THE CONTRIBUTIONS TO IMPLEMENTATION GOOD PRACTICES … · Validation of the process for removing ... Clobetasol ointment manufacturing method and process ... THE CONTRIBUTIONS TO

UNIVERSITY OF MEDICINE AND PHARMACY “GRIGORE T. POPA” IA ŞI FACULTY OF PHARMACY

THE CONTRIBUTIONS TO IMPLEMENTATION

GOOD PRACTICES IN THE TECHNOLOGY OF MANUFACTURE OF

ANTI-INFLAMMATORY DRUGS

Ph.D THESIS ABSTRACT

Scientific adviser PROF. UNIV. DR. MIHAI IOAN LAZ ĂR

Doctor’s degrees candidate

COZMOLICI (BALMO Ş) MIHAELA

IAŞI 2014

CONTENTS

1

CONTENTS INTRODUCTION 7

Reasons for choosing the theme 9

General presentation of the thesis 10

I. CURRENT STATE OF KNOWLEDGE 13

CHAPTER 1 The necessity to know the regulations on good manufacturing practice for medicinal products for human use

13

1.1. The general principles of the Good Manufacturing Practice for medicinal products for human use

13

1.2. Basic concepts relating to quality assurance, good manufacturing practice, quality control and risk management in quality for manufacturing drugs

13

1.2.1. Quality assurance 13

1.2.2. Good manufacturing practice for medicinal products (GMP) 14

1.2.3. Quality control 15

1.2.4. Quality Risk Management in the Pharmaceutical Industry 16

1.2.4.1. Principles of risk management in quality according to GMP 16

1.2.4.2. Steps in Implementing risk management system in quality 17

1.2.4.3 Application of risk management quality in the pharmaceutical industry

19

1.2.5. Conclusions 21

CHAPTER 2 Data from the literature to obtain of two drugs on the same production line

23

2.1. Manufacture drugs in accordance with good manufacturing practice 23

2.1.1. Manufacturing semisolid topical forms according to the rules of good manufacturing practice

23

2.1.2. Prevent cross-contamination in manufacturing 24

2.1.3. Validation of manufacturing processes 25

2.2. Premises and equipment for pharmaceutical production 26

2.2.1. Premises and production equipment 26

2.2.2. Clean rooms 27

2.2.3. Validation of the process for removing impurities and residue from the manufacturing equipment

27

2.2.4. The establishment of accepting the validity of the process of removing the residues and impurities from the production facilities

34

2.3. Anti-inflammatory drugs 37

2.3.1. Anti-inflammatory drugs cortisone derivatives 38

2.3.2. Anti-inflammatory drugs non-derivatives of cortisone (nonsteroidal) 38

2.3.3. The pharmaceutical form of the two anti-inflammatory drugs 40

II PERSONAL CONTRIBUTIONS 43

CHAPTER 3 43

Development of methods for the analysis of phenylbutazone 43

3.1. Introduction 43

CONTENTS

2

3.2. Material and methods 43

3.2.1. The control parameters and admissibility criteria 43

3.2.2. Identification of phenylbutazone - IR method 43

3.2.2.1. Reagents and solvents 43

3.2.2.2. Equipment and experimental conditions 43

3.2.2.3. Method of analysis 44

3.2.2.4. Results and Discussion 44

3.2.3. Determination of related substances of phenylbutazone - HPLC method 44




3.2.3.4. Results 46

3.2.3.5. Discussion 49

3.2.4. Quantitative determination of phenylbutazone - HPLC method 50




3.2.4.4. Results 51


3.2.5. Determination of residual solvents - methanol - GC method 52




3.2.5.4. Results 54


3.2.6. Validation of analytical methods 55

3.3. Conclusions 56

CHAPTER 4 57

Analysis of the pharmaceutical form with phenylbutazone 57




4.2.2. Determination of pH - Potentiometric method 58

4.2.2.1. Solvents 58

4.2.2.2. Aparatura de laborator 58



4.2.3. Determination of related substances of phenylbutazone - HPLC method 58




4.2.3.4. Results 59


4.2.4 Identification and quantitative determination of preservative substances - HPLC method

62




CONTENTS

3

4.2.4.4. Results 63


4.2.5. Identification and determination of phenylbutazone - HPLC method 65




4.2.5.4. Results 66


4.2.6. Determination of menthol and camphor - GC method 67




4.2.6.4. Results 68


4.2.7. Microbial contamination 70

4.2.7.1. Reagents and culture media 70

4.2.7.2. Laboratory equipment 70



4.2.8. Validation procedure of analytical methods 74

4.3. Conclusions 74

CHAPTER 5 77

Development of methods for the analysis of clobetasol propionate 77




5.2.2. Identification of clobetasol propionate - IR spectrometry method 78





5.2.3. Determination of related substances of clobetasol propionate-HPLC method

78




5.2.3.4. Results 79


5.2.4. Quantitative determination of clobetasol propionate - HPLC method 84




5.2.4.4. Results 85


5.2.5. Determination of residual solvents clobetasol propionate - GC method 86




CONTENTS

4

5.2.5.4. Results 88



5.3. Conclusions 93

CHAPTER 6 95

Analysis of the pharmaceutical form of clobetasol propionate 95



6.2.1. The control parameters and admissibility criteria for Clobetasol ointment 95

6.2.2. Determination of related substances of clobetasol propionate-HPLC method

96




6.2.2.4. Results 97


6.2.3. Identification and quantification of clobetasol propionate - HPLC method 99




6.2.3.4. Results 100


6.2.4 Microbial contamination 102

6.2.4.1. Reagents and culture media 102





6.3. Conclusions 106

CHAPTER 7 107

Manufacturing technology of the two anti-inflammatory drugs on the same production line

107



7.2.1. Production facilities 107

7.2.2. Production equipments 108

7.2.2.1. Semisolid preparation equipment type Fryma 108

7.2.2.2. Filling machine in aluminum tubes type CO.MA.DIS SPA 109

7.2.3. Raw materials and packaging 109

7.2.4. Manufacturing methods and process controls during manufacturing process technology

111

7.2.4.1. Clobetasol ointment manufacturing method and process controls 111

7.2.4.2. Phenylbutazone cream manufacturing method and process controls

113

7.2.4.3. Conditioning primary, secondary and batch release 114

7.2.5. Validation process of manufacturing methods 115

7.2.5.1. Validation parameters of the manufacturing process of the drug Clobetasol ointment

115

CONTENTS

5

7.2.5.2. Validation parameters of the manufacturing process of the drug Phenylbutazone cream

116

7.3. Results 116

7.3.1. Results of the validation of the manufacturing process of the drug Clobetasol ointment

116

7.3.2. Results of the validation of the manufacturing process of the drug Phenylbutazone cream

120

7.4. Discussion 124

7.4.1. Evaluation and statistical interpretation of the results of the validation of the manufacturing process for Clobetasol ointment

124

7.4.2. Evaluation and statistical interpretation of the results of the validation of the manufacturing process for Phenylbutazone cream

126

7.5. Conclusions 127

CHAPTER 8 129

Validation of the process for removing impurities and residue from the manufacturing equipment for manufacturing the two products

129


8.2. Selection of anti-inflammatory drugs with different pharmacological potency 129

8.2.1 Material and methods 130

8.2.1.1. Selection of potent active after the validation process was performed to remove residues and impurities

132

8.2.1.2. Selecting low active drug after the validation process was performed to remove residues and impurities

133

8.2.2. Results 134

8.2.3. Discussion 135


8.3. Methods of removing residue and contaminants from the production equipment after the manufacture of the two products

136

8.3.1. Material and methods 136

8.3.1.1. Justification for the choice of the method to remove residues and impurities from the production equipment

136

8.3.1.2. Agenţii de îndepărtare a reziduurilor şi impurităţilor de pe echipamentele de producţie

137

8.3.1.3. Method of removing of residues and contaminants from the production equipment

139

8.3.2. Results and Discussion 141


8.4. The sampling after removing residues and impurities from the production equipment

141

8.4.1. Material and methods 142

8.4.1.1. Sampling methods 142

8.4.1.2. Sampling tools 143

8.4.1.3. Establish sampling points 143

8.4.1.4. The sampling 146

8.4.2. Results 146

8.4.3. Discussion 146


Chapter 9 149

CONTENTS

6

Analysis of samples after the removal process of residues and impurities from the production equipment

149



9.2.1. The control parameters and limits acceptance for validating removal process of residues and impurities

149

9.2.2. Quantitative determination of phenylbutazone trace-HPLC method 150





9.2.3. Quantitative determination of clobetasol propionate trace-HPLC method 156





9.2.4. Quantitative determination of traces of disinfectant agent P3-cosa DES 163





9.2.5. Determinations for microbial contamination 166

9.2.5.1. Culture media 166




9.3. Results 175

9.3.1. Results of the analyzes after removing residues and impurities from production equipment after manufactured the Phenylbutazone cream product

175

9.3.2. Results of the analyzes after removing the residues and impurities from production equipment after manufactured the Clobetasol ointment product

181

9.4. Conclusion 187

III GENERAL CONCLUSIONS 189

BIBLIOGRAPHY 192

Keywords: pharmaceutical form, process technology, strong active, quality risk management, validation process, residues and impurities.

INTRODUCTION

7

INTRODUCTION

In Romania, all drug manufacturers must have authorization manufacturing and GMP

certificate issued by the National Agency for Medicines and Medical Devices (ANMDM) of the Ministry of Health. These authorizations shall be granted only if the manufacturers have implemented and follow the rules of Good Manufacturing Practice (GMP). Drugs prepared industrially must be authorized for placing on the market (APP) by ANMDM based on a very detailed documentation on the quality, effectiveness and their safety.

Due to the fact that drugs are those products that are intended to prevent, treat, cure, or diagnosis of disease, quality, efficacy and safety is a very important issue. In this area low quality deficiencies can bring great damage in terms of their use. Because of this there has been a global concern to create a set of basic rules for drug industry that is widely accepted and respected. For this reason, this branch of industry is governed by a series of laws and rules. The basic rules governing this branch of industry are rules of Good Manufacturing Practice (GMP).

The first version of a guide to good manufacturing practice (GMP) appeared in 1938

in the U.S., issued by the Food and Drug Administration (FDA) in Section 501.B of the law on food, drugs and cosmetics - 21USC351. GMP rules issued by the World Health Organization (WHO) are accepted and used in more than 100 countries worldwide.

Today, these rules are similar to everyone and when speaking of GMP rules, all use similar language and principles could say identical.

In 1997 appeared the first version of the rules of good manufacturing practice in Romania, but were first approved in 1999 by the Scientific Council Decision No 17/09.12.1999 of ANMDM site. And till 31.12.2003 all manufacturers must implement GMP and thus to obtain the manufacturing authorization issued by ANMDM. This guide has been revised regularly so: in October 2001, June 2003, September 2006, March 2009, September 2010 and March 2012.

Quality management system in pharmaceutical development, manufacture and control

of medicinal products in Romania, is held to high standards aligned with EU standards. These standards are regulated by the Ministry of Health - National Agency for Medicines and Medical Devices, by approving the principles and guidelines of good manufacturing practice for medicinal products according to Ministry of Health Order no. 905/2006 which transposed into Romanian legislation Commission Directive 2003/94/EC and Guidance on Good Manufacturing Practice (GMP) in detail and explains the principles and guidelines of GMP.

At present, the latest guidelines on good manufacturing practice for medicinal products for human use in force is approved in March 2012 according to the Decision of the Scientific Council of the National Agency for Medicines and Medical Devices (ANMDM) no. 5 of 07.03.2012.

It is recognized that there are acceptable methods other than those described in

GMP are able to perform quality assurance principles. This guide is not intended to impose any restriction on the development of any new

concepts or technologies that have been validated and provides a level of Quality Assurance at least equivalent to that provided in this Guide.

INTRODUCTION

8

In line with good manufacturing practice, Chapter 3 - Premises and equipment must be located, designed, constructed, adapted and maintained to correspond the operations to be performed. Location and their design should minimize the risk of errors and permit effective cleaning and maintenance in order to avoid cross-contamination, the deposition of dust or dirt and, in general, any adverse effects on product quality.

To minimize the risk of cross-contamination due to serious medical accidents, manufacture of certain medications such as certain antibiotics, certain hormones, certain cytotoxics, certain highly active drugs and non-drug products should not be made in the same facilities. For these products, in exceptional cases, the principle of campaign activity in the same facilities can be accepted provided that special precautions be taken and the necessary validations are made. REASONS FOR CHOOSING THE THEME

The holder of a manufacturing authorization must manufacture drugs: • suitable for the purpose for which they were designed; • comply with the requirements set out in the marketing authorization; • not expose patients to any risk due to deficiencies on safety, quality and effectiveness.

To achieve this objective safely on quality manufacturing plant must have a quality assurance system properly designed and implemented, which includes concepts of good manufacturing practice and quality control.

I chose this topic for the PhD to demonstrate that the same technological production line can produce two consecutive semi-topical nonsteroidal antiinflammatory different pharmacological activity without endangering safety, quality and effectiveness, without risk of cross-contamination ensuring the quality of manufacture of the two drugs.

Demonstration of this and its implementation can bring the following benefits: - Manufacture on the same production line drugs different potency; - Saving outlets designed, built and equipped for production according to the rules of

good manufacturing practice; - Lowering the cost of production of the drug that keeps ȋn while ȋşi safety, quality and

efficacy.

GENERAL PRESENTATION OF THE THESIS

According to GMP, a manufacturer could produce a potent active on the same production line that manufactures drugs that do not contain highly active, such as antibiotics, corticosteroids strong, certain hormones, etc..

For economic reasons, it is not cost effective for a manufacturer to dedicate a separate production line to manufacture only highly active drug. In this case, the manufacturer should produce potent active on the same production line on the principle of campaign activity, but under special precautions and perform all validations necessary.

To be accepted and approved this, the manufacturer must implement a quality management system in the manufacture of drugs, so to show that in the same production departments on the same production line can produce two different potent drugs (highly active and low active) while ensuring the quality of the activities, the manufacturing process involved and the finished product, so that the finished products are fit for the purpose for which it was designed, will be in accordance with the requirements provided the marketing authorization and will not expose patients to any risk due to deficiencies on safety, quality and effectiveness.

INTRODUCTION

9

The real chances of success of such a project must be supported by extensive scientific knowledge, material resources available and practical experience in the field. All this will allow better organization of scientific activities and ultimately achieve favorable results.

The research results can then practical application for the implementation of quality management in the production of drugs from therapeutic classes with different potency on the same production line and can be extrapolated to other drugs belonging to the same class manufacturing pharmaceuticals.

To prove this we need a deep and thorough research involves a number of activities related to quality assurance, quality control and manufacturing processes drugs. These activities are:

- Establishing manufacturing processes for the two drugs; - Validation of manufacturing processes; - Establishing control methodology for the two drugs; - Establishing the parameters and limits of admissibility; - Validation control methodology; - Establishing procedures to remove residues and impurities after highly active drug

manufacture; - Validation process for removing residues and impurities; - Assessing the quality of the two drugs. Proper completion of these steps and demonstrate the effectiveness of each step

leading to quality assurance for production the two drugs with different potencies on the same production line.

For compliance with good manufacturing practice, every manufacturer must carry out and to thoroughly document the activities mentioned above. But according to the purpose and objective of the proposed implementation of the quality management system of validation of manufacturing processes, control methodology, process residues and impurities removal and depending on the results obtained it can be demonstrated that it is performed quality assurance in manufacturing on the same production line of two drugs with different potencies, while ensuring quality activities, processes, and last but not least, ensure the quality of the finished product.

An important role in this activities it have the validation process to remove residues and impurities from the production equipment.

Through validation process residues and impurities removal means: - Validation process to remove residues and impurities from the workplace and - Validation process for removing impurities and residue from the manufacturing

equipment. Depending on the admissibility limits and according to the results obtained after

performing the validation process residues and impurities removal will be considered acceptable or not making the two drugs on the same production line production.

Steps involved in validation process residues and impurities removal, responsibilities and how the actual work must be well established in a validation protocol.

After completing the validation process residues and impurities removal, all data record are written in a validation report. Report validation process to remove residues and impurities contain final conclusions and information on monitoring of residues and contaminants removal over a year.

During the removal of residues and contaminants should consider several aspects that are very important:

- Personnel - that can not be validated, it takes a very good training and effective supervision;

INTRODUCTION

10

- Microbiological aspects - analyzing the risk of contamination; a contaminated product can not be recovered, it is destroyed, which means huge economic loss;

- Methods of sampling - rinsing method, deleting - depending on sampling locations; - Establishing sampling places - places less accessible; - Establishing the parameters that must be analyzed and evaluated; - Setting limits eligibility.

In all the official standards validation process residues and impurities removal is the

requirement: - "Particular Attention Should Be accorded to the validation of ... cleaning Procedures"

(WHO); - "Cleaning Validation Should Be Performed in order to confirm the Effectiveness of

the cleaning procedure" (PIC / S); - "The time SHOULD support the Conclusion That residues have been Reduced to year

'acceptable' level '(FDA), it is not clear described the strategy of the validity process for removing the residues

and impurities. The strategy of validation process for removal residues and impurities and it sets each

manufacturer in part by drugs that are produced on each line technology. Finally, each manufacturer has its merits in its quality assurance for the manufacture of safe medicines.

Conclusions

Manufacture of certain highly active drugs can achieve the same facilities based

activities and processes documented scientific results proving effectiveness and validation. If the manufacturer, the aim and purpose of the proposed quality management system

implementation in the validation process of removal of residues and contaminants, control methodologies and manufacturing processes shows that the production of a highly active drug does not endanger the production of another drug in the same production line, then he can use the same facilities for its entire portfolio of drugs that are manufactured by the same manufacturing technology.

Thus, it combines in a very pleasant professionalism in conducting the business of manufacturing quality medicines with the spirit of business in this area.

THE CONTRIBUTIONS TO IMPLEMENTATION GOOD PRACTICES IN THE TECHNOLOGY OF MANUFACTURE OF ANTI-INFLAMMATORY DRUGS

11

CURRENT STATE OF KNOWLEDGE

CHAPTER 1 THE NECESSITY TO KNOW THE REGULATIONS ON GOOD MANUF ACTURING PRACTICE FOR MEDICINAL PRODUCTS FOR HUMAN USE 1.1. The general principles of the Good Manufacturing Practice for medicinal products

for human use

According to the guideline of Good Manufacturing Practice (GMP), the holder of a manufacturing authorization must manufacture drugs:

- suitable for the purpose for which they were designed; - comply with the requirements set out in the marketing authorization; - not expose patients to any risk due to deficiencies on safety, quality and

effectiveness. To achieve this objective safely on quality manufacturing plant must have a quality

assurance system properly designed and implemented, which includes concepts of Good Manufacturing Practice (GMP), quality control and risk management in quality [1, ch. 1].

In line with good manufacturing practice (GMP) - [1, chapter 1] • the basic concepts related to quality assurance, good manufacturing practice, quality

control and risk management in quality are interrelated. These are described below to highlight the relationship between them and their

fundamental importance of the manufacture and control of medicinal products. 1.2. Basic concepts relating to quality assurance, good manufacturing practice, quality

control and risk management in quality for manufacturing drugs Quality assurance

Quality assurance is a broad concept that encompasses all subjects, individually or collectively, influence the quality of a product; is a set of measures aimed at getting products whose quality suitable for the purpose for which they were designed. Quality Assurance incorporating Good Manufacturing Practice. [1, chapter 1]

Good manufacturing practice for medicinal products (GMP)

Good Manufacturing Practice (GMP) is that part of quality assurance system which ensures that products are consistently produced and controlled to the quality standards appropriate after their intended use and as required by the marketing authorization or product specification. [1, Chapter .1] Quality control

Quality control is the part of GMP dealing with sampling, specifications, testing procedures and organization, documentation and release to ensure that the necessary and relevant tests were performed, the materials are not released for use and finished products are released for sale or distribution until their quality has not been declared as appropriate. [1, chapter 1]


12

Quality Risk Management in the Pharmaceutical Industry

Risk management in quality is a systematic process to assess, control, communication and review of risks to the quality of the product. It can be applied both prospectively and retrospectively. [1, ch. 1, Appendix 20]

In Romania, introduced Annex 20 GMP guide, to align with EU legislation, with new rules of Good Manufacturing Practice approved under the judgment of the Scientific Council of the National Agency for Medicines and Medical Devices (ANMDM), no. 23 of 03.09.2010. Although currently this Schedule 20 is optional, all operators in the pharmaceutical industry should implement new rules on risk management in quality.

The GMP guide says: "However, it is not intended as Annex 20, in itself, lead to new regulatory requirements, thereby providing only an inventory of methods and international instruments related to risk management in quality, while providing a list of possible applications which are available for manufacturers. "

Application of risk management for facilities / equipment / utilities means completing certain mandatory steps, without which it can not pass the drug manufacturing processes. Each manufacturer should draw up a Validation Master Plan which are scheduled qualifications for facilities, utilities, manufacturing equipment and control, followed by validation processes for obtaining purified water, purified air, the compressed air to remove the validation process residues and impurities from the premises and production equipment, validation of physico-chemical and microbiological analysis.

Risk management in the pharmaceutical industry in quality integrated quality system is a process that supports decisions on scientific and practical by competent staff and appointed by the company. Proper use of quality management in the pharmaceutical industry does not negate the obligation to comply with the requirements of GMP and other regulatory requirements. However, an efficient risk management in quality can facilitate better decisions and more informed, can provide greater regulatory authorities regarding the company's ability to address possible risks and possible influence on the extent and level of supervision direct from the regulator. In addition, quality risk management can facilitate the more efficient use of resources by all parties. Conclusions

Risk management in quality must be integrated into all operations / processes / existing activities, mainly for the specific and critical for the pharmaceutical industry and should be properly documented.

Risk management in quality as documented and applied correctly can lead to fewer non-compliance, lower costs of interventions and repairs to streamline specific processes (manufacturing, inspection, cleaning, supply, storage, distribution, maintenance, etc..) To increase production efficiency, increased staff skills, increasing performance indicators, increasing product quality, etc..

Training staff in the pharmaceutical industry on risk management processes in quality ensures a better understanding of decision making processes and gives confidence in the quality of risk management results.

Certainly, after a year of the implementation and application of risk management in the quality of the results will be quite noticeable, and staff involvement will be much higher.


13

CHAPTER 2 DATA FROM THE LITERATURE TO OBTAIN OF TWO DRUGS ON THE SAME PRODUCTION LINE 2.1. Manufacture drugs in accordance with good manufacturing practice

Drug manufacturing operations are carried out in accordance with clearly defined

procedures must conform to the principles of good manufacturing practice to get quality products required and must be in accordance with authorizations of manufacturing and marketing.

Before starting any processing operations must be taken to ensure that the manufacturing and equipment are clean; any raw material, product, residue or document that is required to be removed. Intermediate and bulk products should be kept in appropriate conditions.

Critical processes should be validated. Any necessary process control and environmental control should be performed and recorded. Any significant deviation from the expected yield should be recorded and investigated. Manufacturing semisolid topical forms according to the rules of good manufacturing practice

Topical semi-solid forms (ointments, creams, gels) are particularly susceptible to microbial contamination and other contamination during production. Therefore, special measures must be taken to prevent any contamination.

The use of closed systems manufacturing and transfer is recommended in order to protect the product from contamination. Manufacturing areas where products or containers clean, covered, exposed should normally be effectively ventilated with filtered air.

Tanks, containers, pipes and pumps have to be designed and installed in such a way that they can be easily cleaned and, if necessary, cleaned. In particular, the project equipment must include a minimum of dead spaces or places where they may accumulate residues that contribute to microbial proliferation.

Chemical and microbiological quality of water used in production must be specified and controlled. The quality of materials received in bulk containers should be checked before they are transferred to storage tanks of bulk products.

Materials likely to yield fibers or other contaminants (eg cardboard or wooden pallets) must not enter areas where products or clean containers are exposed.

You must maintain the homogeneity of mixtures, suspensions etc.. during filling. Mixing and filling processes should be validated. Particular care should be taken at the beginning of the filling process, after interruptions and at the end of the process to ensure the homogeneity. Prevent cross-contamination in manufacturing

Contamination of raw material or product with another material or product must be avoided. The risk of accidental cross-contamination occurred originates uncontrolled release of dust, gases, vapors, aerosols or organisms from materials and products in production of residues from clothing, equipment and operators. The significance of the risk varies depending on the type of contaminant and product contamination. Among the most hazardous contaminants are strongly sensitizing materials, biological preparations containing living organisms, certain hormones, cytotoxics, and other highly active substances. Products that avoid contamination is particularly important are the injectable and high doses and / or long time.


14

Validation of manufacturing processes Validation studies should reinforce Good Manufacturing Practice and be conducted in accordance with defined procedures. Requirements and principles of the validation process relates to the manufacture of pharmaceutical dosage forms, comprising the initial validation of processes, the validation process later modified and re-validation. 2.2. Premises and equipment for pharmaceutical production Premises and production equipment Premises and manufacturing equipment shall be:

• Place the technological flow to ensure the activities of production in chronological order; • designed, constructed and adapted to the technology of each manufacturer individually; • maintained on a schedule well documented and justified.

They should ensure that risks are reduced to the maximum error and cleaning them is effective. This prevents cross-contamination, dust or dirt deposit and final product quality assurance is guaranteed [1, Chapter 3]. Cross-contamination must be avoided by appropriate technical or organizational measures, such as:

- Production in campaign, or in separate areas (off-time), followed by a proper cleaning; - Use of procedures to remove residues and impurities and efficient decontamination

known, insufficient cleaning of the equipment is a common source of cross-contamination; [1, Chapter 3].

Manufacture of other medications such as certain antibiotics, certain hormones, certain chemotherapy, certain highly active drugs and non-drug products should not be made in the same facilities. For these products, in exceptional cases, the principle of campaign activity in the same facilities can be accepted provided that special precautions be taken and the necessary validations are made. [1, Chapter 3] Clean rooms Create a free microclimate harmful impurities technique is now done "Clean Rooms" (English: clean rooms, German: reinraum) devised since 1960. As defined by the standards in force, a clean room is an implement which provides a controlled environment space from the point of view of the contamination with particles, temperature, humidity, air pressure and velocity. Clean rooms are designed to ensure a work environment free of aerosol technology or to maintain and control a particular concentration of them. The purity of the clean air required by the conventional class is defined cleaning, which is the maximum number of particles (aerosols) the size of 0,5 mm or more admitted per unit of volume of air (cubic foot, or m3 per liter ). In a clean, ultra-filtered air while eliminating the bulk of aerosol contamination problems. To obtain performance drugs have been introduced nationally and internationally a range of regulations, norms and standards of quality, purity and safety in the manufacture of drugs. In achieving these requirements of modern pharmaceutical industry, a basic role they have clean rooms must provide a number of basic conditions that:

• sterile injectable solutions during processing, packaging; • avoid contamination of product formulations foreign agents; • Protection of operating personnel in handling of assets.


15

Validation of the process for removing impurities and residue from the manufacturing equipment

The validation process for removing residues and impurities must be made to confirm

the effectiveness of the removal of residues and impurities. In this regard, you should use validated analytical methods whose sensitivity to detect

residues or contaminants. The detection limit for each analytical method should be sufficiently sensitive to detect the established acceptable residue or contaminant.

It will be one validation study using case, worst case, "taking into account the critical Testing "to be clean" is not considered a suitable alternative for cleaning validation. [1, Appendix 15] The establishment of accepting the validity of the process of removing the residues and impurities from the production facilities

According to an article, let us review the history of process validation acceptance

limits for removal of residues and contaminants from equipment for active pharmaceutical ingredients (API) and identified the origins of the limits currently used in industry. Current approaches to setting acceptance limits were analyzed and discussed in some of the problems and weaknesses of these approaches.

Table 2.1. Process validation acceptance limits for removal residues and impurities, after Barr The limits on the basis of dose The limits of "implicit"

1/10th of the therapeutic dose Not more than 1 ppm 1/50th of the maxim therapeutic dose 3 ppm (Arsenic-based limit) 1/1,000th of the minim therapeutic dose 10 ppm

Less than the lowest therapeutic dose 22 ppm 30 ppm for cleaning agents

Some other limitations The limit of detection of the method < 5 mg/ swab sampling

As can be seen in Table 2.1., Acceptance limits in use at that time were inconsistent

from one company to another, and in many cases, chosen arbitrarily. There were two models that have emerged; use based limits in a way therapeutic dose and arbitrary limits (or as some call them today workers limits "implicit"). At least one company has tied their limit values for arsenic reasoning implicit in the USP, because if arsenic could be present 3 ppm, the residue of their compounds at lower levels should be considered safe. We note that both types of limits are suggested by Harder or Mendenhall.

At the time this article was a landmark in the world of the validation process for removing residues and impurities as it was the first publication that established specific criteria to determine acceptance limits of the validation process for removing residues and impurities. Underscoring its importance, this article has been quoted in almost every subsequent item on the validation process residues and impurities removal for many years afterwards. Pharmaceutical companies now have something to show in the way they set acceptance limits for the process validation program to remove residues and impurities.


16

2.3. Anti-inflammatory drugs Some of the most prescribed and used drugs are anti-inflammatory. Anti-inflammatory

drugs may be classified according to the chemical structure: - Anti-inflammatory drugs : aspirin, ibuprofen, diclofenac, phenylbutazone,

nimesulide, celecoxib etc. and - Anti-inflammatory steroids , which are also known as corticosteroids, prednisone,

hydrocortisone, dexamethasone, clobetasol, etc., and are synthetic derivatives of natural corticosteroid hormones secreted by the adrenal glands.

Anti-inflammatory drugs cortisone derivatives

Cortisone and cortisol are natural hormones secreted by the adrenal glands, with multiple actions. Anti-inflammatory drugs are cortisone derivatives of the anti-inflammatory activity superior to that of the natural hormone, but with less effect on the water retention. Anti-inflammatory drugs non-derivatives of cortisone (nonsteroidal)

NSAIDs have anti-inflammatory, analgesic and anti-pyretic.

The pharmaceutical form of the two anti-inflammatory drugs Drug cortisteroid - Clobetasol propionate the pharmaceutical form of ointment Clobetasol propionate part of Pharmacotherapeutic group: Corticosteroids for

dermatological use, very potent. Clobetasol ointment is indicated for the following conditions: limited plaque psoriasis resistant to other treatments, particularly localized palmoplantar forms; eczema refractory to treatment; lichen planus; discoid lupus erythematosus and other skin disorders uninfected corticosensibile that do not respond to corticosteroids with lower action.

Clobetasol ointment is an ointment emulsion ointment base because it contains the following ingredients: white petrolatum, cetyl alcohol, propylene glycol and polysorbate 80, but instead contains no water.

NSAID - Phenylbutazone the pharmaceutical form of cream Phenylbutazone cream part of pharmacotherapeutic group topical products for joint

and muscle pain, topical NSAIDs. Phenylbutazone cream is indicated for the treatment of acute gout, rheumatoid arthritis, the sinovitelor, osteoarthritis and ankylosing spondylitis.

Phenylbutazone hydrophilic cream is a cream as the cream base containing the following ingredients: emulsifying type cetylstearyl alcohol, white petrolatum, glycerol, polysorbate 80 and purified water 34%.


17

PERSONAL CONTRIBUTIONS

CHAPTER 3 DEVELOPMENT OF METHODS FOR THE ANALYSIS OF PHENYLBU TAZONE 3.1. Introduction

We aimed to establish the control parameters, limits the admissibility of analysis and

experimental conditions for phenylbutazone. Quantitative determination of phenylbutazone was performed by an HPLC method that we will use later in the quantitative determination of phenylbutazone in the formulation of the cream.

In developing this method, we started from the existing data in the literature and we took into account the physicochemical properties of phenylbutazone.

3.2. Material and methods The control parameters and admissibility criteria

The control parameters and admissibility limits for phenylbutazone we set according to the European Pharmacopoeia (FE) July edition monograph - Phenylbutazone 0422 because the quality of active substances used in the formulation of medicinal products must comply with the European Pharmacopoeia in force.

Tabel 3.I - The physico-chemical properties of phenylbutazone

No. crt.

QUALITY PARAMETERS THE ADMISSIBILITY LIMITS

1. Description - organoleptic White or almost white crystalline powder

2. Solubility Practically insoluble in water, sparingly soluble in alcohol. Is dissolved in alkaline solution

3. Identification: Melting Point (º C); 104 – 107

Spectrometry IR; IR spectra recorded for the sample corresponds to the IR spectrum Phenybutazone CRS;

4.

Related substances, wt% (HPLC) - Impurity A, B; - Impurity C; - Any other impurity; - Total impurities

Max. 0,25 each Max. 0,20 Max. 0,1 Max. 0,5

5. Impurity E, ppm (HPLC) Max. 5,0

6. Content in phenylbutazone, g% (on dry basis) (HPLC)

99,0 – 101,0

7. Residual solvents, ppm - Methanol (GC)

Max. 3 000

9. Particle size, µm Max. 50,0

Identification of phenylbutazone was performed by IR spectrometry method

according to the European Pharmacopoeia Phenylbutazone0422 monograph. The reagent used was potassium bromide, and the equipment and experimental

conditions were used Tensor27 IR spectrophotometer manually operated hydraulic press The sample was prepared by Tabletting potassium bromide using manually operated

hydraulic press and compared spectra obtained for analyte IR spectrum for phenylbutazone CRS.

IR spectra recorded for the sample corresponded to the IR spectrum for phenylbutazone CRS.


18

Determination of related substances of phenylbutazone was performed by HPLC method in accordance with the European Pharmacopoeia Phenylbutazone 0422 monograph, the principle of the separation method is HPLC with UV detection at 240 nm.

The reagents and solvents used are acetonitrile - a solvent HPLC, sodium acetate (R), citric acid (R) Purified water - solvent HPLC impurity C phenylbutazone (1,2-diphenylhydrazine), phenylbutazone, and impurity B CRS benzidine (R) .

We used an Agilent 1100 HPLC machine, pressure liquid chromatograph equipped with UV lamp, recorder, automatic integration of peak areas. Column chromatography using a l = 125m x Ø = 4.0 mm, made of stainless steel with octadecylsilyl silica gel stationary phase chromatography (R), Zorbax Eclipse XDB C18 with 5 micron particle diameter of the interior.

Mobile phase A was made by dissolving 1.36 g of sodium acetate in water and adjusting the solution to pH 5.2 was carried out with a solution of citric acid 52.5 g / l and then made up to 1000 ml with water, and mobile phase B was acetonitrile.

The flow rate was 1.5 ml / min with UV detection at a wavelength λ = 240 nm, at 30 ° C. Sample injection volumes were 20 µl .

For impurity E - chromatographic conditions were modified as follows: UV detection wavelength was λ = 280 nm and injected samples were analyzed solution and reference solution (c). The signal - noise was min. 10 for the principal peak.

Results The required to impurity A is max. 0.25%, and the result was below the detection limit

and within the limits imposed, it is appropriate. The required to impurity B is max. 0.25%, and the result was 0.145% and falls within

the limits, which is appropriate. The amount required for impurity C is max. 0.20%, and the result was below the

detection limit and within the limits imposed, it is appropriate. The amount required for other individual impurity is max. 0.10%, and the result was

0.02% and falls within the limits, which is appropriate. The required total impurities is max. 0.50%, and the result was 0.20% and falls within

the limits, which is appropriate. The amount required for impurity E is max. 5.00%, and the result was below the

detection limit and within the limits imposed, it is appropriate. Related results for impurities of phenylbutazone and impurity E were within the limits

imposed are appropriate. Quantitative determination of phenylbutazone was carried out by HPLC analytical

method, the principle of the separation method is HPLC with UV detection at a wavelength λ = 254 nm.

The reagents and solvents used were methanol (A), acetonitrile, HPLC, doubly distilled water, orthophosphoric acid (R).

HPLC method was used an Agilent 1100 liquid chromatograph equipped with pressure UV lamp, recorder, automatic integration of peak areas. Column chromatography using a 150 x 4.6 mm, stainless steel, solid-phase octylsilane (C8) Zorbax Eclipse XDB C8, 5 mm particle diameter, and the inner mobile phase consisting of a mixture of acetonitrile and water bidistilled HPLC in a ratio of 75: 25 (v / v) and 100 ml of mobile phase was added 0.1 ml of orthophosphoric acid (R). The flow rate was 1ml/min with UV detection at λ = 254 nm wavelength, at a temperature of 25 ° C. Sample injection volumes were 20 µl.


19

Results Chromatogram was considered valid because the resolution between the peaks of

phenylbutazone was greater than 2 (cf. USP and FE provides greater than 1.5). Phenylbutazone the corresponding peak in the chromatogram of the sample solution

had the same retention time and about the same size as the corresponding peak in the chromatogram of the standard solution phenylbutazone.

Values required for phenylbutazone are: 99.0 to 101.0%. The result was 99.13%, which falls within the limits and appropriate.

For analysis of residual solvents - methanol we used a GC analytical method was

examined by gas chromatography injection system "head-space". The reagents and solvents used are Class 2 solvents CRS residual methanol (R),

dimethylformamide (A). We used a Thermo Focus GC gas chromatograph machine with injection system

"head-space" with autoinjector equipped with flame ionization, recorder, automatic integration of peak areas. Column chromatography using a TR-V1 fused silica material, length 30 m, diameter 0.53 mm, film 6% cianopropilfenil-94%-dimethylpolysiloxane with a thickness of 3 mm, and a nitrogen carrier gas, and the flow rate was 5 ml / min.

FID detector was set at the working temperature of 250 ° C, and injector at 140 ° C was set. The injection was split with a split ratio of 1:5, the volume of sample injected was 1 ml.

Autosampler, the headspace was set at a temperature of 105 ° C incubation for 45 minutes. Under these conditions syringe injection temperature was 110 ° C, nitrogen gas was used as conditioning.

Results The amount of methanol required for max. 3000 ppm. The result was 30 ppm, which is

within the limits imposed appropriate.

Validation of analytical methods Since this method of control product was carried out according to methods formalized

reference pharmacopoeias (EF) there was no need to validate all the parameters of the analytical methods validation tests have been carried out only compatibility of the system, making the necessary adjustments .

Substance phenylbutazone, suitabilitatea system (system suitability) determined the reference solution B was an assessment of the conditions for the resolution of the signals corresponding to impurity B and phenylbutazone. According to the method, between the impurity B and phenylbutazone has to be a resolution of at least 2.0. In this case, we obtained a resolution of 7.1362 between corresponding signals phenylbutazone retention time (15.57) and phenylbutazone impurity B (retention time - 17.57). Considering the mixture of impurity B and 1,2-diphenylhydrazine was obtained 14.46 a resolution factor of the impurity phenylbutazone C with a retention time of 20.55 minutes.

For impurity is due to very low concentrations FE monograph requires the system suitabilitatea a minimum limit of 10 for the signal noise. For gas concentrations of 0.05 mg / ml was determined a signal-to-noise of 101.65, which allowed accurate evaluation of possible impurity is characteristic signals of the samples analyzed.


20

3.3. Conclusions The analytical methods used to test the active substance phenylbutazone also complies

with GMP and methods described in the European Pharmacopoeia. The raw material is considered appropriate physico-chemical as results.

HPLC method for content determination phenylbutazone may be used to determine the content in phenylbutazone from pharmaceutical form of cream.

The quality of the pharmaceutical active substance phenylbutazone is analyzed in appropriate according to the European Pharmacopoeia monograph - Phenylbutazone 0422. Due to proven quality, phenylbutazone is considered appropriate for its use in the formulation of medicinal products and ensure the quality of the purpose for which the drug was designed is conditional. CHAPTER 4 ANALYSIS OF THE PHARMACEUTICAL FORM WITH PHENYLBUTA ZONE 4.1. Introduction

We aimed to establish the control parameters, limits admissibility control methodology and experimental conditions for the analysis of product Phenylbutazone cream. In establishing control analytical methodology we have considered the pharmaceutical form in which the active substance is conditional - form semisolid topical cream.

Quantitative determination of phenylbutazone was carried out by HPLC analytical method. HPLC analytical method we will use later to determine traces of phenylbutazone in samples taken from the production equipment after cleaning them.

In developing this analytical methodologies have started from existing data in the literature and we took into account the physicochemical properties of phenylbutazone. 4.2. Material and methods The control parameters and admissibility criteria for Phenylbutazone cream

The control parameters and limits admissibility for Phenylbutazone cream I established according to European Pharmacopoeia edition 7 monograph - Phenylbutazone 0422 monograph "SEMI-SOLID PREPARATIONS FOR CUTANEOUS APPLICATION" and according to "Note for Guidance on Excipients, antioxidants and antimicrobial Preservatives in the dossier for application for marketing Authorisation of a medicinal product "- EMEA CPMP/QWP/419/03 because the quality of medicinal products must comply with the European Pharmacopoeia in force.

Table 4.I - Physicochemical and microbiological properties for Phenylbutazone cream: No. crt.


1 Appearance, color, odor, homogeneity homogeneous cream, white color and characteristic odor of camphor and menthol

2 Particle size below 50 microns 3 pH 4,5 – 5,5

4

Phenylbutazone - related substances (HPLC) Impurities A and B Impurity C Other individual impurities Total impurities

Max. 0,25% Max. 0,20% Max. 0,10% Max. 0,50%

5 Identification and dosage nipagin, nipasol (HPLC) nipagin nipasol 0,054 – 0,066 0,06 ± 10 %

0,036 – 0,044 0,04 ± 10 %


21

No. crt.


6 Identification and dosage phenylbutazone (HPLC) 3,88 – 4,12 (4 g ± 3%) 7 Identification and dosage camphor (g/100 g cream) (GC) 2,7 – 3,3 (3 g ±10%) 8 Identification and dosage menthol (per 100 g cream) (GC) 0,9 – 1,1 (1 g ± 10 %)

9

Microbial contamination • No. Total microorganisms (aerobic bacteria) / g max. 102 UFC • No. Total yeasts and filamentous fungi / g max. 101 UFC • Staphylococcus aureus/g product absent • Pseudomonas aeruginosa /g product absent

Determination of pH was carried out according to the European Pharmacopoeia

potentiometric head. 2.2.3. - Potentiometric determination of pH. Laboratory equipment used was pH Inolab potentiometer 7110, Analytical balance

accurate XB 620C water bath Memert, glass Erlenmeyer flask, magnetic stirrer. It weighed 10.00 g cream and hot-dispersed in a Erlenmeyer flask with 50 ml of water

approximated. After cooling was completed to 100 ml with purified water and pH was determined using potentiometric pH potentiometer Inolab 7110.

Results and Discussion When measuring pH in purified water dispersion of 10% cream potentiometer

WTWpH 7110 Inolab obtained value of 4.99. The result was within limits: 4.5 to 5.5 and appropriate.

Determination of related substances of phenylbutazone was performed by HPLC

method in accordance with the European Pharmacopoeia monograph Phenylbutazone, the principle of the separation method is HPLC with UV detection at 240 nm The reagents and solvents used are acetonitrile - a solvent HPLC, sodium acetate (R), citric acid (R) Water - HPLC solvent, methanol - HPLC solvent, impurity C (1,2-diphenylhydrazine), impurity B CRS. We used an Agilent 1100 HPLC machine, pressure liquid chromatograph equipped with UV lamp, recorder, automatic integration of peak areas. Column chromatography using a 150 x 4.6 mm stainless steel with octadecylsilyl silica gel solid phase (C18) Zorbax Eclipse XDB inner diameter 5 micron particles. Mobile phase A was made by dissolving 1.36 g of sodium acetate in water and adjusting the solution to pH 5.2 was carried out with a solution of citric acid 52.5 g / l and then made up to 1000 ml with water, and mobile phase B was acetonitrile. The flow rate was 1.5 ml / min with UV detection at a wavelength λ = 240 nm, at 30 ° C. Sample injection volumes were 10 µl .

Results and Discussion The validity of the test: The retention times relative to the retention time of

phenylbutazone (about 13 min) were: impurity A = approx. 0.2; impurity B = about. 1.2; impurity C = about 1.3; impurity D = about. 1,7.

The chromatograms were considered valid as between peak and peak phenylbutazone impurity B in the chromatogram of the reference solution ba there was a resolution of at least 2.

Correction factor: for the calculation of a peak area of impurity C multiplied by 0.55. The required to impurity A is max. 0.25%, and the result was below the detection limit

and within the limits imposed, it is appropriate. The required to impurity B is max. 0.25%, and the result was 0.24% and falls within

the limits, which is appropriate.


22

The amount required for impurity C is max. 0.20%, and the result was below the detection limit and within the limits imposed, it is appropriate.

The amount required for other individual impurity is max. 0.10%, and the result was 0.06% and falls within the limits, which is appropriate.

The required total impurities is max. 0.50%, and the result was 0.30% and falls within the limits, which is appropriate.

Related results for impurities of phenylbutazone were within the limits imposed are appropriate.

Identification and quantitative determination of preservative substances was performed by HPLC analytical method, the principle of the separation method is HPLC with UV detection at 254 nm.

The reagents and solvents used were sodium dihydrogen phosphate monohydrate (NaH2PO4) 0.16% w / v phosphoric acid (H3PO4) 0.1% w / v methanol - HPLC solvent acetonitrile - a solvent HPLC.

We used an Agilent 1100 HPLC machine, pressure liquid chromatograph equipped with UV lamp, recorder, automatic integration of peak areas. Column chromatography using a 150 x 4.6 mm stainless steel, with the stationary phase octylsilane (C8) Zorbax Eclipse XDB, 5 mm particle diameter, and the inner mobile phase consisting of a mixture of 56% solvent and 44% methanol could change ratio in order to obtain the necessary performance chromatography, and the solvent was obtained by mixing equal parts of sodium dihydrogen phosphate (NaH2PO4) 0.16% w / v phosphoric acid (H3PO4) 0.1% w / v was adjusted to pH 2.5. The flow rate was 2.0 ml / min with UV detection at a wavelength λ = 254 nm at a temperature of 25 ° C and injection volume of 20 ìl.

Identification: the corresponding peaks of methyl p-hydroxybenzoate and p-hydroxybenzoate, n-propyl in the chromatogram of the sample solution had the same retention time and about the same size as the corresponding peaks of methyl p-hydroxybenzoate and p-n-hydroxybenzoate propyl chromatogram of the standard solution.

Results and Discussion Chromatogram was considered valid because the resolution between the peaks of p-

hydroxybenzoate and p-hydroxy-n-propyl was greater than 2 (cf. USP. FE provides greater than 1.5).

Peaks corresponding to methyl p-hydroxybenzoate and p-hydroxybenzoate, n-propyl in the chromatogram of the sample solution had the same retention time and about the same size as the corresponding peaks of methyl p-hydroxybenzoate and p-hydroxybenzoate of propyl N-the chromatogram of the standard solution.

Values are imposed nipagin: 0.054 to 0.066 g/100g cream. The result was 0.0573%, within the limits imposed appropriate.

Values are imposed nipasol: 0.036 to 0.044 g/100g cream. The result was 0.0400%, within the limits imposed appropriate.

Identification and quantitative determination of phenylbutazone was performed by HPLC analytical method, the principle of the separation method is HPLC with UV detection at a wavelength λ = 254 nm. The reagents and solvents used were methanol (A), acetonitrile, HPLC, doubly distilled water, orthophosphoric acid (R). HPLC method was used an Agilent 1100 liquid chromatograph equipped with pressure UV lamp, recorder, automatic integration of peak areas. Column chromatography using a 150 x 4.6 mm, stainless steel, solid-phase octylsilane (C8) Zorbax Eclipse XDB C8, 5 mm particle diameter, and the inner mobile phase consisting of a mixture of acetonitrile and water bidistilled HPLC in a ratio of


23

75: 25 (v / v) and 100 ml of mobile phase was added 0.1 ml of orthophosphoric acid (R). The flow rate was 1ml/min with UV detection at λ = 254 nm wavelength, at a temperature of 25 ° C. Sample injection volumes were 20 µl.

Results and Discussion Chromatogram was considered valid because the resolution between the peaks of

phenylbutazone was greater than 2 (cf. USP and FE provides greater than 1.5). Phenylbutazone the corresponding peak in the chromatogram of the sample solution

had the same retention time (2.7 minutes) and approximately the same size as the corresponding peak in the chromatogram of the standard solution phenylbutazone.

Values required for phenylbutazone are: 3.88 -4.12 g/100g cream. The result was 3.96% which is within the limits imposed appropriate.

Determination of menthol and camphor has been carried out by a gas

chromatographic method of analysis, separation principle of the method is GC FID detection. We used an Agilent 6890 N gas chromatograph apparatus with autoinjector equipped

with flame ionization, recorder, automatic integration of peak areas. Column chromatography using was a HP-Innowax, 30 m length, 0.32 mm diameter, film thickness 0.25 µm, with polyethylene film (according to USP G16) and nitrogen carrier gas, and the flow rate was 1.9 ml / min.

FID detector was set at 250°C operating temperature, and the injector temperature was set to 125°C. The injection was split with the split ratio of 10:1, the injected sample volume was 1 µl, injection vaporization temperature of 250°C, for 8 minutes.


camphor and menthol was greater than 2 (cf. USP. FE provides greater than 1.5). identification:

Camphor and menthol corresponding to the respective peaks of the chromatogram of the test solution had the same retention time and about the same size as that of menthol and camphor corresponding peaks in the chromatogram of the standard solution.

Values required for menthol are 0.90 to 1.10 g/100g cream. The result of 0.99% within the limits imposed appropriate.

Values required for camphor are 2.70 to 3.30 g/100g cream. The result of 2.96% within the limits imposed appropriate.

Microbial contamination Verification of microbial contamination was performed according to the European

Pharmacopoeia: - Determine the total number of microorganisms (aerobic) and determine the total

number of yeasts and filamentous fungi - as head. 2.6.12. Microbiological Examination of Non-sterile products: microbial enumeration tests.

- Pseudomonas aeruginosa, Staphylococcus aureus - under the head. 2.6.13. Microbiological Examination of Non-Sterile Products: Test for Specified Micro-Organisms. The reagents and culture media used was buffered peptone - sodium chloride (NaCl) at

pH 7, tween 80, sodium hydroxide (NaOH) 1M culture medium (Sabouraud), culture medium (agar) liquid, medium Cetrimide agar - Cetra., nutrient broth and Mannitol salt agar medium - MSA.


24

Laboratory equipment used was a laminar flow hood (Minifil), an electric thermostat Incucell 55, an electric thermostat 111 Incucell, a balance Kern KB 600-2, an colony counter, an magnetic stirrer, a water bath thermostatic Memert, laboratory glassware, Petri plates.

Microbial contamination was performed by plate counting method. The sample was prepared for analysis as follows: they weighed 10 grams of sample

were suspended in 90 ml of buffered peptone - sodium chloride (NaCl) pH 7 and obtained a dilution of 1/10 of the sample taken in it.

Since the analyte is sparingly soluble in water, a surfactant was added (5 g Tween 80). The sample thus prepared was homogenized by stirring carefully on a water bath to a maximum of 40 ° C for up to 30 minutes, then adjusted to pH 6-8 with sodium hydroxide solution (NaOH) 1 M.

Casting method in Petri plates For dilution of 1/10 serial dilutions were prepared: 1/100, 1/1000, using the same

solvent system: pH 7 buffer solution. Petri plates were used with a diameter of 9 cm (two series of three plates) that were

made 1 ml sample of the three dilutions. For each dilution were seeded four Petri dishes. Six petri plates were poured 15-20 ml of culture medium (agar) liquefied and cooled to

45 ° C (bacteria), and the other six were added to 15-20 ml of culture medium (Sabouraud - for fungi).

The plates were incubated at 35 ° C for 5 days and that at 25 ° C for 5 days. Identification of Pseudomonas aeruginosa:

Dilution of 1/10 of the sample buffer pH 7 was inoculated into 100 ml of medium 10 ml nutrient broth. It was stirred and incubated at 30-35 ° C 18-24 hours. After this period of time to perform the transition cetrimide agar medium and incubated at 30-35 ° C 18-72 hours.

Results No microbial growth was detected, the result was negative, the product passed the test. If it was a hit, there is a steering medium beige color yellow - blue - green and then

perform oxidase test, but this has not happened. In this case, a colony isolated on a seed disk is impregnated oxidase. The positive reaction was seen in 5-10 seconds at 25-30 ° C. A delayed reaction may occur in 10-60 seconds, but any change in color after 60 seconds or no change is considered to be a negative reaction. Identification Staphylococcus aureus:

Sample was used and an amount corresponding to 1 g, or 10 mL of the dilution of 1/10 of the sample buffer pH 7 was used to inoculate 100 ml of nutrient broth. It was stirred and incubated at 30-35 ° C 18-24 hours. After this was done passage Mannitol salt agar medium and incubated at 30-35 ° C 18-72 hours.

Results No microbial growth was detected, the result was negative, the product passed the test. If it was a hit, an occurrence of colonies yellow / white surrounded by a yellow zone,

and perform coagulase test using rabbit plasmǎ. This test highlights the species Staphylococcus aureus based on coagulase enzyme production which would lead to clot plasma. The test is negative for other species of the genus Staphylococcus.

Discussion On the blank board Cetrimide specific pathogen Pseudomonas aeruginosa isolated

colonies appeared yellow and sample plate remained unchanged, which means that the product is not contaminated with Pseudomonas aeruginosa.


25

Plate Mannitol salt agar witness the pathogen Staphylococcus aureus specific and changed color from red to yellow, and the sample plate was unchanged, which means that the product is not contaminated with Staphylococcus aureus.

The results were within the limits imposed ie pathogens were absent and no. Total microorganisms (aerobic) / g and no. Total yeast and filamentous fungi / g product was less than 10 CFU. Procedure validation of analytical methods

Since this method of control product was carried out according to methods formalized reference pharmacopoeias (EF) there was no need to validate all the parameters of the analytical methods validation tests have been carried out only compatibility of the system, making the necessary adjustments .

Substance phenylbutazone, suitabilitatea system (system suitability) determined the reference solution B was an assessment of the conditions for the resolution of the signals corresponding to impurity B and phenylbutazone. According to the method, between the impurity B and phenylbutazone has to be a resolution of at least 2.0. In this case, we obtained a resolution of 7.1362 between corresponding signals phenylbutazone retention time (15.57 min.) And phenylbutazone impurity B (retention time - 17.57 min.). Considering the mixture of impurity B and 1,2-diphenylhydrazine, to obtain a resolution factor of 14.46, phenylbutazone impurity C with a retention time of 20.55 minutes.

For impurity E is due to very low concentrations FE monograph requires the system suitabilitatea a limit of at least 10 for the signal / noise. For gasoline concentrations of 0.05 µg/ml was determined ratio signal / noise of 101.65, which allowed accurate evaluation of possible impurity is characteristic signals of the samples analyzed. 4.3. Conclusions

The analytical methods used to test Phenylbutazone cream product also complies with GMP and analytical methods described in the European Pharmacopoeia. The product is properly analyzed in terms of physico-chemical and microbiological according to results.

HPLC method for content determination phenylbutazone assays may be used to process trace phenylbutazone after removal of the residues and impurities from the production facilities.

Phenylbutazone product quality cream is appropriate according to the European Pharmacopoeia monograph - Phenylbutazone 0422 monograph "SEMI-SOLID PREPARATIONS FOR CUTANEOUS APPLICATION" and according to "Note for Guidance on Excipients, antioxidants and antimicrobial Preservatives in the Dossier for Application for Marketing Authorisation of a medicinal product "- EMEA, CPMP/QWP/419/03.

Drug Phenylbutazone cream, according to the results obtained, the purpose it was designed. CHAPTER 5 DEVELOPMENT OF METHODS FOR THE ANALYSIS OF CLOBETAS OL PROPIONATE 5.1. Introduction

We aimed to establish control parameters, limits and experimental conditions admissibility analysis for clobetasol propionate. Quantitative determination of clobetasol propionate was performed by an HPLC method that we will use later in the quantitative determination of clobetasol propionate ointment in the formulation.


26

In developing this method, we started from the existing data in the literature and we took into account the physicochemical properties of clobetasol propionate. 5.2. Material and methods Control parameters and criteria for eligibility for clobetasol propionate

Control parameters and limits eligibility for clobetasol propionate I determined according to the European Pharmacopoeia edition 7 monograph - Clobetasole propionate 2127, because the quality of active substances used in the formulation of medicinal products must comply with the European Pharmacopoeia in force.

Table 5.I - The physico-chemical properties of clobetasol propionate

No. crt.


1. Description - organoleptic White or almost white crystalline powder

2. Solubility Practically insoluble in water, slightly soluble in

acetone, poorly soluble in ethanol (96%)

3. Identification IR spectrophotometry

According to the corresponding spectrum clobetasol propionate CRS

4. Specific optical rotation, º + 112 → + 118

5.

Related substances (HPLC),% - Impurity E - Impurity D - Impurities B, C - Impurities A, L, M - Unspecified impurities - Total impurities

Max. 0,7 Max. 0,5 Max. 0,3 Max. 0,2 Max. 0,1 Max. 2,0

6. Dosage clobetasol propionate, on the dried basis,%

97,0 – 102,0

7.

Residual solvents (GC), ppm - Methylene chloride - dimethylformamide - tetrahydrofuran - acetone - methanol

Max. 600 ppm Max. 880 ppm Max. 720 ppm Max. 5000 ppm Max. 3000 ppm

8. Particle size, µm Max. 50,0

Identification of clobetasol propionate was performed by IR spectrometry method

according to the European Pharmacopoeia monograph "Clobetasole propionate." The reagent used was potassium bromide, and the equipment used were IR

spectrophotometer Tensor27 manually operated hydraulic press. The sample was prepared by Tabletting potassium bromide using manually operated

hydraulic press and compared spectra obtained for the analyte with clobetasol propionate IR spectrum for CRS.

IR spectra recorded for the sample corresponds to the IR spectrum for clobetasol propionate CRS.

Determination of related substances of the clobetasol propionate was carried out by HPLC method in accordance with the European Pharmacopoeia monograph "Clobetasole propionate", the principle of the separation method is HPLC with UV detection at 240 nm.

The reagents and solvents used were methanol (A), acetonitrile (R), sodium dihydrogen phosphate monohydrate (R), sodium hydroxide (A) and clobetasol propionate, clobetasol impurity J CRS.


27

We used an Agilent 1100 HPLC machine, pressure liquid chromatograph equipped with UV lamp, recorder, automatic integration of peak areas. Column chromatography using a l = 150cm x Ø = 4.6 mm, made of stainless steel with octadecylsilyl silica gel stationary phase chromatography (R) C18 Zorbax Eclipse XDB inner diameter 5 micron particles.

Mobile phase I obtained by mixing 10 volumes of methanol (A), a solution of 42.5 volumes of 7.85 g / l solution of sodium dihydrogen phosphate monohydrate (R), which was adjusted to pH - 5.5 with a solution 100 g / l sodium hydroxide solution (R) and 47.5 volumes of acetonitrile (a);

The flow rate was 1.0 ml / min with UV detection at a wavelength λ = 240 nm at room temperature. The volumes of solution to be analyzed (a), reference (b), (c) and (d) were injected 10 ml. Analysis time was 3 times the retention time of clobetasol propionate.

Results and Discussion In order to identify the appropriate impurities using the chromatogram of the reference

solution (C) containing clobetasol to identify the peak (peak for identification) for identifying peaks due to the impurities A, B, C, D, E, L and M.

Validity of the test Retentions in relation to the relative retention time of clobetasol propionate (TR -

approx. 10.0 min) were as follows: Impurity A - approx. 0.4; Impurity B - approx. 0.6; Impurity C - approx. 0.9; Impurity J - approx. 1.1; Impurity D - approx. 1.2; Impurity L - approx. 1.3; Impurity M - approx. 1.6; Impurity E - approx. 2.1.

Suitabilitatea system: Resolution was minimum 2.0 between the peaks due to clobetasol propionate and J impurity in the chromatogram obtained with reference solution (b). Corresponding chromatogram of the reference solution (c) was similar to the corresponding standard chromatogram peak identification (clobetasol for peak identification).

The required to impurity A is max. 0.20%, and the result was 0.17% and falls within the limits, which is appropriate.

The required to impurity B is max. 0.30%, and the result was 0.13% and falls within the limits, which is appropriate.

The amount required for impurity C is max. 0.30%, and the result was below the detection limit and within the limits imposed, it is appropriate.

The amount required for impurity D is max. 0.50%, and the result was 0.21% and falls within the limits, which is appropriate.

The amount required for impurity E is max. 0.70%, and the result was 0.01% and falls within the limits, which is appropriate.

The amount required for impurity L is max. 0.20%, and the result was below the detection limit and within the limits imposed, it is appropriate.

The amount required for impurity M is max. 0.20%, and the result was 0.14% and falls within the limits, which is appropriate.

Setpoint for unspecified impurities is max. 0.10%, and the result was below the detection limit and within the limits imposed, it is appropriate.


Related results for impurities of clobetasol propionate were within the limits imposed are appropriate.

Quantitative determination of the clobetasol propionate was carried out by HPLC analytical method, the principle of the separation method is HPLC with UV detection at a wavelength λ = 240 nm.


28

The reagents and solvents used were methanol (A), acetonitrile (R), sodium dihydrogen phosphate monohydrate (R), sodium hydroxide (R), clobetasol propionate CRS.


Mobile phase A I obtained by mixing 10 volumes of methanol (A), a solution of 42.5 volumes of 7.85 g / l solution of sodium dihydrogen phosphate monohydrate (R), which was adjusted to pH - 5.5 with a solution 100 g / l sodium hydroxide solution (R) and 47.5 volumes of acetonitrile (R);

The flow rate was 1.0 ml / min with UV detection at a wavelength λ = 240 nm at room temperature. Volumes of test solution (b) and reference (a) were injected 10 µl. Analysis time was 3 times the retention time of clobetasol propionate.

We used the same method of analysis to determine the related substances with the following modification: only injected test solution (b) and reference solution (a).


clobetasol propionate was greater than 2 (cf. USP and FE provides greater than 1.5). Clobetasol propionate corresponding peak in the chromatogram of the sample solution

had the same retention time and about the same size as the corresponding peak CRS clobetasol propionate in the chromatogram of the standard solution.

Clobetasol propionate imposed values are: 97.0 to 102.0%. The result was 99.64%, which falls within the limits and appropriate.

For analysis of residual solvents we used a GC analytical method was examined by

gas chromatography injection system "head-space". The reagents and solvents used are Class 2 solvents residual CRS, purified water (R),

methanol (R), acetone (A), dimethylformamide (A), 1,3-dimethyl-imidazolinone (R), tetrahydrofuran (R,) methylene chloride (R), 1,3-dimethyl acetamide (R).

We used a Thermo Focus GC gas chromatograph machine with injection system "head-space" with autoinjector equipped with flame ionization, recorder, automatic integration of peak areas. Column chromatography using a TR-V1 fused silica material, length 30 m, diameter 0.53 mm, film 6% cianopropilfenil-94%-dimethylpolysiloxane with a thickness of 3 mm, and a nitrogen carrier gas, and the flow rate was 5 ml / min.

FID detector was set at the working temperature of 250 ° C, and injector at 140 ° C was set. The injection was split with a split ratio of 1:5, the volume of sample was injected every 1 µl each. Autosampler, the headspace was set at a temperature of 105 ° C incubation for 45 minutes. Under these conditions syringe injection temperature was 110 ° C, nitrogen gas was used as conditioning.

Results The amount of methylene chloride is required for max. 600 ppm. The result was 260

ppm, which is within the limits imposed appropriate. The required to dimethylformamide is max. 880 ppm. The result was 403 ppm, which

is within the limits imposed appropriate. The amount required for tetrahydrofuran is max. 720 ppm. The result was 5.56 ppm,

which is within the limits imposed appropriate. The amount of acetone is required for max. 5000 ppm. The result was 792 ppm, which

is within the limits imposed appropriate.


29

The amount of methanol required for max. 3000 ppm. The result was 148 ppm, which is within the limits imposed appropriate.

Procedure validation of analytical methods Since this method of control product was carried out according to methods formalized

reference pharmacopoeias (EF) there was no need to validate all the parameters of the analytical methods validation tests have been carried out only compatibility of the system, making the necessary adjustments 5.3. Conclusions

The analytical methods used to test the active substance clobetasol propionate comply with GMP standards and methods of analysis described in the European Pharmacopoeia. The raw material is considered appropriate physico-chemical as results. HPLC method for the determination of the clobetasol propionate can be used to determine the content of the pharmaceutical form clobetasol propionate ointment.

The quality of the active substance clobetasol propionate is analyzed pharmaceuticals, is appropriate according to the European Pharmacopoeia monograph - Clobetasole propionate 2127. Due to proven quality, clobetasol propionate is considered appropriate for use in the formulation of medicinal products and ensure quality for purpose designed drug that is conditioning. CHAPTER 6 ANALYSIS OF THE PHARMACEUTICAL FORM OF CLOBETASOL PROPIONATE 6.1. Introduction

We aimed to establish control parameters, limits admissibility control methodology and experimental conditions for the analysis of product Clobetasol ointment. In establishing control analytical methodology we have considered the pharmaceutical form in which the active substance is conditional - form semisolid topical ointment.

Quantitative determination of the clobetasol propionate was carried out by HPLC analytical method. HPLC analytical method we will use later to determine trace clobetasol samples taken from production equipment after cleaning them.

In developing this analytical methodologies have started from existing data in the literature and we took into account the physicochemical properties of clobetasol propionate. 6.2. Material and methods Control parameters and eligibility criteria for Clo betasol ointment

Control parameters and limits eligibility for Clobetasol ointment I established according to European Pharmacopoeia edition 7 monograph - Clobetasole propionate 2127 monograph "SEMI-SOLID PREPARATIONS FOR CUTANEOUS APPLICATION", British Pharmacopoeia monograph - Clobetasol Ointment edition 2004, according "Note for Guidance on Excipients, antioxidants and antimicrobial Preservatives in the dossier for Application for marketing Authorisation of a medicinal product" - EMEA CPMP/QWP/419/03 because the quality of medicinal products must comply with the European pharmacopoeia force.


30

Table 6.I – The physico-chemical and microbiological properties of Clobetasol ointment: No. crt.


1 Appearance, color, odor and consistency Table semi-solid, homogeneous, oily to the touch, odor characteristic white excipients

2

Related substances (HPLC) - Individual - Impurity A (clobetasol) - Total impurities

Max. 0,5% Max. 0,5% Max. 2%

3 Identification of clobetasol - HPLC

The chromatogram obtained with solution (2) has a peak with the same retention time as the peak of clobetasol propionate in the chromatogram obtained with solution (1).

4 Determination of clobetasol propionate (0.05 g per 100 g of said ointment) (HPLC method)

0,05 ± 5% 0,0475 – 0,0525

5

Microbial contamination - No. Total microorganisms (aerobic bacteria) / g max. 102 - No. Total yeasts and filamentous fungi / g max 101 - Staphylococcus aureus/g product Absent - Pseudomonas aeruginosa/g product Absent

Determination of related substances of clobetasol propionate was performed by

HPLC method according to the British Pharmacopoeia ed. 2004 - Monograph - Clobetasol Propionate Ointment and Clobetasole propionate, the principle of the method was isocratic elution with UV detection at 240 nm.

The reagents and solvents used were sodium dihydrogen (R) solution of 0.05 mol / l, methanol (R), orthophosphoric acid (R) absolute ethanol (R) acetonitrile (R), HPLC water.

We used an Agilent 1100 HPLC machine, pressure liquid chromatograph equipped with UV lamp, recorder, automatic integration of peak areas. Column chromatography using a stainless steel backing with a length of 100 mm and an internal diameter of 2.1 mm containing octadecylsilane (C18) Hypersil the inner diameter of the particles is 3 microns.

Mobile phase A was made by mixing the following components: 425 by volume solution of 0.05 mol / l sodium dihydrogen the pH adjusted to 2.5 with orthophosphoric acid (A) and 475 volume of acetonitrile, and after cooling the mixture was added 100 volume of methanol (a). Report constituents of the mobile phase was adjusted as appropriate.

Type type elution was isocratic flow rate was 0.4 ml / min, detection UV spectrophotometer at the wavelength λ = 240 nm at room temperature. Sample injection volumes were 10 µl.

Results and Discussion The assay was valid because the chromatogram obtained with solution (2), the

resolution of two peaks corresponding to impurity A and clobetasol propionate is clobetasol propionate was less than 1.5.

The required to impurity A is max. 0.50%, and the result was below the detection limit and within the limits imposed, it is appropriate.

The amount required for other individual impurity is max. 0.50%, and the result was 0.41% and falls within the limits, which is appropriate.


Identification and determination of the clobetasol propionate was carried out by

HPLC method according to the British Pharmacopoeia monograph 2004 - Ointment Clobetasol, the principle of the method was isocratic elution with UV detection at 240 nm.


31

The reagents and solvents used were HPLC water, absolute ethanol (R), clobetasol propionate, s.r.

HPLC method was used an Agilent 1100 liquid chromatograph equipped with pressure UV detector, recorder, automatic integration of peak areas. Column chromatography using a 100 x 2.1 mm, stainless steel, solid-phase ODS, inner diameter 3 mm, and the particles mobile phase that consisted of a mixture of absolute ethanol HPLC and HPLC water in a proportion of 45 : 55 (v / v). The sequence of injections was standard solution, sample test, benchmark, with UV detection at λ = 240 nm wavelength, at 25 º C. Sample injection volumes were 20 µl.

Results and Discussion Chromatograms were considered valid because the relative standard deviation for a

subsequent injection was not less than 2%. Clobetasol propionate corresponding peak in the chromatogram of the sample solution

had the same retention time (14.3 minutes) and about the same size as the corresponding peak in the chromatogram of clobetasol propionate is the standard solution.

Clobetasol propionate imposed values are 0.0475 to 0.0525 g/100g ointment. The result was 0.0492% falling within the limits and appropriate.

Microbial contamination Verification of microbial contamination was performed according to the European Pharmacopoeia:

- Determine the total number of microorganisms (aerobic) and determine the total number of yeasts and filamentous fungi - as head. 2.6.12. Microbiological Examination of Non-sterile products: microbial enumeration tests.

- Pseudomonas aeruginosa, Staphylococcus aureus - under the head. 2.6.13. Microbiological Examination of Non-Sterile Products: Test for Specified Micro-Organisms.

The reagents and culture media used are peptone buffer - pH 7 sodium chloride, polysorbate 80 (Tween 80), 1 M sodium hydroxide, culture medium (Sabouraud), culture medium (agar) gas, cetrimide agar environment, nutrient broth medium, Mannitol salt agar medium. Laboratory equipment was used laminar flow hood (Minifil), electric thermostat Incucell 55, electric thermostat 111 Incucell, balance Kern KB 600-2, colony counter, magnetic stirrer, water bath thermostatic Memert, glassware and petri plates. Microbial contamination was performed by plate counting method. The sample was prepared for analysis as follows: they weighed 10 grams of sample were suspended in 90 ml of buffered peptone - sodium chloride pH 7 and obtained a dilution of 1/10 of the sample taken in the work. Since the analyte is sparingly soluble in water, a surfactant was added (5 g polysorbate 80). The sample thus prepared was homogenized by stirring carefully on a water bath to a maximum of 40 ° C for up to 30 minutes, then adjusted to pH 6-8 with sodium hydroxide solution 1M

Casting method in Petri plates For dilution of 1/10 serial dilutions were prepared: 1/100, 1/1000, using the same

solvent system: pH 7 buffer solution. Petri plates were used with a diameter of 9 cm (two series of three plates) that were

made 1 ml sample of the three dilutions. For each dilution were seeded four Petri dishes.


32

Six petri plates were poured 15-20 ml of culture medium (agar) liquefied and cooled to 45 ° C (bacteria), and the other six were added to 15-20 ml of culture medium (Sabouraud - for fungi).

The plates were incubated at 35 ° C for 5 days and that at 25 ° C for 5 days. Identification of Pseudomonas aeruginosa:

Dilution of 1/10 of the sample buffer pH 7 was inoculated into 100 ml of medium 10 ml nutrient broth. It was stirred and incubated at 30-35 ° C 18-24 hours. After this period of time to perform the transition cetrimide agar medium and incubated at 30-35 ° C 18-72 hours.

Results No microbial growth was detected, the result was negative, the product passed the test. If it was a hit, there is a steering medium beige color yellow - blue - green and then

perform oxidase test, but this has not happened. In this case, a colony isolated on a seed disk is impregnated oxidase. The positive reaction was observed in 5-10 seconds at 25-30 ° C. A delayed reaction may occur in 10-60 seconds, but any change in color after 60 seconds or no change is considered to be a negative reaction. Identification Staphylococcus aureus:

Sample was used and an amount corresponding to 1 g, or 10 mL of the dilution of 1/10 of the sample buffer pH 7 was used to inoculate 100 ml of nutrient broth. It was stirred and incubated at 30-35 ° C 18-24 hours. After this was done passage Mannitol salt agar medium and incubated at 30-35 ° C 18-72 hours.

Results No microbial growth was detected, the result was negative, the product passed the test. If it was a hit, an occurrence of colonies yellow / white surrounded by a yellow zone,

and perform coagulase test using rabbit plasmǎ. This test highlights the species Staphylococcus aureus based on coagulase enzyme production which would lead to clot plasma. The test is negative for other species of the genus Staphylococcus.

Discussion On the blank board Cetrimide specific pathogen Pseudomonas aeruginosa isolated

colonies appeared yellow and sample plate remained unchanged, which means that the product is not contaminated with Pseudomonas aeruginosa.

Plate Mannitol salt agar witness the pathogen Staphylococcus aureus specific and changed color from red to yellow, and the sample plate was unchanged, which means that the product is not contaminated with Staphylococcus aureus.

The results were within the limits imposed ie pathogens were absent and no. Total microorganisms (aerobic) / g and no. Total yeast and filamentous fungi / g product was less than 10 CFU.

Procedure validation of analytical methods Since this method of control product was carried out according to methods formalized

reference pharmacopoeias (EF) there was no need to validate all the parameters of the analytical methods validation tests have been carried out only compatibility of the system, making the necessary adjustments .

However, the following analytical methods were validated: • Determination and identification of active substances in the finished product (HPLC) • Determination and identification of related substances in the finished product (HPLC)


33

6.3. Conclusions The analytical methods used to test Clobetasol ointment product also complies with

GMP and methods described in the British Pharmacopoeia. The product is properly analyzed in terms of physico-chemical and microbiological as obţonute results.

HPLC method for the determination of the clobetasol propionate can be used to determination of trace amounts of clobetasol propionate after removal process residues and impurities from the production facilities.

Quality medicine Clobetasol ointment is appropriate according to the European Pharmacopoeia monograph - Clobetasole propionate 2127 monograph "SEMI-SOLID PREPARATIONS FOR CUTANEOUS APPLICATION" British Pharmacopoeia monograph - Clobetasol Ointment edition 2004 according to "Note for Guidance on Excipients, antioxidants and antimicrobial Preservatives in the dossier for Application for marketing Authorisation of a medicinal product "- EMEA, CPMP/QWP/419/03.

Clobetasol ointment medicine, according to the results obtained, the purpose it was designed. CHAPTER 7 MANUFACTURING TECHNOLOGY OF THE TWO ANTI-INFLAMMATO RY DRUGS ON THE SAME PRODUCTION LINE 7.1. Introduction Manufacturing processes of the two anti-inflammatory drugs with different pharmacological activity were performed on a production line for the production of semisolid topical formulations sterile located in an area classified Class D production cleaning in accordance with good manufacturing practice .

Manufacturing processes have been validated prior to their routine after routine use / during routine use trend study process, study their robustness and reproducibility.

We aimed to establish the manufacturing technology of the two anti-inflammatory drugs and manufacturing equipment because of their thorough knowledge was important in this research and has been input for choosing the method of removing the residues and impurities from the production equipment , selection of sampling points and the choice of sampling methods for removing residues and impurities from the production equipment. Method of application and their achievement has influenced the final outcome of this work. 7.2. Material and methods Production facilities

In accordance with good manufacturing practice, the production of drugs took place inside of " clean room / zone", defined as "the area with defined environmental control in terms of particles and microbial contamination, made and used so as to reduce the introduction, generation, and retention of contaminants in the area ".

Production capacity semisolid forms - threads has been split into two distinct areas: the area classified Class D cleaning (clean) and the unclassified (social clean). Table 7.I – Parameters air for cleanliness class D

Air cleanliness

class

The concentration of the particles suspended in the air (ISO 14644-1:1999)

Contaminare microbiană (UE cGMP, Anexa 1, 2008)

ISO 8 (UE cGMP

Class D)

≥ 0.5 µm, ≤ 3,520,000 particles/m3

air ≥ 5.0 µm, ≤ 29,000 particles/m3 air

Active sampling: 200 UFC/ m3 air Passive sampling (Petri plates with diameter of 90mm): 100 UFC/ 4h


34

Production facilities, particularly "clean areas" were equipped with ventilation type HVAC (Heating, Ventilating, and Air Conditioning) which ensured a purified air quality standards required by good manufacturing practice, treatment systems water (purified water - only water used in drug formulation to remove residues and impurities from production equipment and reagent mixing control laboratories) and secondary areas conditioning (packaging) and control laboratories were equipped with air conditioning.

The temperature in the areas of production and control laboratories was maintained at a comfortable climate in the range 18-25 ° C in both temperature regimes (summer and winter). Production equipment

The production equipment of the station topical semi-solid forms (ointments, creams,

gels) that have been produced the two products were as follows: • Preparation equipment type Fryma - which was intended for the actual preparation of

ointments, creams, gels; • Filling Machine type CO.MA.DIS tubes. SPA - which was intended primary

conditioning tubes bulk product. Raw materials and packaging

Raw materials, both active substances and auxiliary substances used in the

manufacture of these drugs have been pharmaceutical and were supplied by authorized manufacturers. The manufacturer of the active substance possessed Good Manufacturing Practice Certificate and / or Certificate (s) of Suitability to the Monographs of the European Pharmacopoeia (CEPs).

Manufacturing methods and process controls during the technological process of production of the two drugs.

Before each stage of manufacturing process technology have made certain checks to

ensure good manufacturing practice of the two drugs. Check the status of operation of HVAC, process performance was examined residues and impurities removal and disinfection of premises and equipment production was examined health and hygiene staff has verified the presence of documents required for the manufacturing of the product launch, was verified absence of any document, material or packaging material from the product previously manufactured, balance calibration was checked and check environmental conditions: temperature, humidity and pressure in each room. Validation process manufacturing methods

The validation of the manufacturing processes was carried out on three consecutive

series of products. Any method of manufacture involves a number of factors that may affect product quality. These factors have been identified in the development period of the product (preformulation formulation, scale - up) and facilitate process optimization studies. After completing the development phase and after optimization, validation process provided a structured way methodical assessment of the factors that impact on the final product.

Validated preparation was preceded by an analysis of critical points in the manufacturing process.


35

The results of manufacturing process validation of the drug Clobetasol ointment Table 7.IV – The results of validation parameters 1-7

Parameter Expected

working time (min.)

Working time determined (min.)

Series 001 Series 002 Series 003

Parameter 1 of validation - During the preparation of lipophilic phase

35-45 35 40 45

Parameter 2 of validation - propylene glycol heating during the duplicator 5 5 5 5

Parameter 3 of validation - the time of dissolution of clobetasol propionate in propylene glycol

5 5 5 5

Parameter 4 of validation - During the emulsification

10 10 10 10

Parameter 5 of validation - the time of removal of trace amounts of clobetasol propionate

15 15 15 15

Parameter 6 of Validation - During the cooling ointment

40-50 40 45 50

Parameter 7 of validation - During the homogenization of ointment

15-20 20 20 20

Parameter 7 of validation - During the homogenization of ointment For the assessment of homogeneity were determined content of clobetasol propionate

in different locations of the facility to 3 times by homogenization. Sampling was conducted at 10, 15 and 20 minutes of mixing. Location where sampling was done is described below:

S – The sample taken from the top of the preparation vessel, next to the anchor axis A1 M - Samples taken from the middle of the cooking vessel in the vicinity of the wall (M1 and M3) and near the impeller (M2) I - Sample taken from near the bottom of the drain installation. T1 =10 min.; T2 = 15 min.; T3 = 20 min.

Parameter 8 of validation - control of the finished product Table 7.VIII. The results obtained in control of the finished product Clobetasol ointment

No. crt.

Characteristics Admissibility limits on release Results - of the 3 series prepared in

installation type Fryma Series 001 Series 002 Series 003

1 Appearance, color, odor, homogeneity

semisolid mass, homogeneous, unctuous to the touch, white with

a characteristic odor of excipients

coresp. coresp. coresp.

2 Identification of clobetasol

- HPLC

The chromatogram obtained with solution (2) has a peak with the same retention time as the peak of clobetasol propionate in the chromatogram obtained with

solution (1).


3

Related substances. (HPLC) - Individual Max. 0,5% 0,39 0,33 0,35 - Impurity A (clobetasol) Max. 0,5% ND ND ND - Total impurities Max. 2% 0,39 0,65 0,35

4 Determination of clobetasol propionate (0.05 g/100 g ointment declared)

0,0475 – 0,0525 0,0496 0,0491 0,0493

A1

A3

A2

M1

M2

M3

I

S


36

No. crt.



(method HPLC)

5

Microbial contamination - No. Total microorganisms (aerobic bacteria) / g

max. 102 UFC < 10 < 10 < 10

- No. Total yeasts and filamentous fungi / g

max 101 UFC < 10 < 10 < 10

- Staphylococcus aureus/g product

Absent Absent Absent Absent

- Pseudomonas aeruginosa/g product

Absent Absent Absent Absent

6 Total weight g / tube The average mass min. 20g 20,000 20,001 20,000 Individual min. 20g - 10%

(min.18 g) Coresp. Coresp. Coresp.

ND – not detected The results of manufacturing process validation of the drug Fenilbutazonă cremă Table 7.IX – The results of validation parameters 1-8

Parameter Expected

working time (min.)

Working time determined (min.)

Series 001 Series 002 Series 003

Parameter 1 of validation - during melting and preparation of lipophilic phase

40-60 50 50 50

Parameter 2 of validation - during the preparation of the hydrophilic phase

5-10 5 5 5

Parameter 3 of validation - the time of dissolution of the preservatives into the hydrophilic phase

5 5 5 5

Parameter 4 of validation - phase during emulsification hydrophilic lipophilic phase

10 10 10 10

Parameter 5 of validation - during homogenization and cooling emulsion

20-30 30 25 30

Parameter 6 of validation - the time of dissolution of camphor and menthol

20-30 20 20 20

Parameter 7 of validation - during the suspension of the active substance

15-20 15 15 15

Parameter 8 of validation - the time of incorporation of the alcoholic solution of menthol and camphor and suspension

10-15 5 5 5

Parameter 9 of validation - during the homogenization of the cream For the assessment of homogeneity were determined phenylbutazone content in

different locations of the installation for preparing the homogenizer 3 times. Sampling was conducted at 10, 15 and 20 minutes of mixing. Location where sampling was done is described below:

S – The sample taken from the top of the preparation vessel, next to the anchor axis A1 M - Samples taken from the middle of the cooking vessel in the vicinity of the wall (M1 and M3) and near the impeller (M2) I - Sample taken from near the bottom of the drain installation. T1 =10 min.; T2 = 15 min.; T3 = 20 min.

Parameter 10 of validation - control of the finished product

A1

A3

A2

M1

M2

M3

I

S


37

Tabelle 7.XIII. The results obtained in control of the finished product Phenylbutazone cream

No. crt.



1 Appearance, color, odor and consistency

homogeneous cream is yellowish-white color and characteristic odor of camphor and

menthol coresp. coresp. coresp.

2 Particle size Below 50 microns coresp. coresp. coresp.

3 The pH of the aqueous dispersion 10%

4,5 – 5,5 5.00 4.98 5.00

4 Identification camphor, menthol (GC)

corresponding peak menthol, camphor that the chromatogram of the sample solution

should be similar to the corresponding peak of menthol, camphor that the chromatograms

of the standard solution


5 Identification preservatives (HPLC)

corresponding peaks preservative substances on the chromatogram of the sample solution

were similar retention time and size approximately equal to the corresponding

peaks preservative substances on chromatograms of standard solutions


6 Quantitative Determination of phenylbutazone (g/100 g cream) (HPLC)

3,88-4,12 3.92 3.95 3.93

7 Quantitative Determination preservatives - nipaesteri (g/100g cream) (HPLC)

1. methyl p-hydroxybenzoate 0,054 – 0,066 g 0.0587 0.0585 0.0585

2. p-hydroxybenzoate n-propyl 0,036 – 0,044 g 0.0407 0.0402 0.0408

8 Dosing camphor (g/100 g cream) (GC) 2,70-3,30 2.98 2.99 2.98 9 Dosing menthol (g/100 g cream) (GC) 0,90-1,10 1.00 1.00 0.99

10

Phenylbutazone related substances (HPLC): - Impurity A - Impurity B - Impurity C - Other individual impurities - Total Impurities

Max. 0,25% Max. 0.25% Max. 0,20% Max. 0,10% Max. 0,50%

ND 0.20 subD 0.07 0.27

ND 0.21 subD 0.07 0.28

ND 0.21 subD 0.06 0.27

11

Microbial contamination • no. Total microorganisms

(aerobic bacteria) / g cream • no. Total yeasts and filamentous

fungi / g cream • Staphylococcus aureus / g cream • Pseudomonas aeruginosa / g cream

max. 102 UFC max. 101 UFC

absent absent

< 10

< 10

absent absent

< 10

< 10

absent absent

< 10

< 10

absent absent

12 Masa totală, g/tub The average mass min. 35 g

Individual min. 35 g-10% (31,5 g) 35.001 coresp.

35.00 coresp.

35.00 Coresp.

ND – not detected, subD – below detection limit Discussion Evaluation and statistical interpretation of the results of the validation of the manufacturing process for Clobetasol ointment Statistical study of homogenization time Tabelul 7.XIV. Descriptive statistical parameters of the process of homogenization (the concentration of clobetasol propionate)

The mixing time (min.) Series

15 20 25 Statistical parameter

Series 001 0.04944 0.05008 0.04974 Medium content in clobetasol propionate Series 002 0.0491 0.04946 0.04972


38

Series 003 0.04964 0.04944 0.04972 (g/100 g ointment)

Series 001 0.00052249 0.00046043 0.000541 Standard deviation Series 002 0.00035355 0.000152 0.000217

Series 003 0.000251 0.000416 0.000335 Series 001 1.05682447 0.91939812 1.088248

Coefficient of variation Series 002 0.72006801 0.306627 0.436031 Series 003 0.5056366 0.841288 0.673097

For each series included in the validation study, it was observed that with increasing homogenization time there has been a decreasing trend coefficients of variation calculated for values obtained from the concentration of clobetasol propionate in all five points of the ointment. It was noted that the value determined for clobetasol was within the specified limits, the coefficient of variation of the data was below 2 all three times (10, 15 and 20 minutes). Interpretation results of finished product control Clobetasol ointment Table 7.XV. Descriptive statistical parameters of control of the finished product

Parameter analyzed Parameter statistically

Results - of the 3 series prepared in installation type Fryma

001 002 003 Quantitative determination clobetasol propionate (g/100 g ointment)

Media 0,049333 Standard deviation 0,000252 Coefficient of variation 0,510124

Quantitative determination of the impurities (g/100 g of ointment)

Imp. Individual Total imp. Media 0,356667 0,463333 Standard deviation 0,030551 0,1628905 Coefficient of variation 8,565562 35,156235

Total weight, g / tube Media 45,001 Standard deviation 0,001 Coefficient of variation 0,002222

The statistical analysis of the control of the finished product Clobetasol ointment was observed that there was no statistically significant variation between the series 001, 002 and 003 produced the results that were obtained were within the specified limits. Evaluation and statistical interpretation of the results of the validation of the manufacturing process for Phenylbutazone cream Statistical study of homogenization time Table 7.XVI. Descriptive statistical parameters of the process of homogenization (phenylbutazone concentration)

The mixing time (min.)

Series 10 15 20 Statistical parameter

Series 01.05.13 3.946 3.942 3.942 Medium content in phenylbutazone (g/100 g cream)

Series 002.05.13 3.942 3.94 3.94 Series 003.05.13 3.94 3.932 3.946 Series 001.05.13 0.02073644 0.008367 0.008367

Standard deviation Series 002.05.13 0.0083666 0.012247 0.01 Series 003.05.13 0.01581139 0.01095445 0.011402 Series 001.05.13 0.52550536 0.212243 0.212243

Coefficient of variation Series 002.05.13 0.21224252 0.310849 0.253807 Series 003.05.13 0.40130427 0.27859744 0.288945

For each series included in the validation study, it was observed that with increasing homogenization time there has been a decreasing trend coefficients of variation calculated for values obtained from the concentration of phenylbutazone in all five points of the cream. It was noted that the value determined for phenylbutazone was within the specified limits, the coefficient of variation of the data was below 2 all three times (10, 15 and 20 minutes).


39

Interpretation results of finished product control Phenylbutazone cream Table 7.XVII. Descriptive statistical parameters of the finished product control Phenylbutazone cream

Parameter analyzed Parameter statistically

Results - of the 3 series prepared in installation type Fryma

001 002 003

The pH of the aqueous dispersion 10%

Media 4.993333 Standard deviation 0.011547 Coefficient of variation 0.231248

Quantitative Determination of phenylbutazone (g/100 g cream)


Quantitative Determination of camphor (g/100 g cream)


Quantitative Determination of menthol (g/100 g cream)


Quantitative Determination preservative (g/100g cream)

Nipagin Nipasol Media 0.058567 0.040567 Standard deviation 0.000115 0.000321 Coefficient of variation 0.19716 0.792412

Total weight, g / tube


The statistical analysis of the finished product Phenylbutazone control cream was observed that there was no statistically significant variation between the series 001, 002 and 003 yielded results that were within the specified limits.

Conclusions

Manufacturing processes of the two different potent anti-inflammatory drugs were made in production facilities medications on line semisolid topical preparation on specific production equipment pharmaceutical industry according to good manufacturing practice.

Validation studies have confirmed that the method of manufacture of drugs Clobetasol ointment and Phenylbutazone cream is consistent and robust and resulted in a finished product that met the quality requirements.

We have found that the manufacturing process was stable since there were no significant differences between the series included in the validation study in which the check of the control parameters of the finished product.

Validation studies have verified the methods of manufacturing processes by performing test conditions and / or extreme point possible during manufacturing processes and controls, thus demonstrating that it retains control of the process, even in extreme conditions. Once validated, it is considered that maintaining control processes given that their parameters remain unchanged.

On the basis of the obtained results in the technological processes for the two drugs were consistent, thus ensuring reproducibility of the production batch and quality of the finished product, and constantly uniform quality in accordance with the product.


40

CHAPTER 8 VALIDATION OF THE PROCESS FOR REMOVING IMPURITIES A ND RESIDUE FROM THE MANUFACTURING EQUIPMENT FOR MANUFACTURING THE TWO PRODUCTS 8.1. Introduction

Due to the fact that pharmaceutical active substances and pharmaceutical products (FAS) can be contaminated by other pharmaceuticals or other FAS, the cleaning agents, the micro-organisms or other materials (eg, dust, dirt, lubricants, raw materials, intermediates, auxiliaries) and in many cases, the same equipment (machinery) is used to cook (process) different products, waste removal procedures and appropriate impurities are essential to avoid contamination of pharmaceutical products in question.

In order to validate the process of removing the residues and impurities from production equipment, we intend to establish medicines by which to perform validation process to remove residues and impurities removal processes residues and contaminants, sampling points to perform physicochemical and microbiological analyzes to determine traces of active substances, cleaning agents, microbiological contaminants.

The purpose of the validation of the cleaning was to confirm the veracity of the procedure for removing the residues and impurities, so that the analytical monitoring may be omitted or minimized to play routine. Selection of anti-inflammatory drugs with different pharmacological potency

Antiinflammatory drugs with different pharmacological activities have been studied semi-solid pharmaceutical forms for non-sterile topical (creams and ointments).

To demonstrate that the same production line technology can be manufactured two drugs with different pharmacological potency, firstly, these drugs were selected from a list of drugs that produce sterile semisolid topical on the same production line. To make this selection was necessary scientific evaluation of all drugs are manufactured in the production department of semisolid topical sterile, applying well-chosen selection method.

Selection and two anti-inflammatory drugs with different pharmacological activity represented a very important step, is just one of the critical points in the scientific research, since this stage depended on the final outcome of the research and therefore its application in practice.

Material and methods Method of selection of both drugs was performed after the removal process validation

residues and impurities was made on the principle of "worst case" using the method of "bracketing".

Objective method of "bracketing" is to demonstrate that there is a scientific justification for the "worst" drug evaluation program validation process of removing impurities from waste and production equipment. The first thing that was done was to make groups and sub-groups - which we have called "bracketing" which were subsequently selected "worst cases" based on evaluation results.

The two drugs were selected from the list of products that are manufactured in the same production departments at the time when we started this study. Selection of potent active after the validation process was performed to remove residues and impurities

Selection was made pursuing the "worst", and the activity of the active substance was first selection criterion.


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Potent active then became one of the validation process for removing residues and impurities was Clobetasol ointment. Selecting drug that performed poorly active after validation process residues and impurities removal

Selection was made pursuing the "worst case" and dispersive method based medicine was first selection criterion.

Medicines that performed poorly active after a second validation process residues and impurities removal was Phenylbutazone cream.

Results Using this method of selecting "bracketing" on the "worst case", the following

products were selected: The first product selected highly active drug, was:

• Clobetasol propionate ointment pharmaceutical form (Clobetasol ointment) The other product selected poorly active drug, was:

• Phenylbutazone in a cream formulation (Phenylbutazone cream)

Discussion Clobetasol propionate was selected in two of the four "worst case" required: the

activity of the active substance, the solubility of the active substance in water and the NaOH solution 0.1 mol / lg / ml, in addition,

Clobetasol ointment product besides contains a highly active substance - strong steroid formulation of semisolid topical ointment is harder than the other formulations washable semisolid topical cream and gel.

Clobetasol ointment is part of highly active corticosteroid drugs, and in accordance with good manufacturing practice should be made in the campaign or technological production line dedicated to this category of product.

Phenylbutazone cream was selected in three of the four "worst case" requirements: the amount of active substances / product Serial solubility of active substances in water and NaOH solution 0.1 mol / lg / ml and how dispersion of the active substance into the cream / ointment / gel, in addition,

Phenylbutazone cream product besides phenylbutazone contains and camphor and menthol.

Phenylbutazone cream is part of the NSAIDs drugs which according to the rules of good manufacturing practice could be manufactured on a production line of a factory production which is highly active cortosteroid.

Conclusions From a total of 14 drugs, using the method for selecting "bracketing" on the "worst

case" has selected two anti-inflammatory drugs with different pharmacological activity: • one of the drugs after I performed the validation process for the removal of waste and

debris production equipment was Clobetasol ointment. • the second product after I performed the validation process for the removal of waste

and debris production equipment was Phenylbutazone cream. As the activity of the active substance outweigh the other three "worst" Clobetasol

ointment is the drug target validation process after which we performed to remove residues and impurities from production equipment and Phenylbutazone cream is the product after I


42

performed another validation removal process residues and impurities to demonstrate that both drugs can be manufactured on the same production line without sacrificing quality.

The validation process for removing residues and impurities such equipment we made after 3 cycles to remove residues and impurities after 3 consecutive series of product selected. Methods of removing residue and contaminants from the production equipment after the manufacture of the two products

Material and methods The choice of methods to remove residues and impurities was based on the

composition of the products manufactured production equipment (heavy or light skimmed solubility of active substances in cleaning agents), depending on how the manufacture of these drugs and depending on how operation of production equipment, including we took into account the materials they are made of these machines.

We chose a method of removing waste and impurities taking into account the physico - chemical properties of active substances and pharmaceutical form which are made on the equipment manufacturing.

Total removal of traces of active substances and trace cleaner trace determination demonstrated by analysis of active substances and cleaning agents. Method for removal of residues and contaminants was accepted following test results that were within the limits of admissibility imposed.

Time to remove residues and impurities was determined according to the workload of the equipment, the agent's instructions for cleaning and was mentioned in the description of how to remove residues and impurities for each piece of equipment.

Temperature was between 50-60 � C for roughing according to the data sheet from the manufacturer of the cleaning agent, between 20-25 � C for Disinfectants stage equipment according to manufacturer data sheet cleaning agent and between 20 - 25 � C with purified water rinsing step.

Concentration cleaners, detergent and disinfectant, was according to the manufacturer's data sheet. The detergent was used at a concentration of 2%, the disinfectant year was used in a concentration of 0.2%.

The removal of residues and dirt (cleaning) was made by immersion stirred for equipment for the preparation and static immersion of the parts of the divided tube, the transfer pump components have been removed and cleaned separately.

The number of cycles was three times rinsing with purified water were sampled for physico-chemical and microbiological analysis after each rinse and after they have completed their analyzes and interpreted the results, it was determined a two rinse cycles.

The degree of removal: The preparation equipment is not removed, remove residues and impurities was

performed according to the method of removing semi residues and impurities determined. Equipment division in aluminum tubes and transfer pumps were disassembled and cleaned manually to the method of removing the residues and impurities determined.

Results and Discussion The removal of residues and impurities from the production equipment was

appropriate, all production equipment, parts removable Annex became clean dishes were prepared for sampling to assess the process of removing the residues and impurities.

Large vessels with flat surfaces were cleaned automatically or running water, and for some loose parts was required manual cleaning using brushes.


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Were used detergents and disinfectants suitable for pharmaceutical and medicinal products which have characteristics produced by the production equipment.

The residue and debris removal was performed until he secured a proper wash each piece of equipment, waste removal process requirements and impurities are observed. Time to remove residues and impurities, temperature, concentration of cleaning agents stated in the method of removal of waste and debris from the work equipment has been strictly adhered to.

Conclusions At the end of the process of removing impurities and residue from the manufacturing

equipment, due to the fact that there were no deviations from the working method and the process according to the over and fixed been observed parameters (temperature, contact time solution concentration, etc.), we considered making the removal process residues and impurities from the work equipment as appropriate.

The removal of residues and dirt was applied to three consecutive series of the product and samples were taken after each application three times with purified water rinsing. The sampling after removing residues and impurities from the production equipment

Sampling was carried out from points that were chosen taking into account the equipment surfaces that come in contact with the product in production. These areas have been divided in two categories:

accessible surfaces; inaccessible areas.

Material and methods Sampling technics used were swab method and rinsing method. The swab method of sample was used for surface grinding. This method of sampling

using hydrophobic sterile swab is effective for (remove) very poorly soluble substances and cleaning agents used in this validation (P3-cosa products, according to the manufacturer).

The advantages of this method are easy availability flat surface, the mechanical removal of residues from the production equipment possible.

The rinsing method was used for hard to reach areas. Sampling method rinsing means another extra rinse after removing the residues and impurities was considered complete. The advantage of this method is that a very large area can be covered. The solvent used for the final rinsing should ensure absorption of all the residues from the production equipment. It was necessary to determine the recovery rate because the calculation was based on the acceptance criterion samples buffer method.

The sampling Samples were taken after completion of removal of the residues and impurities from

the points indicated by means of the rinsing and swab method. Individual samples were taken for each parameter analyzed, namely:

• samples for microbiological analysis (for total and pathogenic strains germeni/cm2) • samples for physico-chemical analysis (for the quantitative determination of traces of

active and traces of cleaning agent) Samples were taken after the first rinse cycle (time I) with purified water, after rinsing

the second time (time II), and after a third cycle rinse time (time III ) with purified water. For sample no. 5 sampled by rinsing method for microbiological analyzes was collected and purified water sample before making rinsing machine parts.


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Results The result of this process was the appropriate sampling, all samples were taken from

the points indicated, observing the quantity and surface sampling. There are a total of 12 samples taken 6 samples to determine the physical-chemical

and microbiological determinations 6, additionally a sample was collected and purified water. Discussions The points from which samples were taken are indicated. Sampling was done by the

rinsing and swab method as indicated. Samples were taken from the first rinse cycle (time I), after the second flush cycle

time (time II) after the third rinsing step (time III ). A rinse meant rinsing for 10 minutes with the indicated amount of purified water to remove residues and the instructions for each machine individually impurities.

The samples by rinsing method for microbiological analyzes were collected and purified water samples before making the first rinse cycle to make a comparison between the microbiological quality of purified water before and after rinsing.

Conclusions At the end of the sampling process, because there were no deviations from the work

instructions and that this process went according to and follow all parameters were fixed (points, surface sampling, sample materials and tools, amount of samples, etc.), we considered as appropriate sampling.

CHAPTER 9 ANALYSIS OF SAMPLES AFTER THE REMOVAL PROCESS OF RE SIDUES AND IMPURITIES FROM THE PRODUCTION EQUIPMENT 9.1. Introduction

After samples were taken after the removal process residues and impurities from production equipment, they were analyzed for physico-chemical and microbiological. in order to assess the effectiveness of the removal of the residues and impurities, the following tests were performed: determinarea cantitativă a urmelor de substanţă activă

• Microbiological determinations � determine the total number of germs / cm2 � determining the pathogenic strains

• Qquantitative determination of traces of disinfectant P3-cosa DES We aimed to establish control parameters, limits the admissibility and experimental

conditions of analysis for determining trace active substance (phenylbutazone and clobetasol propionate), the determination of traces of disinfectant (P3-cosa DES) and to determine microbial contamination. Quantitative determination of trace amounts of active substance (phenylbutazone, clobetasol propionate) was conducted by HPLC method and the determination of traces of disinfectant was carried out by three methods specified by the manufacturer: determining the conductivity of P3-cosa the quantitative determination and the determination acid DES titrimetrically peracetic "analytical test strip for peroxide".

Material and methods

The control parameters and limits acceptance for validating removal process of residues and impurities

Regarding the extent of the work and the prospects of a successful outcome in the validation process for removing residues and impurities, determination of appropriate limits


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for residues allowed on production equipment has a very important role. For this reason, in determining the acceptance limits for the validation of the residue removal process residues and impurities have required the strict criteria.

Eligibility limits for microorganisms were: o For total germeni/cm2 = 1 CFU / 1 cm2 o For pathogenic strains = ABSENT

Eligibility limit for trace was active = ABSENT Limits eligibility for traces of the cleaning agent are:

o determination of traces of disinfectant P3-cosa = DES by determining conductivity below 4 ppm

o determination of traces of disinfectant P3-cosa DES by "analytical test strip for Peroxide" = less than 1 ppm

o determination of traces of disinfectant P3-cosa DES (titrimetrically) = ABSENT

Quantitative determination of phenylbutazone trace-HPLC method

Quantitative determination of the trace phenylbutazone was carried out by HPLC analytical method, the principle of the separation method is HPLC with UV detection at a wavelength λ = 254 nm.

The reagents and solvents used were methanol (A), acetonitrile, HPLC, doubly distilled water, orthophosphoric acid (R).

HPLC method was used an Agilent 1100 liquid chromatograph equipped with pressure UV lamp, recorder, automatic integration of peak areas. Column chromatography using a 150 x 4.6 mm, stainless steel, solid-phase octylsilane (C8) Zorbax Eclipse XDB C8, 5 mm particle diameter, and the inner mobile phase consisting of a mixture of acetonitrile and water bidistilled HPLC in a ratio of 75: 25 (v / v) and 100 ml of mobile phase was added 0.1 ml of orthophosphoric acid (R). The flow rate was 1ml/min with UV detection at λ = 254 nm wavelength, at a temperature of 25 ° C. Sample injection volumes were 20 ¶.

For samples swab method After the samples have been taken, the hydrophobic sterile cotton swab was brought in

a 100 ml volumetric flask. Hydrophobic sterile cotton swab was washed with 20 ml methanol 4 times, then brought to volume with methanol. 2.5 ml of the solution thus obtained was transferred into a 25 ml volumetric flask and brought to volume with mobile phase.

Of this solution was injected into the column 20 � it octylsilane (C8) Zorbax Eclipse XDB C8 particle diameter inner 5 mm, with a flow rate of 1mL/min., � = 254 nm. The retention time was 2.75 minutes.

The samples by rinsing method After they were taken were taken and 2.5 ml of this solution were transferred to a 25

ml volumetric flask and brought to volume with mobile phase. Of this solution was injected into the column 20 � it octylsilane (C8) Zorbax Eclipse

XDB C8 particle diameter inner 5 mm, with a flow rate of 1mL/min., � = 254 nm. The retention time was 2.75 minutes.

Results and discussion Determination of traces of phenylbutazone result was below the limit of detection for

all times to rinse. In the chromatogram, the retention time of phenylbutazone (2.7 minutes) has occurred

during a detection signal from the flush I, II and III, and the times did not show any signal detection (below detection limit), the the specific signal peak occurred only for injection to 1.5 minutes.


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Active trace detection results (phenylbutazone) at times II, III rinsing were within the limits imposed - absent. Quantitative determination of clobetasol propionate trace-HPLC method

Quantitative determination of trace amounts of clobetasol propionate was carried out by HPLC analytical method, the principle of the separation method is HPLC with UV detection at a wavelength λ = 240 nm.

The reagents and solvents used were methanol (A), acetonitrile (R), sodium dihydrogen phosphate monohydrate (R), sodium hydroxide (R), clobetasol propionate CRS.


Mobile phase I obtained by mixing 10 volumes of methanol (A), a solution of 42.5 volumes of 7.85 g / l solution of sodium dihydrogen phosphate monohydrate (R), which was adjusted to pH - 5.5 with a solution 100 g / l sodium hydroxide solution (R) and 47.5 volumes of acetonitrile (a);

The flow rate was 1.0 ml / min with UV detection at a wavelength λ = 240 nm at room temperature. Volumes of test solution (b) and reference (a) were injected 10 ml. Analysis time was 3 times the retention time of clobetasol propionate.

For samples swab method After the samples have been taken, the hydrophobic sterile cotton swab was brought

into a 10 ml volumetric flask. Hydrophobic sterile cotton swab was washed with 2 ml of methanol and was brought to the mark with mobile phase.

Were injected separately each 10 ml of the standard solution and the sample solution to be analyzed.

The samples by rinsing method After samples were taken were taken and 2 ml of this solution were transferred to a 10

ml volumetric flask and brought to volume with mobile phase. Were injected separately each 10 ml of the standard solution and the sample solution

to be analyzed. Results and discussion Result clobetasol propionate trace determination was below the detection limit for all

times to rinse. The chromatograms, the retention time of clobetasol propionate (14.3 minutes) did not

show any signal detection (below detection limit), but other evidence under 13 minutes, possibly detected traces of disinfectant as chromatograms. Active trace detection results (clobetasol propionate) at times I, II, III rinsing were within the limits imposed - absent. Quantitative determination of traces of disinfectant agent P3-cosa DES Quantitative determination of traces of disinfectant agent was performed by three analytical methods specified by the manufacturer: determining conductivity quantitative determination of P3-cosa DES titrimetrically and determination of peracetic acid "analytical test strip for peroxide". The reagents and solvents used had been purified water, potassium iodide (solid), 25% sulfuric acid solution, 3% ammonium molybdate, 1% starch solution, a solution of 0.1 N sodium thiosulfate.


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Laboratory apparatus used was conductivity, titrimetru automatically Merckoquant Test Peroxide (1.10011.0001) (analytical test strips for detection and semiquantitative determination of peroxides), magnetic stirrer and glassware.

Preparation of samples For samples swab method

Determination of traces of disinfectant by determining the conductivity Purpose: This method was tested using aqueous samples containing very small

amounts of P3-cosa DES. The method is selective for the particular all substances which have a conductivity (ionic substances dissolved in water may interfere with the test example).

After the samples have been taken, the hydrophobic sterile cotton swab was brought in a volumetric flask of 250 ml of water. Hydrophobic sterile cotton swab was washed with 50 ml of water 4 times, then brought to volume to 250 ml with purified water. This quantitative solution was put in a 2000 ml flask and brought to volume. The sample thus obtained was stirred in a magnetic stirrer for 3 minutes at 400 rpm. Then immediately conductivity was measured using conductometric.

The result was inserted into the equation: y = 0,159x + 0,3705,

where y is the conductivity in mS / cm, and x is the concentration in ppm P3-cosa. Determination of traces of disinfectant by quantitative determination of P3-cosa DES (titrimetrically)

After the samples have been taken, the hydrophobic sterile cotton swab was brought into a 25 ml volumetric flask. Hydrophobic sterile cotton swab was washed with 5 ml of water 4 times, then brought to volume to 25 ml with purified water. Determination:

Titration: 10 ml of solution taken from a hydrophobic sterile cotton swab was brought in an

Erlenmeyer flask, and were added 20 ml of 25% sulfuric acid. Was added a spatula tip of potassium iodide and 1 ml of 3% ammonium molybdate solution was left in place for 1 to 2 minutes.

Then it was titrated with 0.1 N sodium thiosulfate until the blue color disappeared. Calculation of concentration:

Volume of 0.1 N sodium thiosulphate used in the titration (n/10) x 0.064 =% P3-cosa DES. Determination of traces of disinfectant by determination of peracetic acid "analytical test strip for peroxide"

Purpose: This method was tested using aqueous samples containing small amounts of P3-cosa DES. The assay is useful concentrations of P3-cosa Des between 0.8 to 50.8 mg / l (ppm).

For this determination were used Merckoquant Test Peroxide (1.10011.0001) (analytical test strips for detection and semiquantitative determination of peroxides)

The method of determination was based on the peroxidase. The enzyme OXYGEN transfer of hydrogen peroxide (P3-cosa ingredient DES) in an organic redox indicator, which is then passed into an oxidation product of a blue color.

After the samples have been taken, the hydrophobic sterile cotton swab was brought into a 25 ml volumetric flask. Hydrophobic sterile cotton swab was washed with 5 ml of water 4 times, then brought to volume to 25 ml with purified water.


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Test strip was inserted deep into the solution for 1 second and then removed from the solution and to remove excess fluid and compared the reaction zone scale color vision after 15 seconds.

The samples by rinsing method Determination of traces of disinfectant P3-cosa DES through the conductivity

After the samples have been taken, the solution was brought to quantitatively to a 2000 ml flask and brought to volume. The sample thus obtained was stirred in a magnetic stirrer for 3 minutes at 400 rpm. Then immediately conductivity was measured using conductometric.

The result was introduced in the equation: y = 0,159x + 0,3705,

where y is the conductivity in mS / cm, and x is the concentration in ppm P3-cosa DES. Determination of traces of disinfectant by quantitative determination of P3-cosa DES (titrimetrically)

After the samples were taken, 10 ml were taken and brought in an Erlenmeyer flask and added 20 ml of 25% sulfuric acid. Was added a spatula of potassium iodide and type 1 ml of 3% ammonium molybdate solution was left in place for 1 to 2 minutes. Determination:

Titration: Then it was titrated with 0.1 N sodium thiosulfate until the blue color disappeared.

Calculation of concentration: Volume of 0.1 N sodium thiosulphate used in the titration (n/10) � 0.064 =% P3-cosa

DES. Determination of traces of disinfectant P3-cosa frequently through the peracetic acid "analytical test strip for peroxide"

Once you have collected samples were taken 10 ml solution and test strip inserted deep into the solution for 1 second and then removed from the solution and to remove excess fluid and compared the reaction zone by visual color scale 15 seconds.

Results and discussion Determination of traces of disinfectant P3-cosa DES - titrimetrically

- The addition of 0.1 N sodium thiosulfate aqueous solution was decolorized (at time I), which indicates that traces of disinfectant solution

- The addition of 0.1 N sodium thiosulfate aqueous solution was not changed the color of the blue (at time II and III ), which indicated that the solution did not present evidence of a disinfectant.

Calculation of concentration: The volume of sodium thiosulfate 0.1 N used for the titration (n/10) × 0,064 = % P3-cosa DES

Vblank=0,10 ml For time I : Vcons was: 0,11 ml, 0,12 ml, 0,13 ml and 0,14 ml The results were between → (0,001, 0,002, 0,003 şi 0,004) × 0,064 = 0,000064% = 0,64ppm, 1,28ppm, 1,92ppm and 2,56ppm For time II şi III : Vcons was 0,10 ml The results were → 0,00 × 0,064 = 0,00% = 0ppm


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Determination of traces of disinfectant P3-cosa DES - conductivity - For the time I observed that the conductivity was higher, which led to results between

3.33 ppm - 4.59 ppm (below and above 4ppm); - For the time II was observed that the conductivity decreased, which led to results

ranging from 0.18 ppm - 2.07 ppm (below 4ppm). - For the time III has been observed that the conductivity decreased more, which led to

the result of 0ppm (below 4ppm). The result was inserted into the equation:

y = 0,159x + 0,3705, where y is the conductivity in mS / cm, and x is the concentration in ppm P3-cosa DES. The conductivity of the water is 1,3 µS/cm For time I The conductivity was: y = 2,2 - 2,3 şi 2,4 µS/cm → y = 0,159x + 0,3705 → The results were: x = 3,33ppm, 3,96ppm şi 4,59ppm; For time II The conductivity was: y = 1,7 – 1,8 – 1,9 – 2,0 µS/cm → y = 0,159x + 0,3705 → The results were: x = 0,18ppm, 0,81ppm, 1,44ppm şi 2,07ppm For time III The conductivity was: y = 1,3 µS/cm → y = 0,159x + 0,3705 → The results were: x = 0 ppm; Determination of traces of disinfectant P3-cosa DES - Analytical test strip peroxide.

- was observed a weak blue color of the test strip for time I, corresponding to 1.3 ppm standard strip - more than the permissible limit

- was observed that the test strip for time II and III remained white even after 20 minutes (below 0.8 ppm)

The determinations for microbial contamination

The culture media used were melted and cooled agar medium at 45 � C (for bacteria), Sabouraud (for fungi) Petrii cast slabs, Escherichia coli - MacConkey broth medium, Staphylococcus aureus - mannitol agar medium, Pseudomonas aeruginosa Cetrimide-environment agar.

Laboratory apparatus used was a laminar flow hood (Minifil), electric thermostat Incucell 55, electric thermostat 111 Incucell, balance Kern KB 600-2, colony counter, magnetic stirrer, water bath thermostatic, glassware.

The method of analysis for samples by swab method

Determining the total number of germs / cm2

After the samples have been taken, the hydrophobic sterile cotton swab was placed in a test tube with 9 ml of 0 1% peptone water was allowed to stand for 15 minutes to vigorously stirred to disperse the bacterial concentration. Thus obtained crude suspension (dilution 1/10).

The crude suspension was made of 1/100 dilution (1 ml crude was taken over which water was added 9 ml peptone 1% 0).

From each dilution 1 ml was incorporated in the agar medium melted and cooled to 45 � C (for bacteria) and Sabouraud (for fungi) Petrii cast slabs.

Agar plates were incubated in the thermostat at 37 � C for 48 hours, after which the counter to count colonies colonies.


50

Sabouraud plates were incubated in the thermostat at 25 � C for 7 days, after which colony counter counting colonies. Determination of the pathogenic strains

From dilution 1/10 to 1 ml seeded on enrichment media: • Escherichia coli - MacConkey agar medium • Staphylococcus aureus - mannitol agar medium • Pseudomonas aeruginosa Cetrimide agar-medium

Was thermostated at 37 � C, 48 h, was to increase the potential pathogenic strains. Then passages were made of each strain specific agar media.

The method of analysis for the samples by rinsing method Determining the total number of germs

After sampling was performed to determine the total number of bacteria on membrane filtration method. 10 ml of sample was filtered through membrane filters with a pore diameter of 0.45 � m After filtration, the filter was transferred to the surface of media: agar (for bacteria) and Sabouraud (for fungi)

Petrii plates were incubated in the thermostat at 37 � C (agar) for 48 hours at 25 � C (Sabouraud) for 7 days. Colonies were counted and are reported in 1 ml. Determination of the pathogenic strains After sampling was performed to determine the pathogenic strains by the membrane filtration method. 10 ml of sample was filtered through membrane filters with a pore diameter of 0.45 � m After filtration, the filter was transferred to the surface of specific culture media:

• Escherichia coli - MacConkey agar medium • Staphylococcus aureus - mannitol agar medium • Pseudomonas aeruginosa Cetrimide agar-medium

Was thermostated at 37 � C, 48 h, was to increase the potential pathogenic strains. Results and discussion

To determine the pathogenic strains - No pathogenic strains developed (for the time I, II, III ) - The samples by rinsing method was performed and analysis of purified water before

inserting pieces of equipment. This analysis was performed to make a comparison of purified water before rinsing and after rinsing. As can be seen, the values of the results are the same, so purified water has not changed microbiological parameters - pathogenic strains - after rinsing.

To determine the total number of germs / cm2 or / 1ml - Total number of germs / cm2 was within permissible limits (for the time I, II, III ) - Total number of germs / 1 ml was within the permissible limits (for the time I, II, III ) - The samples by rinsing method was performed and analysis of purified water before

inserting pieces of equipment. This analysis was performed to make a comparison of purified water before rinsing and after rinsing. As can be seen, the values of the results are almost identical, so purified water has not changed microbiological parameters - total number of bacteria / 1 ml - after rinsing.


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Conclusions The results of measurements performed to validate the process of removing impurities

from waste and production equipment for the manufacture of anti-inflammatory drug Phenylbutazone cream and Clobetasol ointment were within the permissible limits as long validation times second and third rinse. For this reason, the process of removing residue and contaminants from the production equipment to two cycles of rinsing with purified water for 10 minutes has been found effective to remove residues of the degradation products and cleaners, so that there is no any risk associated with cross-contamination of active substances.

Monitoring the removal of residues and impurities from production equipment could be minimized routine phases so that proposed making these determinations quarterly and at the end of these results will be included in an evaluation report annually the process of removing the residues and impurities.

Applying this method to remove residues and impurities from production equipment and strictly observing all stages of removing all residues and impurities imposed parameters (time, temperature, concentration of cleaning agents, number of rinse cycles) corresponding results were obtained within the eligibility process, demonstrating the reproducibility of the process of removing the residues and impurities, and by imposing limits acceptance to validate the removal process residues and impurities from production equipment to avoid cross contamination and microbial product of the next production cycle.


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GENERAL CONCLUSIONS Manufacture of drugs of the same pharmaceutical form, but with different

pharmacological activities can be done in the same facilities and activities based on well-documented scientific trials proving efficacy results and their validation.

If the manufacturer, the aim and purpose of the proposed implementation of the quality management system for compliance with good manufacturing practice in the validation process for removing residues and impurities control methodology and processes and the removal of waste and debris from production facilities shows that the production of a highly active drug does not jeopardize the manufacture of another drug in the same production line active low, then he can use the same facilities for its entire portfolio of drugs that are manufactured by the same manufacturing technology. Thus, it combines in a very pleasant professionalism in conducting the business of manufacturing quality medicines with the spirit of business in this area.

1. Analytical methods used to test active substances phenylbutazone and clobetasol propionate also complies with GMP and methods described in the European Pharmacopoeia. The raw material is considered appropriate physico-chemical. HPLC methods for the determination of the clobetasol propionate phenylbutazone and were unable to utilize in order to determine the content of active substance in the formulation of the cream / ointment.

The quality of active substances phenylbutazone and clobetasol propionate are analyzed pharmaceuticals are suitable according to the European Pharmacopoeia monographs - Phenylbutazone 0422 and respectively Clobetasole propionate 2127. Due to proven quality, active substances analyzed are suitable for use in the formulation of medicinal products, ensuring the quality and the purpose for which they were designed drugs that are conditional.

2. The analytical methods used to test anti-inflammatory drug Phenylbutazone cream and Clobetasol ointment complies with GMP standards and methods described in the European Pharmacopoeia. Products reviewed are appropriate physico-chemical and microbiological.

HPLC methods for the determination of phenylbutazone and the clobetasol propionate can be used to determination of trace phenylbutazone, clobetasol propionate, respectively after removal of the residues and process impurities from the production facilities.

Quality drug Phenylbutazone cream and Clobetasol ointment are appropriate according to the European Pharmacopoeia monograph - Phenylbutazone 0422/ and Clobetasole propionate 2127 monograph "SEMI-SOLID PREPARATIONS FOR CUTANEOUS APPLICATION" according to the British Pharmacopoeia monograph - Clobetasol Ointment edition 2004 according to "Notes for Guidance on Excipients, antioxidants and antimicrobial Preservatives in the dossier for Application for marketing Authorisation of a medicinal product "- EMEA, CPMP/QWP/419/03.

Antiinflammatory drugs Phenylbutazone cream and Clobetasol ointment, according to the results obtained correspond to the purpose for which they were designed.

3. Manufacturing processes of the two different potent anti-inflammatory drugs were made in production facilities for the preparation of topical semi-solid forms, the special production equipment for the pharmaceutical industry according to good manufacturing practice.

Validation studies confirmed that drug manufacturing methods Phenylbutazone cream and Clobetasol ointment, are consistent and robust and resulted in a finished product that met the quality requirements.

We have found that the manufacturing process was stable since there were no significant differences between the series included in the validation study in which the check of the control parameters of the finished product.


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Validation studies have verified the methods of manufacturing processes by performing test conditions and / or extreme point possible during manufacturing processes and controls, thus demonstrating that it retains control of the process, even in extreme conditions. Once validated, it was considered that maintaining control processes given that their parameters remain unchanged.

On the basis of the obtained results in the technological processes for the two anti-inflammatory drugs have been consistent, thus ensuring reproducibility batch to batch and product quality, steadily and uniformly in accordance with the quality of the product.

4. Method for removal of residues and impurities from the production equipment has been accepted as test results were within the limits of admissibility imposed in the validation process.

The results of measurements performed to validate the process of removing impurities from waste and production equipment were manufactured after the two anti-inflammatory drugs with different pharmacological activities (Clobetasol ointment and Phenylbutazone cream) were within the permissible limits on during the whole process of validating the removal of the residues and impurities from the production facilities for the times the second and third rinse. For this reason, the process of removing residue and contaminants from the production equipment to two cycles of rinsing with purified water for 10 minutes is regarded as effective for the removal of waste, degradation products and cleaners, so that there is no risk associated with cross-contamination of active substances.

Monitoring can be minimized routine phases, so it is proposed to conduct quarterly these determinations, which are recorded in the books of record of tests and at the end of these results will be integrated into the annual evaluation report process removal of the residues and impurities.

Re-validation process to remove impurities and residues from the production facilities will be made when there are changes in protocol validation, such as

- Changing the process for removing residues and impurities or - Change agents to remove residues and impurities or - Changing the product selected for validation of the process of removing impurities

and residue from the manufacturing equipment or - Change the cooking equipment or - Deviations from the permissible limits imposed in the validation protocol. Implementing good practices in the manufacture of drugs with great strictness in every

activity and process leads to quality, effectiveness, efficiency and safety. Documentation of implementation of good manufacturing practices based on scientific results of medicament ensures quality, secure and consistent purpose for which they were designed. The implementation of good manufacturing practices have taken into account all the factors that can influence the quality, safety and efficacy of a product, factors imposed by the rules of good manufacturing practice, but I've adapted to the formulation studied the methodology to control technological process, workspaces, production equipment used in this paper and the main objective of this thesis. And analysis of all these factors and setting clear limits strict admissibility of each process conducted I managed to get the desired results.

Finally, the validation process of removing impurities from waste and production

equipment by setting certain specific eligibility limits, stringent and well selected validation and application of risk management also demonstrated that the manufacturing quality on the same line technological semisolid pharmaceutical drugs as highly active topical sterile (corticosteroids) with different pharmacological activity other drugs (NSAIDs) can not influence the quality of products manufactured on the production equipment and at the same time is accepted and there is no risk of contamination cross-active substances.


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THE CONTRIBUTIONS TO IMPLEMENTATION GOOD PRACTICES … · Validation of the process for removing ... Clobetasol ointment manufacturing method and process ... THE CONTRIBUTIONS TO