solved:

1. Bring in the Rules of carrying out researches of biological medicines of the Eurasian Economic Union approved by the Decision of Council of the Eurasian economic commission of November 3, 2016 No. 89, of change according to appendix.

2. This Decision becomes effective after 180 calendar days from the date of its official publication.

Members of council of the Eurasian economic commission:

From the Republic of Armenia

M. Grigoryan

From the Republic of Belarus

I. Petrishenko

From the Republic of Kazakhstan

S. Zhumangarin

From the Kyrgyz Republic

A. Kasymaliyev

From the Russian Federation

A. Overchuk

 

Appendix

to the Decision of Council of the Eurasian economic commission of July 4, 2023 No. 77

The changes made to Rules of carrying out researches of biological medicines of the Eurasian Economic Union

1. In the offer the second paragraph two of Item 2.3.2.1 of Chapter 1 of the word "or in or others" shall be replaced with words "or in others".

2. In the offer the first the paragraph of the seventh subsection 1.1 of Section 1 of Chapter 5.4 of the word of "three R" (to reduce/optimize/replace "reduce/refine/replace") "shall be replaced with words" 3R (replacement, improvement and reducing (replacement, refinement, reduction)".

3. In the offer third paragraph one of subsection 4.3 of Section 4 of Chapter 15.2 of the word" (replacement, improvement, reducing) (replacement, refinement, reduction)" shall be replaced with words "(replacement, improvement and reducing (replacement, refinement, reduction))".

4. In the offer the fifth paragraph one of subsection 4.3 of Section 4 of Chapter 15.3 of the word" (reduce - refine - replace, reducing - improvement - replacement)" shall be replaced with words "(replacement, improvement and reducing (replacement, refinement, reduction))".

5. State Chapter 15.6 in the following edition:

"Chapter 15.6. Preclinical and clinical trials of biosimilar (biosimilar) medicines on the basis of heparins of low molecular weight

1. Introduction

Heparin is highly sulfonated and heterogeneous member of family of the glikozaminoglikanovy carbohydrates consisting of different disakharidny units. Treat the most widespread disugars: 2-O-sulfate - L-iduronic acid and 6-O-sulfate, N-sulfate and - D - glycosamine and IdoA (2S) - GlcNS (6S). Endogenous heparin is produced in granules of corpulent cages (labrocytes) and has the highest density of negative charge among all biological molecules.

Heparin oppresses formation of several serinovy proteases of system of fibrillation by means of antithrombin activation. The main role in linkng of heparin with antithrombin is played by the pentasakharidny sequence containing 3-O-sulfate glyukozaminovy remaining balance. After linkng with antithrombin heparin causes conformational change of its molecule that leads to activation of the area responsible for inhibition of the activated folding factors. Besides, heparin acts as the catalyst, connecting inhibitor and the activated serine proteases, such as thrombin (factors of II and IIA), factors of IXa and XIa. This property of heparin depends mainly on number of monosaccharides and provision of sulphatic groups in heparin molecule.

The heparin molecules containing less than 18 monosaccharides do not catalyze thrombin inhibition, but oppress action of factor of folding of Xa. After serinovy proteases begin to affect specific peptide communication of Arg-Ser (arginine-serine) of the active center of molecule of antithrombin, heparin increases reaction speed between thrombin and antithrombin at least in one thousand times, with formation of stable complex 1:1. Besides, the contribution to antitrombotichesky effect of heparin is made also by other mechanisms independent of antithrombin. Similar mechanisms include release endoteliy vessels of inhibitor of way of fabric factor, significant natural inhibitor of system of coagulation, interaction with cofactor of the II heparin, inhibition of prokoagulyantny effects of leukocytes, stimulation of fibrinolysis, and also influence on endoteliya of vessels (as mediated by receptors, and receptor - independent).

Low-molecular heparins receive from unfractionated heparin in the course of chemical or enzymatic depolymerization. As a result of process of depolymerization, low-molecular heparins mainly consist of the molecules containing less than 18 monosaccharides. This decrease in the size of molecules is followed by loss thrombin - the inhibiting activity and strengthening of inhibition of factor of Xa in comparison with unfractionated heparin.

All low-molecular heparins which are available now in the world market turn out from mucous membrane of intestines of pigs. Observed heterogeneity of composition of low-molecular heparins is caused by the nature of unfractionated heparin, and also technology of process of depolymerization (chemical or fermental).

The original low-molecular heparins which are available in the market differ on pharmacokinetic and the phracodynamic to properties. Because of complexity of determination of low-molecular heparins it is impossible to estimate pharmacokinetic properties of medicines on the basis of heparins at blood. However it is possible to estimate absorption and elimination of low-molecular heparins with use of phracodynamic of tests, the most important of whom is determination of anti-Xa and anti-IIa of activity.

All original low-molecular heparins have therapeutic indications for treatment of venous thromboses (treatment and prevention of venous thrombembolia, and some also have the additional indications connected with arterial thrombosis and sharp coronary syndrome and myocardial infarction.

The most frequent undesirable reaction in case of use of heparins is bleeding, and heparin - the induced thrombocytopenia of the II type which develops under the influence of formation of antibodies to new antigens which are formed when forming the complex containing heparin and trombotsitarny factor 4 (HPF4) belongs to the most serious undesirable reaction seldom observable. Linkng of antibodies with new antigen (the complex of heparin and trombotsitarny factor 4 (HPF4)) causes activation of platelets with the subsequent formation of trombogenny microaggregates of platelets. At patients with immuno-modulated heparin - the induced thrombocytopenia increases risk of arterial and venous tromboembolichesky complications (heparin - the induced thrombocytopenia and thrombosis). Though, in comparison with unfractionated heparin, the frequency of emergence of antibodies to complex of heparin and trombotsitarny factor 4 (HPF4) and developments heparin - the induced thrombocytopenia of the II type is lower against the background of use of unfractionated heparin, in case of purpose of medicines of low-molecular heparin it is regularly necessary to control content of platelets at all patients, and at those patients at whom thrombocytopenia or tromboembolichesky complications is revealed, determination of antibodies to the complex containing heparin and trombotsitarny factor 4 is necessary (HPF4).

This Chapter is applied in addition to requirements to establishment of similarity (similarity) of two medicines according to Chapter 15 of these rules. Requirements of this Chapter need to be considered together with other relevant requirements and acts, bodies of the Union in the field of drug circulation.

2. Scope of application

This medicine - specific Chapter contains preclinical and clinical requirements to confirmation of bioanalogousness (biosimilarity) of two medicines containing low-molecular heparin, at the same time it is necessary to consider the following specific aspects of quality within comparative development:

1) information on biological source of biosimilar (biosimilar) low-molecular heparin, production process of unfractionated heparin, the mode of depolymerization and the corresponding conditions for this process shall be available. Biosimilar (biosimilar) and original (reference) low-molecular heparins needs to be characterized and compared in details, using modern methods. Compliance to requirements of the Pharmacopoeia of the Union is the minimum standard requirement;

2) comparative analysis of physical and chemical and biological parameters of biosimilar (biosimilar) and original (reference) low-molecular heparin shall show high degree of comparability in the relation:

distributions of molecular weight and general chemical composition;

initial material (type of fabric and species) and depolymerization mode;

construction blocks of disaccharides, profiles of comparison of fragments and the sequences of the chosen not fragmented oligosaccharides;

biological and biochemical methods of the analysis.

3. Communication with other Chapters

In Chapters 15 - 15.2 these rules contain general instructions for development of biosimilar (biosimilar) medicines.

4. Preclinical researches

Before clinical trials preclinical researches shall be conducted. Preclinical researches have comparative character and are directed to detection of distinctions between effect of biosimilar (biosimilar) medicine and original (reference) low-molecular heparin, but not to studying of the pharmacological (clinical) response as such to medicine introduction. The choice of approach shall be completely reasonable in the overview of preclinical data.

Pharmakodinamika research

Research in vitro

For comparison of pharmakodinamichesky activity of similar medicine and original (reference) low-molecular heparin these several comparative biological analyses (based on modern data on clinically significant pharmakodinamichesky influence of low-molecular heparins, including at least assessment of anti-Xa and anti-IIa of activity) shall be provided. For measurement of activity it is necessary to use the standardized methods (for example, according to the Pharmacopoeia of the Union). Such data can be obtained in the course of studying of quality of medicine earlier.

Research in vivo

If in case of the characteristic of physical and chemical and biological properties with use of highly sensitive modern methods high degree of similarity biosimilar (biosimilar) and original (reference) medicines is established, carrying out the researches in vivo as parts of studying of comparability is not required. In other cases of in vivo comparative quantitative studying of pharmakodinamichesky activity biosimilar and original (reference) low-molecular heparins includes (biosimilar):

use of the pharmakodinamichesky in vivo model developed taking into account the most modern data on clinically significant pharmakodipamichesky effects of low-molecular heparins which includes at least assessment of anti-Xa and anti-IIa of activity, and also assessment of extent of release of inhibitor of way of fabric factor;

use of suitable model of venous, or arterial thrombosis on animals, according to clinical indications.

Toxicity research

As a rule, studying of toxicity in case of repeated introductions of one dose is not required.

In certain cases (for example if the composition of medicine includes new or poorly studied excipient), carrying out additional researches of toxicity is necessary.

Carrying out comparative researches of nonspecific toxicity only for assessment of the established distinctions as a part of impurity should not be carried out. The best strategy for decrease in the risks caused by impurity (for example, proteins) are data of content of impurity to minimum according to requirements of pharmacopoeian Article (monograph) of the Pharmacopoeia of the Union, and in case of absence in it - with requirements of pharmacopeias of state members.

If the immunogenicity is not estimated within clinical trial, the immunogene potential of biosimilar (biosimilar) and original (reference) low-molecular heparin needs to be compared in the corresponding preclinical researches. The predictive value of researches on animals concerning immunogenicity at the person usually is considered low. However to characterize physical and chemical properties of complexes of heparin and trombotsitarny factor 4 (HPF4), is allowed to conduct the researches in vitro. Modern methods of the analysis allow to determine the connecting affinity of low-molecular heparin to trombotsitarny factor 4, stekhiometriya, charge and the size of final complexes and change in the frequency of emergence of secondary structural elements (alpha spirals and beta sheets) in protein of trombotsitarny factor 4. In this regard these characteristics of complex heparin - trombotsitarny factor 4 shall be determined as function of concentration and the relation of content of low-molecular heparin and trombotsitarny factor 4. Besides, it is necessary to consider the possibility of studying of capability of complexes of heparin and trombotsitarny factor 4 (HPF4) to contact earlier created antibodies against complexes of heparin and trombotsitarny factor 4 (HPF4) and to activate platelets, with use of serum from patients about heparin - the induced thrombocytopenia of the II type.

Feasibility of any used method shall be appropriately proved. To show sufficient sensitivity of the corresponding methods, as positive control it is allowed to use unfractionated heparin (which has bigger immunogenicity in comparison with low-molecular heparins). Researches of pharmacological safety, reproductive toxicity are not obligatory in case of comparative research biosimilar (biosimilar) and original (reference) low-molecular heparins. Researches of local portability are not conducted if the composition of medicine does not include excipients for which there is no sufficient documentary confirmed experience of use in case of this way of introduction of medicine or it is experience of use is limited. If other researches in vivo were carried out, then assessment of local portability is allowed to be carried out as part of such researches.

5. Clinical trials

Research of pharmacokinetics and pharmakodinamika

Heterogeneity of low-molecular heparins does not allow to conduct regular research of pharmacokinetic properties. Instead comparison of pharmakodinamichesky activity, first of all concerning anti-Xa and anti-IIa, between biosimilar (biosimilar) and original (reference) low-molecular heparin shall be carried out. Besides, it is necessary to compare ratio of activity of anti-Xa and anti-IIa of factors, as well as activity of inhibitor of way of fabric factor. Studying of similarity (similarity) biosimilar (biosimilar) and original (reference) medicines on phracodynamic to indicators is carried out in randomized cross and it is desirable double blind research in two groups of healthy volunteers at hypodermic introduction of medicines. As hypodermic introduction of medicine allows to characterize both absorption, and elimination of low-molecular heparin, additional pharmacological researches for intravenous or intra arterial application (when applicable), are not required.

The chosen dose shall correspond to sensitive (abrupt) part of curve dependence "dose - effect". Equivalence limits also shall be determined and are properly proved in advance.

Efficiency research

Key evidence of comparable efficiency shall be obtained based on the similarity shown in physical and chemical, functional and pharmacological comparative researches. Performing separate clinical trial of comparative efficiency is not required.

Safety research

Biosimilar (biosimilar) and original (reference) low-molecular heparins shall show convincing similarity of physical and chemical and functional characteristics and phracodynamic of profiles. In that case it is expected that the side effects connected with it is excessive the expressed pharmacological effects (for example, bleeding), will be observed with similar frequencies. If in addition the profile of impurity and the nature of excipients of biosimilar (biosimilar) medicine does not cause uncertainty concerning their influence on safety and (or) immunogenicity, research of safety and (or) immunogenicity it is allowed not to carry out. In this case, it is necessary to carry out further studying of immunogene potential within the preclinical researches as it is described in Section 4 of this Chapter.

Otherwise comparative data on safety and (or) immunogenicity at patients shall be received before registration. In such clinical trial, assessment of immunogenicity shall include determination of antibodies to complex of heparin and trombotsitarny factor 4 and obligatory monitoring of number of platelets for the purpose of identification of cases heparin - the induced thrombocytopenia of the II type. Besides, extensive and clinically significant uncomprehensive bleedings shall be registered and described. At the same time it is necessary to use the approved and clinically relevant classification of bleedings. Optimum approach is approach in case of which the decision on the hemorrhagic phenomena is passed by the central committee of independent experts based on the blinded data.

6. Plan of pharmakonadzor

In case of registration of biosimilar (biosimilar) medicine it is necessary to provide the risk management plan according to Rules of practice of pharmakonadzor. The revealed and potential risks of use of original (reference) medicine according to the instruction for use of original (reference) medicine, and also action for tracking of parameters of safety of application for the corresponding indications of original (reference) medicine on which extrapolation of results of researches on other indications was carried out shall be reflected in the risk management plan. Besides, it is necessary to provide the risk management plan, connected with the serious undesirable phenomena in case of administration of drugs of low-molecular heparins (for example, heparin - the induced thrombocytopenia of the II type, anaphylactic and anaphylactoid reactions).

7. Extrapolation of indications

Confirmation of the biosimilarity based on physical and chemical and functional characteristics, phracodynamic profiles and, data of research of safety and (or) immunogenicity (where it is necessary), allows to extrapolate results to other ways of introduction and the indication to application registered at original (reference) medicine, when applicable and is properly proved.".

6. Add with Chapters 24 - 30 following contents:

"Chapter 24. Instructions for assessment of production process of medicines from plasma of blood of the person concerning risk of prion infection

1. General provisions

1. The alternative form of disease of Kreytstfeld - Jacoba (vBKYa) was for the first time registered in 1996. It is authentically determined that alternative disease of Kreytstfeld - Jacoba calls the causative agent of spongy encephalopathy of cattle which is transferred to the person. The most probable cause of disease consider consumption of products, kontaminirovanny transmissible agents of spongy encephalopathy of cattle.

2. Now incidence of alternative disease of Kreytstfeld - Jacoba does not give in to forecasting. Unlike sporadic odds alternative form of disease of Kreytstfeld - Jacoba, is characterized by extensive involvement in pathological process of limforetikulyarny system that causes probability of transfer of disease through the blood or medicines received from blood of the person who is in the incubatory period of disease. Prionny danger of components of blood is confirmed when conducting pilot studies on rodents. Also infectivity of leykotsitarny film of gray mouse lemur (Microcebus murinus) who was infected in vitro with the strain of spongy encephalopathy adapted for macaques was found.

3. When carrying out trans-species hemotransfusions it is determined that as disease the spongy encephalopathy caused in experimental conditions and natural infection of skreypa can be transmitted in case of blood transfusion at sheep. In other researches it is shown that blood transfusion of the person to wild and transgene mice and monkeys does not cause disease, but researches are not completed yet. In Great Britain 2 cases of alternative disease of Kreytstfeld - Jacoba which possible reason consider transfusion of erythrocytes of donors in which symptoms of alternative disease of Kreytstfeld - Jacoba are retrospectively found are registered. Today any case of this disease connected with introduction of medicines of blood is not registered (researches were conducted in groups of recipients of high risk, such as patients with hemophilia). Cumulative epidemiological data on alternative disease of Kreytstfeld - Jacoba is not enough for risk assessment of transfer of transmissible agents of spongy encephalopathy in case of use of medicines of blood. As precautionary measure plasma of donors from Great Britain is not used for production of medicines of blood as in Great Britain the greatest number of cases of spongy encephalopathy and considerably bigger, than in any other country was registered, quantity of cases of alternative disease of Kreytstfeld now - Jacoba.

2. Scope of application

4. Modern production technologies of medicines of blood provide considerable decrease in infectivity of the agents who are perhaps present at plasma of blood of the person.

5. Producers shall estimate contribution of stages of production process to decrease in infectivity with use of complementary evaluation methods.

6. This Chapter contains instructions for assessment of production process of medicines of blood concerning risk of transfer of alternative disease of Kreytstfeld - Jacoba.

3. Researches on assessment of procedures of cleaning of transmissible agents of spongy encephalopathy

3.1. General principles

7. The Instructions given in Chapter 3 of these rules extend also on the transmissible agents of spongy encephalopathy (TASE).

8. Material with content no more than 10% of certain transmissible agent is added to intermediate products. Researches on assessment of procedures of cleaning need to be conducted in the conditions of the reduced scale of the production process modeling real production process. Researches shall be conducted by the personnel having proper qualification in specially equipped laboratory with careful documentation of all procedures.

9. It is necessary to prove validity (suitability) of the reduced scale of production and to confirm the maximum compliance of level of cleaning in the reduced production process scale to full-scale process of industrial production.

10. Only production stages which are considered effective for inactivation and (or) removal of transmissible agents of spongy encephalopathy are subject to research.

11. Transmissible agents of spongy encephalopathy are steady against the majority of the physical and chemical procedures of inactivation which are traditionally used in production process of medicines of blood. With respect thereto the special attention needs to be given to such stages of removal (separation) as fractionation by ethanol at low temperatures, sedimentation by polyethyleneglycol, chromatography, deep filtering or nanofiltering. Validation researches of the specified stages need to be conducted not only concerning viruses, but also for assessment of removal of transmissible agents of spongy encephalopathy.

12. Researches of procedures of cleaning of transmissible agents of spongy encephalopathy more labor-consuming and expensive, in comparison with researches of traditional viruses therefore carrying out theoretical data analysis, confirming robustness of production process or use of the validirovanny in vitro methods is allowed.

13. Changes of parameters of production process exert impact on removal of transmissible agents of spongy encephalopathy. Therefore they need to be considered in case of development of design of validation researches of stages of separation.

14. Validation researches shall include assessment by in the vitro methods of separation of prionny particles in intermediate fractions. If the coefficient of decrease in one stage makes 1,0 of lg or less, then he is recognized insignificant, and is not considered in decrease total calculation of factor (coefficient). The caption of the transmissible agent of spongy encephalopathy in the material added when carrying out validation researches can be rather high for assessment of contribution of two and more stages. Therefore the design of validation researches shall allow to estimate decrease coefficients for each stage separately and at combinations in case of assessment of intermediate products. The received decrease coefficients for each stage are summed up for reasons for general contribution of all stages to process of removal of transmissible agents of spongy encephalopathy, including stages which made insignificant contribution.

15. Validation researches of several stages are suitable also in case of possible change of physical and chemical properties of the transmissible agent of spongy encephalopathy at certain production phase which can exert impact on efficiency of the following stage of removal (for example, when processing by detergent before filtering stage).

16. It is necessary to aim at carrying out validation researches of all stages of production process according to capability to delete transmissible agents of spongy encephalopathy.

17. However there are restrictions of researches connected with insufficient content (caption) of the transmissible agent of spongy encephalopathy in the added material for decrease assessment during two and more stages of production process.

18. The major factors requiring attention are:

the reduced scale of production (model);

the choice of the added transmissible agent of spongy encephalopathy;

the choice of evaluation method of quantitative content of the added transmissible agent of spongy encephalopathy;

choice of stages of production process;

interpretation of data and restriction of researches;

repeated researches on assessment of procedures of cleaning of transmissible agents of spongy encephalopathy;

sanitary cleaning of industrial equipment.

3.2. The reduced scale of production (model)

19. The principle of modeling of large-scale production in the reduced scale in vitro used for carrying out validation researches on inactivation and (or) elimination of viruses is applicable also concerning transmissible agents of spongy encephalopathy.

20. Producers shall represent the data on exit of ready-made product, quality and composition of medicine of blood or intermediate products received in the reduced scale comparable to real production process.

3.3. The choice of the added transmissible agent of spongy encephalopathy

21. In case of hit of the transmissible agent of spongy encephalopathy in organism of animals in experimental conditions the greatest number of the infecting particles is found in blood with presence of the activator in half of animals in plasma, and in other half of animals - in leykotsitarny film. Content of infectious particles in plasma is 10 000 times lower, than it is found out in tissue of brain of animals that determines it the most suitable material for carrying out validation researches. The maximum content of transmissible agents in the added material for pilot studies shall not exceed 10% of total amount.

22. The major factors influencing the choice of the added transmissible agent of spongy encephalopathy to which it is necessary to pay attention are:

the transmissible agent of spongy encephalopathy in the added material and type of animal from which it was received;

physical and chemical properties of the transmissible agent of spongy encephalopathy in the added material.

Type of animal and the transmissible agent of spongy encephalopathy in the added material

23. The choice of the transmissible agent of spongy encephalopathy in the added material depends on the following factors:

obtaining source;

availability of method of quantitative assessment;

similarity of the infecting properties with the potential activator which is present at plasma of blood of the person.

Receipt of material from patients with alternative disease of Kreyttsfeld - Jacoba is limited for ethical and other reasons and its use is optional. Use of tissues of brain of cattle is limited in connection with complexity of receipt of material of proper quality and complexity of assessment of quantitative content of transmissible agents of spongy encephalopathy.

24. For confirmation of removal during production process not only the added transmissible agents, but also the causative agent of alternative disease of Kreyttsfeld - Jacoba, it is reasonable to use laboratory strains of transmissible agents of spongy encephalopathy (for example, fasten, heritable disease of Kreyttsfeld - Jacoba, spongy encephalopathy of cattle or alternative disease of Kreyttsfeld - Jacoba).

25. As pathogenic properties and characteristics of strains differ, for researches it is necessary to use several strains. Methods of indication of the specified laboratory strains are available. It is necessary to represent reasons for the choice of the used laboratory strains.

Physical and chemical properties of the transmissible agent of spongy encephalopathy in the added material

26. Physical and chemical properties of transmissible agents of spongy encephalopathy are up to the end not studied in spite of the fact that their infecting capability is proved in pilot studies on animals. Tissue of brain of animals is recognized as acceptable source of accumulating of transmissible agents of spongy encephalopathy.

27. Now as the main four material types received from homogenate of brain of hamster are considered:

a) crude homogenate of brain. According to the published scientific data, the crude homogenates of tissue of brain contain the highest concentration of transmissible agents. The Gomogenost and availability of receipt of the specified material promotes its choice for validation researches. Presence at material of transmissible agents of the different sizes allows to use physical methods of their removal;

b) microsomal fraction of tissue of brain. Microsomal fractions receive by centrifuging of homogenates of tissue of brain with department and removal of agents of the large sizes. Transmissible agents on cellular membranes contain the remaining microsomal fractions. Level of their capability to cause disease below, than in the crude homogenates of brain, but it is sufficient for inclusion in validation researches;

c) kaveolopodobny domains of membrane (CLD). Material is received ultracentrifuging of lizirovanny homogenates of tissues of brain;

d) the purified protein of PrPSc. The purified protein of PrPSc is received consecutive extraction of homogenates of brain with the subsequent salt sedimentation and ultracentrifuging. The crude homogenate, microsomal fraction and kaveolopodobny domains of membrane (CLD) of tissue of brain can be used for validation researches of stage of sedimentation.

3.4. Choice of methods of quantitative analysis

28. Assessment of capability of transmissible agents to cause development of disease of spongy encephalopathy now is "gold standard" of similar researches. Presence of the transmissible agent at fabric or liquid is confirmed by development of neurologic disease in laboratory animal upon termination of the incubatory period, and also titration method to final point.

29. Duration of the incubatory period is also used for assessment of the infecting capability of the researched material together with infectivity determination by titration method to final point. Existence of specific and shtammovy distinctions of transfer of disease limits use of materials for infection.

For example, materials, the containing transmissible agents of sporadic disease of Kreyttsfeld - Jacoba of the person seldom use for infection of wild mice, at the same time they are suitable for transgene mice. The specified fact needs to be considered in case of the choice of the added transmissible agent.

30. Biological methods of the analysis are long in case of reproduction that is connected with duration of the incubatory period and possibility of receipt of results upon termination of 6 - 9 months of observations and clinical monitoring of the infected animals (for example, hamsters of the line 263K) and up to 15 - 18 months of not transgene mice.

31. Biological methods shall be reproduced in specially equipped rooms for animals with observance of rules of works with microorganisms of the corresponding group of pathogenicity.

32. Now standard in vitro test for determination of presence of transmissible agents and assessment of their quantitative content is absent. Several cellular lines (N2a, GT1) are unstable and can be infected with the separate laboratory strains of spongy encephalopathy adapted for mice, at the same time some strains, transfitsirovanny by PrP gene, can duplicate separate strains fasten.

33. The method of detection of protein PrPSc is alternative method of quantitative determination of transmissible agents of spongy encephalopathy. It is experimentally determined that transmissible agents of spongy encephalopathy consist of protein with conformational matrix of PrPSc which physical and chemical properties are still not established. The protein having pathogenic conformation (PrPSc) is rather steady against proteinase K (it can konformirovatsya in protease - steady protein of PrPres) and denaturant agents in different concentration, such as guanidine hydrochloride.

3.5. Choice of production stages

34. Considering resistance of transmissible agents of spongy encephalopathy to traditional methods of inactivation of viruses (for example, heat treatment), for research it is necessary to choose such production stages during which it is possible to expect partial removal or separation of transmissible agents. For carrying out validation researches of stage of processing by solvent-detergent and heat treatment are not chosen.

35. Such stages of production process as fractionation by ethanol, sedimentation, chromatography and filtering according to different researchers showed considerable efficiency of transmissible agents of spongy encephalopathy at a distance.

36. Producers shall estimate critically production processes according to provisions of this Chapter.

3.6. Interpretation of data and restriction on carrying out researches

37. To delete validation researches on assessment of capability of production process transmissible agents of spongy encephalopathy have the following restrictions:

a) process of modeling of full-scale production can be imperfect. It is difficult to simulate the technological stages based on physical separation in laboratory scale when carrying out validation researches. Especially it concerns fractionation stage ethanol which makes the significant contribution to removal of transmissible agents of spongy encephalopathy;

b) the general contribution to removal of transmissible agents of spongy encephalopathy needs to be estimated totally for at least two effective stages of production process, but such approach is not applicable in case of use of the different added agents;

c) preliminary processing can influence extent of cleaning of the added agent. For example, if the researched material is processed by detergent, it can pass the following stage, such as filtering, it is much easier, than raw;

d) methods of quantitative determination of the added agents long, labor-consuming and imperfect;

e) the transmissible agent of spongy encephalopathy and type of animal from which it was received determine the choice of method of quantitative determination. Despite the absence of proofs of significant influence of origin of the added material with the transmissible agent of spongy encephalopathy on removal stages, there is probability that extent of its removal depends on origin of the added material;

e) physical and chemical properties of the added transmissible agent of spongy encephalopathy can influence removal process. Now physical and chemical properties of transmissible agents of spongy encephalopathy are not determined. There are proofs that the different added materials with the membrane-bound transmissible agent of spongy encephalopathy were removed equally at all studied sedimentation stages. Unlike it, the added materials with not membrane-bound transmissible agent of spongy encephalopathy managed to be deleted only at certain stages of sedimentation;

g) content of transmissible agents in blood of experimental animal can be low, and in the added material the highest. There is assumption that removal of the added material takes place less effectively in case of low concentration of the transmissible agent, than in case of high;

h) assessment of processes of inactivation and (or) removal of viruses includes assessment of robustness of process (for example, studying of influence of changes of parameters of production process). Complexity of carrying out similar researches with transmissible agents of spongy encephalopathy is connected with lack of possibility of carrying out repeated researches.

38. With respect thereto, reliability of assessment of removal of the transmissible agent of spongy encephalopathy at any stage of production process is lower, than when studying possibility of removal of model virus.

3.7. Repeated assessment of extent of cleaning of transmissible agents of spongy encephalopathy

39. In case of entering of essential changes into production process carrying out repeated validation researches on assessment of extent of cleaning of transmissible agents of spongy encephalopathy perhaps will be required. At the same time use of new scientific developments in the field of research of methods of quantitative assessment of transmissible agents of in vitro other than the methods given in this Chapter is allowed.

3.8. Sanitary processing of the equipment

40. The samples containing the causative agent of spongy encephalopathy long time keep pathogenicity in the environment in connection with difficult giving in inactivation of infectivity of the activator. Use of the majority of traditional methods of deactivation for inactivation of prions (for example, use of alkylating agents and detergents) is insufficiently effective.

41. Only separate traditional methods of deactivation are rather effective (for example, soaking in bleach solution with concentration> = 2% or 1 - 2 H solution of sodium of hydroxide (NaOH) within 60 min., autoclaving at temperature of 134 - 138 °C in case of certain mode of maintenance of pressure and time of exposure).

42. The separate procedures recognized as reference methods or recommended to WHO are applied to processing of medical products or production wastes. Application of such procedures for production of biological medicines has number of restrictions. The majority of methods are quite tough on the impact and can destroy the majority of biological medicines, cause corrosion of production equipment or have insufficient efficiency concerning other infectious agents (for example, NaOH solution is considered inefficient in the relation dispute). Use of some methods of processing (for example, alkaline cleaners, proteases, etc.) is at stage of experimental assessment of suitability of their use for cleaning and deactivation.

43. Due to the resistance of causative agents of spongy encephalopathy to inactivation and capability to attachment to stainless steel and other materials, it is necessary to estimate contribution of procedures of sanitary processing and cleaning to processes of inactivation or removal of causative agents of spongy encephalopathy. It is necessary to estimate influence of procedures of sanitary processing and regeneration of hromatografichesky columns on decrease in infectivity of causative agents of spongy encephalopathy. The majority of processes of fractionation come to an end with deep filtering and removal of the used filter. If the specified stage is acknowledged effective, the risk of contamination of medicine any other infectious sources connected with the equipment also decreases.

44. Creation of model for studying of inactivation or removal of the transmissible agents of spongy encephalopathy attached to metal surfaces is complicated. In standard model the steel wire is immersed in sample, the containing transmissible agent of spongy encephalopathy, and implanted into brain of sensitive animal at which the disease develops afterwards.

45. Studying of influence of sanitary processing on decrease in infectivity on the specified model is possible in case of immersion of steel wire in samples with different cultivations of the transmissible agent before carrying out processing of the equipment.

46. In spite of the fact that experimental solutions for disinfection (such as NaOH solution) are used for other options of cleaning of production equipment, approach with direct data transfer of the new methods of disinfection of the medical equipment published when studying, kontaminirovanny prions is not applicable without additional researches concerning the equipment of industrial pharmaceutical production.

47. It is determined that processing 0, the M NaOH solution turns protein of PrPSc in protease - sensitive form both in solution and on metal surface. The provided results need to be confirmed in researches on assessment of infectivity of the activator.

48. Proceeding from provisions of Items 40 - the 47th this Chapter does not exist single reliable instructions for sanitary processing of the industrial equipment used for plasma conversion now.

49. All producers of medicines of blood shall analyze critically production processes according to provisions of this Chapter. The choice of measures for removal of transmissible agents of spongy encephalopathy needs to be performed directly for specific production process and modeling of the worst possible conditions.

Chapter 25. Instructions for assessment of immunogenicity of therapeutic proteins

1. General provisions

1. The amount of the proteins used as therapeutic medicines steadily grows.

For the purposes of this Chapter the concept which means the following is used:

"therapeutic protein" - the proteins, polypeptides and their derivatives received with use of recombinant or not recombinant systems of expression.

2. In general, the majority of undesirable reactions (side effects) in case of use of therapeutic proteins is connected with pharmacological effects of therapeutic proteins. One of exceptions is the capability of therapeutic proteins to induce the undesirable immune answer. The risk of immunogenicity varies between separate medicines and groups of medicines, on the one hand, and between certain patients and groups of patients, on the other hand. The list of the questions on immunogenicity which are subject to consideration in the summary of the module 2 registration files of medicine is provided in this Chapter. This summary reflects justification of approach on risk assessment concerning immunogenicity, confirms that the amount and type of researches of immunogenicity before registration of medicine and the program of the post-registration researches included in the risk management plan are made taking into account risk of immunogenicity and gravity of its potential or observed clinical effects.

3. From the regulatory point of view the prognostic importance of results of researches on animals for assessment of immunogenicity of biological medicine for the person is low in connection with distinction between immune system of the person and animals and inevitable origin at animals of the immune answer to proteins of the person. Development of adequate screening and confirmatory methods for studying of the immune answer to therapeutic protein is the key moment in immunogenicity assessment. Applicants shall show that methods of determination of antibodies are applicable for the proof of correlations of the revealed induced antibodies with clinical effects.

4. The purpose of researches of immunogenicity is detection of the immune answer to therapeutic protein and its influences on clinical effects. Thus, assessment of immunogenicity shall be based on the complex analysis immunological, pharmacokinetic, phracodynamic of indicators, and also the this clinical efficiency and safety of medicine. Questions of immunogenicity shall be reflected in the risk management plan.

5. The provisions of this Chapter including general approaches to studying of immunogenicity need to be adapted for the program of pharmaceutical development of specific type of biological medicine. According to Item 26 of Rules of registration and examination the applicant has the right to address for scientific consultation in authorized bodies (the expert organizations) of state members for such adaptation.

6. Therapeutic proteins are distinguished by immune system of the person. After recognition often on them the immune answer is created. This potentially dangerous immune answer is complex and, in addition to formation of antibodies to medicine, includes activation of T-cages and the answer of inborn system of immunity.

7. Effects of the immune answer to therapeutic protein vary from short-term tranzitorny emergence of antibodies without any clinically significant phenomena to serious, life-threatening conditions. Potential clinically significant effects of development of the undesirable immune answer include decrease in efficiency of therapeutic protein, heavy acute immune reactions, such as anaphylaxis, and for the therapeutic proteins applied as replacement therapy - cross reactivity with endogenous protein analog.

8. Multiple factors which are divided into the factors depending on the patient and factors mediated by disease or medicine exert impact on immunogenicity of therapeutic proteins. The factors depending on the patient can contribute to development of the immune answer in the subject, belong to them: genetic features (heritable predisposition), the preexisting immunity, the immune status, including therapy by immunomodulatory medicines. The mode of dosing and way of introduction of medicine belong to the factors connected with treatment. The factors mediated by medicine which influence probability of development of the immune answer include the characteristics of medicine caused by production process, composition of medicine and its stability.

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