Influence of process variables and product variables on

stability

Mariana Matias Quintela Domingues Ponte

Thesis to obtain the Master of Science Degree in

Pharmaceutical Engineering

Supervisors: Eng. Isabel Margarida Mendes CostaProf. Margarida Maria Portela Correia dos Santos Romao

Examination Committee

Chairperson: Dr. Jose Monteiro Cardoso de MenezesSupervisor: Eng. Isabel Margarida Mendes Costa

Members of the Committee: Dr. Joao Pedro Martins de Almeida LopesDr. Micul Hasmuklal Mulchande

October 2019

Acknowledgments

Primeiro de tudo e importante realcar que passou uma fase, uma fase importante, contruıda com

dedicacao e resiliencia.

Gostaria de expressar o meu profundo agradecimento a minha orientadora do Grupo Medinfar, Engª

Isabel Costa por me ter possibilitado o estagio na empresa e pela constante partilha de conhecimentos

e dedicacao do seu tempo tanto profissional como pessoal, obrigada.

Quero tambem agradecer a alguns colaboradores do Grupo Medinfar pela disponibilidade e ajuda

demonstrada nos 8 meses de estagio, um sincero obrigada ao Dr Micul, Sonia Roque, Ana Magalhaes

e Ines Fonseca. Gostaria tambem de agradecer a minha orientadora Margarida Romao pela sua con-

stante disponibilidade.

Agora passando para uma esfera mais pessoal quero agradecer as minhas colegas de estagio que se

tornaram amigas, pelo enorme apoio, incentivo e pela partilha diaria da mesma realidade que foi a da

escrita da Tese, obrigada, Anita, Mafalda, Mariana, Liliana e Rita.

As minhas amigas mais antigas e chegadas, Ana, Carolina e Margarida, obrigada pela amizade con-

stante, sao palavras que dizem tudo.

A famılia do meu namorado, que e e sempre foi incansavel comigo, apoiando-me em todas as eta-

pas da minha vida, Maria Joao, Jose, Teresa e Irene.

Ao meu pai, Mario, que me apoiou sempre e me transmitiu parte dos valores pelos quais me rejo.

Ao meu namorado, Ze. As palavras sao poucas para agradecer o que fez, apoiou e amou, obrigada.

Agora agradeco as estrelas que nao estao em presenca mas que me acompanham desde que

partiram. A minha avo Jacinta que e das pessoas mais importantes e que sei que seguramente estaria

orgulhosa de me ver passar esta fase. Por fim mas mais importante, a minha mae. Nao a sinto em

corpo mas todos os dias tudo o que faco se move por ela, esta seria uma fase em que ela estaria a

dizer-me o quao orgulhosa estava e o quanto me amava, continuo a ouvir isso mesmo sem ela comigo.

Preface

The work presented in this thesis was performed at the company Group Medinfar (Amadora, Portugal),

during the period March-September 2019, under the supervision of Eng. Isabel Costa. The thesis was

co-supervised at Instituto Superior Tecnico by Prof. Margarida Portela Romao.

i

Abstract

The present work is designed to study the process and the product in order to determine the causes that

lead to problems of chemical stability in a specific product. The main objective is to determine potential

factors that contribute to the instability of the product in order to obtain a more stable formulation, i.e.

with lower percentage of related substances. The manufacturing process was investigated in order to

identify possible process variables that had a direct impact on product stability. The history of several

batches was analyzed and a cross-check between the stability results and the manufacturing data was

performed. Another evaluation was the raw materials used on the manufacturing of the finished product,

namely the API, so the certificate of analysis of each raw material was also analyzed and again these

data were crossed with the stability tests results. The first part of the project consisted of an analysis

of company history data to identify process and product variables. Since the data history was limited,

the results obtained are not reproducible for future batches. The second part of the project comprised

the development of formulations with different antioxidants in order to ascertain if the stability of the

product improved. Several tests were performed by different techniques, HPLC, forced degradation and

exposure to atmospheric air.

Regarding the raw materials, no conclusive results were obtained, in the manufacturing process data,

it was possible to identify a variable that could be related to the related substances which was cooling

rate. The pharmaceutical development of different formulations allowed to identify two formulations that

presented better results than the initial formulation.

Keywords

Stability, related substances, process variable, product variable, data analysis, pharmaceutical develop-

ment, antioxidants.

iii

Resumo

O presente trabalho destina-se a estudar o processo e o produto de forma a determinar as causas

que conduzem a problemas de estabilidade quımica de um produto especıfico. O objetivo central e

determinar potenciais fatores que contribuem para a instabilidade do produto no sentido de obter uma

formulacao mais estavel, ou seja, com menor conteudo de substancias relacionadas. O processo de

fabrico foi investigado no sentido de identificar possıveis variaveis de processo que tivessem impacto

direto na estabilidade do produto uma vez que os varios lotes nao apresentavam uma tendencia relativa-

mente a percentagem de substancias relacionadas O historico de varios lotes foi analisado e realizou-se

um cruzamento entre os resultados de estabilidade e os respetivos dados de fabrico. Outra avaliacao

efetuada foi as materias primas utilizadas no processo de fabrico, nomeadamente o API, foram por isso

analisados os certificados de analise de cada materia prima. A primeira parte do projeto consistiu numa

analise de dados do historico da empresa para identificar variaveis de processo e produto, uma vez que

o historico de dados era limitado os resultados obtidos nao sao reprodutıveis para lotes futuros. A se-

gunda parte do projeto compreendeu o desenvolvimento de formulacoes com diferentes antioxidantes

de forma a perceber o impacto na estabilidade do produto. Foram realizados varios ensaios por difer-

entes tecnicas, HPLC, testes de degradacao forcada e exposicao ao ar atmosferico. Relativamente as

materias primas nao se obteve resultados conclusivos, nos dados de producao foi possıvel indentificar

uma variavel que podera ter impacto na degradacao do API. O desenvolvimento farmaceutico permitiu

identificar duas formulacoes que apresentaram melhores resultados do que a formulacao inicial.

Palavras Chave

Estabilidade, impurezas, variavel de processo, variavel de produto, analise de dados, desenvolvimento

farmaceutico, antioxidantes.

v

Contents

1 Introduction 1

1.1 Stability study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Regulatory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Batch Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.4 Climatic Zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.5 Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6 Testing Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.7 Evaluation of stability data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.7.1 Long-term and accelerated data showing little or no change over time and little or

no variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.7.2 Long-term or accelerated data showing change over time and/or variability . . . . 8

1.8 Ongoing Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.9 Topical semi-solid dosage forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.9.1 Ointments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.9.1.A Hydrocarbon Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.9.1.B Absorption Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.9.1.C Water-removable Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.9.1.D Water-soluble Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.10 Product Quality Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.10.1 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.10.2 Related substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.10.3 Microbiological Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.10.4 Antioxidant and preservative content . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.10.5 Viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.10.6 Tube Content Uniformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.11 Chemical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.11.1 Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

vii

1.11.2 Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.11.3 Photodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.12 Physical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.12.1 Viscosity/rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.12.2 Appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.12.3 Syneresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2 Objectives 16

2.1 Raw materials approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.2 Manufacturing process approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.3 Pharmaceutical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3 Material and Methods 19

3.1 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.2 Analytical method for identification of related substances . . . . . . . . . . . . . . . . . . . 21

3.2.1 Techniques for separation and characterization of related substances . . . . . . . 21

3.2.2 High-Performance Liquid-Chromatography . . . . . . . . . . . . . . . . . . . . . . 22

3.3 Forced degradation studies and impurity profiling . . . . . . . . . . . . . . . . . . . . . . . 23

3.3.1 Hidrolytic study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.3.2 Oxidation study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.3.3 Photostability study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.3.4 Thermal study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.4 Atmospheric air exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.5 Pharmaceutical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.5.1 Sampling procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.5.1.A System Suitability Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.5.1.B Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.5.1.C Reference sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.5.1.D Test sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4 Ointment 31

4.1 Product characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.1.1 Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.1.1.A API . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.1.1.B Excipient A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.1.1.C Excipient B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.1.1.D Excipient C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.1.1.E Excipient D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

viii

4.2 Manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.3 Product stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

5 Strategy 37

5.1 Prediction Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.1.1 Curve fitting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.1.2 Predictive models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.1.2.A Approach I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.1.2.B Approach II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.2 Risk analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

5.2.1 Raw materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

5.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5.2.3 Human Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5.2.4 Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5.2.5 Manufacturing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5.2.5.A Non uniform parameters: . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

5.2.5.B Uniform parameters: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.3 Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

5.4 Formulation with antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.4.1 Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.4.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.4.3 Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.5 Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.5.1 Butylated hydroxytoluene (BHT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

5.5.2 Butylated Hydroxyanisole (BHA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

5.5.3 Alpha Tocopherol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.5.4 Other antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

6 Results and Discussion 49

6.1 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.2 Raw Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

6.2.1 Raw materials CoA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

6.3 Manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

6.4 Stability tests measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6.5 Nitrogen application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6.5.1 Manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6.6 Pharmaceutical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

ix

6.6.1 HPLC analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

6.7 Production scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

6.8 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

7 Conclusions 71

7.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

7.2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

x

List of Figures

1.1 Stability testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2 Principles of stability testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Integrated system ICH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.4 Climatic Zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.5 Related substances analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.6 Types of related substances and sources . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.1 Adopted approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.1 HPLC column methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

3.2 Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.3 Forced Degradation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.4 Stress Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.5 Forced degradation method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.6 Sampling steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4.1 Unit operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

5.1 Model predictive approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.2 Parameters LT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.3 Parameters AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.4 Approach I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.5 Approach II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

5.6 Ishikawa diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

5.7 Process variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.8 Possible variables affecting the product quality . . . . . . . . . . . . . . . . . . . . . . . . 45

5.9 Control points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

6.1 Long term stability results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

xi

6.2 Batches distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

6.3 Related substances API vs related substances FPP. . . . . . . . . . . . . . . . . . . . . . 53

6.4 Differences between API batches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

6.5 Raw materials CoA (A, B and C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

6.6 Differences between raw materials batches . . . . . . . . . . . . . . . . . . . . . . . . . . 55

6.7 Raw materials CoA (E and F) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

6.8 Stirring speed for the five analyzed batches . . . . . . . . . . . . . . . . . . . . . . . . . . 57

6.9 Viscosity for the five analyzed batches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

6.10 Cooling rate for the five analyzed batches . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

6.11 Holding time for the five analyzed batches . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

6.12 Comparative study-LT (Batch I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

6.13 Comparative study-IC (Batch I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

6.14 Comparative study-AC (Batch I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

6.15 Increase percentage of related substances . . . . . . . . . . . . . . . . . . . . . . . . . . 62

6.16 Nitrogen application results (Batch II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

6.17 SST chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

6.18 Solvent chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

6.19 Reference solution chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6.20 Test solution chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6.21 Total related substances percentage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

6.22 Total Related Substances-Forced Degradation . . . . . . . . . . . . . . . . . . . . . . . . 67

6.23 Total Related Substances-(Atmospheric air exposure) . . . . . . . . . . . . . . . . . . . . 68

7.1 Summary of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

xii

List of Tables

1.1 Stability tests (General case) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2 Acceptance criteria for microbiological quality of non-sterile dosage forms . . . . . . . . . 12

1.3 Chemical stability tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.4 Physical stability tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3.1 Typical stress study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.2 Pharmaceutical formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.3 HPLC solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.4 Running HPLC sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

4.1 Product Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

5.1 Antioxidant uses of butylated hydroxytoluene . . . . . . . . . . . . . . . . . . . . . . . . . 47

5.2 Antioxidant uses of butylated hydroxyanisole . . . . . . . . . . . . . . . . . . . . . . . . . 47

6.1 Critical points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

6.2 Preventive measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

6.3 Increase percentage of related susbtances . . . . . . . . . . . . . . . . . . . . . . . . . . 66

6.4 Increase percentage of related susbtances (Forced degradation tests) . . . . . . . . . . . 67

6.5 Increase percentage of related substances (Atmospheric air exposure) . . . . . . . . . . 68

xiii

Acronyms

AC Accelerated Conditions

API Active Pharmaceutical Ingredient

BHA Butylated Hidroxyanisole

BHT Butylated Hydroxytoluene

CoA Certificate of Analysis

FPP Finished Pharmaceutical Product

HPLC High Performance Liquid Chromatography

IC Intermediate Conditions

ICH International Conference on Harmonization

LT Long term

USP United States Pharmacopeia

WHO World Health Organization

xvii

1Introduction

Contents

1.1 Stability study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Regulatory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Batch Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.4 Climatic Zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.5 Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6 Testing Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.7 Evaluation of stability data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.8 Ongoing Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.9 Topical semi-solid dosage forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.10 Product Quality Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.11 Chemical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.12 Physical Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1

Stability represents a critical quality attribute of pharmaceutical products and is influenced by a num-

ber of factors, including the Active Pharmaceutical Ingredient (API) of the Finished Pharmaceutical Prod-

uct (FPP), the excipients used in the formulation, the manufacturing process and the container closure

system [1]. The purpose of the stability test is to provide data that supports understanding of the quality

variation of an API or FPP over time under the influence of a group of factors, such as humidity, tem-

perature and light exposure, and establish a period of retest or shelf life for the FPP, as well as the

intended storage conditions [2] [3]. Stability testing is universal at all stages of the product life cycle. The

objectives, regulatory requirements vary depending on the type of product, phase of the program and

markets for product introduction.

The demonstration of stability, mainly chemistry, is one of the main challenges in the development of

a pharmaceutical product. The estimation of shelf-life of the product is based on the time that the FPP

comply the specifications under the recommended storage conditions [1].

The aim of stability testing is to ensure the triad that supports the pharmaceutical industry, quality,

safety and efficacy of the products until their expiry date. Stability tests can be segmented into several

categories, Organoleptical, Chemical, Physical and Microbiological as shown in figure 1.1 and the results

of these tests must comply the specifications agreed for a particular product until the end of its shelf-life.

The pharmaceutical industry comprises a number of challenges, one of which is the development of

stable formulations. This is especially demanding as many APIs respond unstably to oxidation, moisture

or pH [2].

Figure 1.1: Stability testing

The stability program involves several steps, the most important of which to consider for FPP are as

follows:

• Accelerated and long-term tests;

• Ongoing stability testing;

3

Each stage covers eleven basic principles shown in figure 1.2.:

Figure 1.2: Principles of stability testing

1.1 Stability study design

The design of a stability study has as main objective of establishing, based on a batch standard test of an

API or FPP, a retest period or shelf life applicable to all future batches in the same circumstances. The

tested batches must be representative in all aspects, such as formulation, manufacturing process and

site, container closure system, source and quality of the bulk material of the population of all production

batches and following the specification of the FPP [1]. The stability study should be well designed so

that the shelf life of the product can be estimated with the high degree of accuracy and precision.

1.2 Regulatory Requirements

The purpose of the guidelines is to give relevance to the core of the stability data that is required for

registration of an API or a FPP. The design of the stability studies for the FPP should be based on

knowledge of the behaviour and properties of the API, information from stability studies on the API and

4

on experience obtained from preformulation studies and similar marketed formulations [4]. The likely

changes during storage and the selection of attributes to be tested in the stability studies should be

stated. For a clear and thorough understanding of stability testing of a product, an integrated approach

to the International Conference on Harmonization (ICH) guidelines recommendations is necessary as

shown in figure 1.3. Guideline Q1 presents general aspects of the stability tests [2]. However since

stability is a quality attribute it also makes sense to have a knowledge of other recommendations such

as the Q9 and Q10 guideline, the Q9 guideline describes quality risk management tools that aim to

continuously improve product quality and mitigate all risks associated with processes inherent in each

product [4]. The Q10 guideline addresses several systems that can be used to support the various

practices involved in the development and production of a pharmaceutical product [5]. The Q12 guideline

assists in the changes in the product or production in order to improve the quality of these [6].

Figure 1.3: Integrated system ICH

1.3 Batch Selection

The number of batches used for stability studies differs depending on the product type, e.g. new API or

an existing API. In case FPP contains a new API, stability data should be provided from at least three

primary batches. Two of these three batches should be pilot scale, for each FPP strength. For a FPP

with an existing API stability data should be provided on less than two batches of at least pilot scale.

Primary batches should be a replica of the product to be marketed for the formulation, final container

closure system. The manufacturing process used for the development of primary batches shall simulate

that applied in production batches and ensure that the product meets the quality and specifications that

5

are proposed and required for marketing [2].

1.4 Climatic Zones

The shelf-life of a product depends on its storage conditions of temperature and humidity. Conditions

of temperature and humidity diversify between countries. In order to avoid carrying out Long term (LT)

stability testing under different conditions for each country, and to simplify the LT stability testing of a

product for the global market, four climatic zones have been established. The worldwide climatic zones

recognized by World Health Organization (WHO) as shown in figure 1.4. Climatic Zone I, Temperate

climate. Climatic Zone II, Subtropical and Mediterranean climate. Climatic Zone III, Hot and dry climate.

Climatic Zone IV, Hot and humid climate [7]. These are based on observed temperatures and relative

humidities, both outside and inside buildings, from which mean temperatures and average humidity

values are calculated. For this purpose, WHO has split Climatic Zone IV into two: A, Hot and humid

climate and B, Hot and very humid climate.

Figure 1.4: Climatic ZonesSource:https:q1scientific.com/ich-quality-guidelines

6

1.5 Specification

It is recommended that stability tests include stability-indicating attributes of the FPP, i.e. attributes that

are most likely to change during storage that may have an influence on quality, safety and/or efficacy.

Tests should study various parameters of the product including the physical, chemical, biological and

microbiological attributes, preservative content (e.g. antioxidant or antimicrobial preservatives).

All analytical techniques must be validated and stability-indicating. Shelf-life acceptance criteria shall be

based on all information relating to the stability of the product under analysis [2].

1.6 Testing Frequency

An FPP must be subjected to stability testing under various conditions to define its behaviour regarding

stability. For stability studies, the frequency of testing should be sufficient to provide data to create a

stability profile of the FPP. Generally, in LT studies the test frequency is usually every 3 months in the first

year, every 6 months in the second year and annually thereafter, the proposed shelf life. In Accelerated

Conditions (AC), data is reached at least three time points, including the start and end time points (0,

3, and 6 months). In the case of the FPP has a high likelihood of change over time, time points can be

added to the study. In general, FPP should be evaluated under storage conditions which test its thermal

stability and its sensitivity to humidity. LT studies should include a minimum duration of 12 months in

at least three primary batches at the time of submission and should continue for a sufficient period to

cover the proposed expiry date [2]. The storage conditions and lengths of studies chosen should allow

to cover the scenarios of storage, shipment, and subsequent use. Data from the LT, AC, and, where

appropriate, Intermediate Conditions (IC) for FPP are detailed in table 1.1 [8]. .

Study Storage condition Minimum time period covered by data at submission

Long term25°C± 2°C/60% RH ± 5% RH or30°C ± 2°C/65% RH ± 5% RH or

30°C ± 2°C/75% RH ± 5% RH12 months or 6 months

Intermediate 30°C ± 2°C/65% RH ± 5% RH 6 monthsAccelerated 40 °C ± 2 °C/75% RH ± 5% RH 6 months

Table 1.1: Stability tests (General case)

7

1.7 Evaluation of stability data

After obtaining the stability data, it is necessary to know how to interpret them to define if the product

follows the standards of efficacy, safety and quality. For any FPP a significant change is considered a

failure of the FPP to comply with the specifications. In this sense, the specifications are defined by the

type of product and its characteristics.

Failure to comply with the specification for related substances, physical appearance, moisture content

and other similar tests are viewed as a significant change in pharmaceutical products.

1.7.1 Long-term and accelerated data showing little or no change over time and

little or no variability

Where the LT data and AC data for an attribute show little or no change over time and little or no vari-

ability, suggests that the FPP will comply with specifications during shelf life. In such cases a statistical

analysis of the data is not considered necessary but the lack of such analysis must be justified. Extrap-

olation of the shelf life beyond the period covered by LT data may be a possibility. The proposed shelf

life can be up to twice, but should not exceed 12 months, the period with available LT data. [2].

1.7.2 Long-term or accelerated data showing change over time and/or variabil-

ity

In the case of LT or accelerated data for an attribute shows changes over time and/or variability in one or

more parameters, a statistical analysis of the data should be performed, which can be useful for defining

shelf life. If there are differences on stability data between batches or parameters or combinations, the

combining of data is required, the proposed shelf life should not exceed the shortest period supported

by any batch, other factor, or factor combination. Instead, if the differences were related to a particular

parameter, different shelf lives can be assigned to different levels within the parameter. In this sense,

the cause of the observed differences and the potential impact on the FPP should be discussed [2].

1.8 Ongoing Stability

After the marketing authorization, the stability of the FPP must be continuously monitored in order to al-

low the detection of any stability problem. The main objective of an ongoing stability program is to make

continuous monitoring of the product during its shelf-life and to determine if the product will comply with

the specifications if stored under the indicated conditions [2].

8

The protocol for an ongoing stability program shall extend to the end of its shelf-life and include, but

not be limited to, the following parameters:

1) number of batch(es) per strength and different batch sizes, if applicable.;

2) relevant physical, chemical, microbiological and biological test parameters with acceptance criteria or

reference to the attached specifications;

3) reference to test methods;

4) characterization of the container-closure system(s);

5) testing frequency;

6) description of the conditions of storage (standardized conditions for LT testing as described in these

guidelines, and consistent with the product labelling, should be used);

7) other applicable parameters specific to the FPP.

1.9 Topical semi-solid dosage forms

Topical semi-solid dosage forms can be marketed in various forms, gels, creams, pastes or ointments.

This type of product is formed by one or more APIs dissolved or dispersed on an appropriate base

and some excipients with distinct functions such as emulsifiers, viscosity-increasing agents, stabilizing

agents or antioxidants [9]. Pharmaceutical development involves several studies, including the influence

of excipients on the physical and chemical stability of FPP or the availability of the active substance at the

site of action. Another important study is to ensure that there is no incompatibility between formulation

components. One of the most important step to take in pharmaceutical development is the detailed study

of the characteristics of the chosen API, i.e. its sensitivity to light, oxygen, water in order to develop a

formulation and manufacturing process that minimize the risks involved for the stability of the FPP [10].

1.9.1 Ointments

Ointments are homogeneous, semi-solid formulations designed for external application to the skin or

mucous membranes. Ointment bases recognized for use as vehicles can be split into four general

classes: hydrocarbon bases, absorption bases, water-removable bases, and water-soluble bases.

The choice of a base for semi-solid dosage forms is determined by different factors: the nature of

API(s), the therapeutic effect desired, the availability of API(s) at the site of action, the shelf-life of the

FPP and the climatic conditions in which the product is intended to be administered. In certain cases it

is mandatory to make changes in order to achieve the required stability.

9

1.9.1.A Hydrocarbon Bases

Hydrocarbon bases, known also as “oleaginous ointment bases,” are represented by White Petrolatum.

These bases only allow the incorporation of small amounts of aqueous components. This type of base

is used when the ointment is intended to be in contact with the skin for a long period. These bases are

used for their emollient effects and are difficult to remove from the skin. They do not dry out or change

considerably on aging [9].

1.9.1.B Absorption Bases

This class of bases can be split into two groups: the first allows the incorporation of aqueous solutions

forming a water-in-oil emulsion, and the second group consisting of water-in -oil emulsions which allows

the inclusion of additional quantities of aqueous solution. This bases are also useful as emollients. [9].

1.9.1.C Water-removable Bases

Water-removable bases are oil-in-water emulsions. Because the outer phase of the emulsion is water,

it is easily removed from the skin and objects, an attribute that makes them more suitable for cosmetic

products. Some APIs may be more effective on these bases than on hydrocarbon bases. [9].

1.9.1.D Water-soluble Bases

This group of called “greaseless ointment bases” contain water-soluble constituents. Bases of this

type have many of the advantages of the water-removable bases and, in addition, comprise no water-

insoluble substances such as petrolatum, anhydrous lanolin, or waxes [9].

10

1.10 Product Quality Tests

To ensure that the product comply with all specifications, several quality tests are required. Various

quality tests, physical and chemical are described below.

1.10.1 Assay

A specific and stability-indicating test should be used to determine the strength (content) of the drug

product. This test is essential to ensure that the product comply with the specifications for the content of

API.

1.10.2 Related substances

Process related substances, synthetic by-products, and other inorganic and organic related substances

may be present in the API and excipients used in the manufacturing process of the FPP. These re-

lated substances are controlled by the API and excipients monographs. Organic related substances

arising from the degradation of the API and those arising during the manufacturing process of the FPP

should be monitored. There are different sources of related substances in API and FPP. It comprises

crystallization, stereochemistry, residual solvents, and synthetic intermediate and by- products related

substances. In addition it also includes formulation, impurity arising during storage, method related,

mutual interaction between components [11] [12]. Related substances are classified into various types

based on their ’common names’, ICH Terminology United States Pharmacopeia (USP). The analysis of

related substances depends on the nature of the sample, i.e. API or FPP as shown in figure 1.5.

Related substances can be broadly divided into four types which are discriminated in figure 1.6 [11] [13].

Figure 1.5: Related substances analysis

11

Figure 1.6: Types of related substances and sources

1.10.3 Microbiological Control

The type of microbiological test(s) and acceptance criteria it is dependent on the nature of the API,

manufacturing process, and the intended use of the FPP. The degree of microbial contamination is reg-

ulated and is based on acceptance criteria stipulated in the Pharmacopoeia monographs [14]. In table

1.2 is represented the acceptance criteria for non-sterile dosage forms concerning microbiological qual-

ity, analysing factors such as total aerobic microbial count (TAMC) and total yeast/ mold count (TYMC).

Route of administration TAMC TYMC Specified microorganismsNon-aqueous preparations for oral use 103 102 Absence of Escherichia coli (1g or 1ml)

Aqueous preparations for oral use 102 101 Absence of Escherichia coli (1g or 1ml)Oromucal useCutaneous use

Nasal use102 101

Absence of Staphylococcus aureus (1g or 1ml)Absence of Pseudomonas aeruginosa (1g or 1ml)

Table 1.2: Acceptance criteria for microbiological quality of non-sterile dosage forms

1.10.4 Antioxidant and preservative content

If antioxidant and/or preservative are present in the FPP, the assay of their content normally should be

determined.

12

1.10.5 Viscosity

There are several properties of FPP that may compromise its effectiveness. Rheological properties such

as viscosity of semisolid dosage may impact drug delivery. This highlights the importance of rheologic

properties of semisolid dosage forms namely viscosity, on FPP performance.

An essential factor is to maintain reproducibility of a product’s flow behavior at the time of release

to demonstrate consistency between batches. Moreover, various factors influence the viscosity of a

semisolid FPP, such as the inherent physical structure of the product, product-sampling technique,

sample temperature for viscosity testing, container size and shape, and specific methodology employed

in the measurement of viscosity. [15]. The rheological properties of the FPP can influence its shelf life.

1.10.6 Tube Content Uniformity

Tube uniformity is a test that defines the degree of uniformity of the amount of API in the containers. The

uniformity of dosage is demonstrated by assay of top, middle, and bottom samples obtained from a tube

cut open to remove respective samples for API assay [15].

1.11 Chemical Stability

Chemical degradation of the API is often the critical factor that limits the shelf-life of a pharmaceuti-

cal product. The API degradation and the degradation of other ingredients in the formulations such as

antimicrobial preservatives or antioxidants, may also play a critical role in stability. Reduction in API as-

say/increase in degradation product must be evaluated. Due to the degradation of the API after chemical

reaction with other ingredients in the formulation, or environmental instability (e.g. thermal, oxidative or

light-induced degradation) becomes important quantification and evaluation of the degradation products

originated [16]. In order to ascertain the chemical stability of the product several tests are carried out as

shown in table 1.3.

Tests FunctionAssay Quantification of API, preservative or antioxidant content

Related substances Quantification of known, unknown and total related substances

Table 1.3: Chemical stability tests

Chemical stability is a complex field, and several phenomena are responsible for product degradation

and consequent chemical instability. Several concepts related to chemical stability will be introduced

below.

13

1.11.1 Hydrolysis

This process consists of the breaking of a molecular bond by reaction with water. Water is a common

ingredient or contaminant in pharmaceutical products. Hydrolysis reactions are the most common cause

of chemical degradation [16].

1.11.2 Oxidation

Oxidation reactions involve an increase in the number of carbon-to-oxygen bonds in a molecule or a

reduction in the number of carbon-to-hydrogen bonds. These reactions are a common cause of chemical

instability of drugs [16]. Oxidation reactions tend to be complex, giving a variety of degradation products.

1.11.3 Photodegradation

Photodegradation is another degradation pathway, being known as a major pathway in many products,

it is a complex mechanism [16]. This type of degradation can be decreased using protected container to

avoid the exposition to the light.

1.12 Physical Stability

The established physical stability tests for this type of product are enunciated in table 1.4. A number of

tests are performed to determine the physical stability of the product [16].

Tests FunctionAppearance Evaluation of organoleptic characteristics

Viscosity Viscosity measurementpH pH measurement

Table 1.4: Physical stability tests

1.12.1 Viscosity/rheology

Due to either structure buildup or breakdown within the formulation:

• Structure buildup (thickening) could indicate crystallization or hardening of waxes;

• Structure breakdown (thinning) could indicate micelle coalescence, phase separation, polymer chain

scission.

14

1.12.2 Appearance

This parameter is evidenced by the occurrence of certain chemical reactions within the formulation (e.g.,

reaction of ingredients with each other, oxidation of ingredients, light-induced degradation).

1.12.3 Syneresis

This type of physical instability is seen as liquid separating out from the formulation (commonly seen

within air bubbles or at the air/product or container/product interfaces).

Evidence of physical instability is demonstrated by:

- a noticeable change in consistency, such as excessive ”bleeding” (separation of excessive amounts of

liquid) or formation of agglomerates and grittiness;

- discoloration;

- emulsion breakdown;

- crystal growth;

- shrinking due to evaporation of water;

- evidence of microbial growth.

15

2Objectives

Contents

2.1 Raw materials approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.2 Manufacturing process approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.3 Pharmaceutical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

16

The present project focuses on the study of a semi solid product, an ointment with the need for opti-

mization and stability improvement, namely the content of related substances.

For the purpose of the project two main approaches were used (figure 2.1), i) the study of history

data relating to stability test results, raw materials, manufacturing process and nitrogen application on

the process and ii) the development of various formulations with the inclusion of antioxidants.

These two approaches aim to minimize the total percentage of related substances on the FPP.

Figure 2.1: Adopted approaches

One of the main objectives is to understanding the product characteristics, namely the API, in order

to understand which parameters are the most limiting and which influence the stability of the API. The

Certificate of Analysis (CoA) will be analyzed in detail in order to identify any data that denounces a

possible cause. The manufacturing process will also be investigated and all production variables will

be analyzed in order to define or identify any variable that may be responsible for the reduced product

stability. One possibility that may optimize and improve product stability is formulation.

Pharmaceutical development requires a search for the most suitable components for the required

purpose. In order to be able to discriminate formulations which show improvement over the control

formulation (initial formulation), it will be necessary to perform several trials in which batches are placed

under stability under two conditions, LT and AC. To test the various formulations will also be performed

forced degradation tests and exposure to atmospheric air. Moreover these batches will be analyzed on

High Performance Liquid Chromatography (HPLC) to quantify the related substances.

Since the product does not show a trend towards stability, it was interesting to try to predict from a few

points in time whether the end-of-shelf life product would comply with specifications.

17

2.1 Raw materials approach

Raw materials may represent the solution to optimize the problem if it is possible to verify that some

raw material has a different parameter between the batches with higher degradation rate and the remain

batches.

2.2 Manufacturing process approach

The manufacturing process is composed of several steps and unit operations, in which several param-

eters must be controlled, the objective will be to investigate the process and identify the variables that

have the greatest impact on the degradation rate of batches and thus be a variable that when modified

leads to improved final product stability.

2.3 Pharmaceutical development

This phase aims to improve the initial formulation, for this purpose will be defined several trials where

different types of antioxidants will be included. It is necessary to investigate which antioxidants are most

suitable for the API and the other components of the formulation.

18

3Material and Methods

Contents

3.1 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.2 Analytical method for identification of related substances . . . . . . . . . . . . . . . 21

3.3 Forced degradation studies and impurity profiling . . . . . . . . . . . . . . . . . . . . 23

3.4 Atmospheric air exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.5 Pharmaceutical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

19

20

The case to be studied is a semi solid product, an ointment. In order to obtain results concerning the

product under study, several tests have been carried out, namely in the quantification of the percentage

of related substances. It will be described all the tests performed in the sense of realizing its usefulness

for obtaining the results.

3.1 Software

For the analysis of the manufacturing process and raw materials data were used three softwares, R

(3.5.0 version, New Zealand) SIMCA (Umetrics, Sweden) and OriginPro (9.1 version, OriginLab, Mas-

sachusetts). R and SIMCA softwares were used to perform multivariate analysis of manufacturing and

raw materials data. OriginPro was used to build a prediction model for related substance values based

on given time points.

3.2 Analytical method for identification of related substances

Isolation and identification of related substances is a mandatory action since their most accurate mon-

itoring is required and related substances are often estimated based on the material of the API which

may contain several errors. These estimates are sustained on the principle that related substances are

structurally linked to the material of interest and thus, have the same detector response. The amount

of methods and techniques used is important for the isolation and consequent identification of related

substances. There are different techniques for isolation, quantification and characterization of related

substances.

3.2.1 Techniques for separation and characterization of related substances

One of the growing needs of the pharmaceutical industry is the monitoring and control of API and

FPP related substances. Hence, API impurity profiling, which consists of identification, isolation and

characterization, is required. Limits and specifications should be in accordance with standards dictated

by regulatory authorities. ICH defines the related substances as ”substances in the API that are not the

API itself”.

The related substances can be classified in different fields, the most important, being one of the

product’s focus, are related substances called degradation products which are compounds produced

by the action of external factors such as heat, moisture, oxygen and light. During manufacturing of bulk

drugs degradation of FPP results in the formation of related substances. Degradation products arise

from diffent sources e.g. synthetic process, storage, formulation of dosage form and aging.

21

The estimation of the impurity profile is achieved by the multifaceted use of chromatographic and

spectroscopic methods [17].

3.2.2 High-Performance Liquid-Chromatography

The technique used in the project was HPLC. HPLC is one of the most commonly used and reported

chromatographic technique with regard to the separation, quantification and identification of related sub-

stances. The compounds are identified based on various parameters, namely retention time and direct

comparison with previously known standards. This technique requires the use of a column (stationary

phase), a suitable mobile phase and a software to analyse the data obtained [17].

HPLC depends on interaction of sample analytes with the stationary phase and the mobile phase to

obtain the components separation as shown in figure 3.1.

Figure 3.1: HPLC column methodology

The chromatographic system consisted of a Prodigy ODS3 100A, 3µm, 150 * 4.6mm column (Wa-

ters). HPLC (Merck), UV detector with wavelength at 235 nm. In the product were used two mobile

phases with the same components in different proportions. Mobile phase A is composed of methanol,

acetonitrile and 85% 5g/L orthophosphoric acid (16:36:48). Mobile phase B is composed of methanol,

acetonitrile and 85% 5g/L orthophosphoric acid (20:70:10). For HPLC analysis two solvents were used,

n-Hexane and ethanol.

All HPLC analysis were performed on the same equipment to minimize errors associated with mea-

surements.

22

A chromatogram consists of a graphical representation of the separation of the components which

are represented by peaks with a certain area and a specific retention time as illustrated in figure 3.2.

Figure 3.2: Chromatogram

Most HPLC analysis in the pharmaceutical industry are isocratic analysis which use the same mobile

phase during sample elution. This type of analysis is common in quality control testing since it use

simpler HPLC equipment and premixed mobile phases. On the other hand, gradient analysis, in which

the mobile phase force increases during sample elution, it is suitable for complex solutions and those

containing broad polarity analytes. [18].

The purpose of the analysis was to separate and quantify related substances, so the gradient analysis

was selected.

3.3 Forced degradation studies and impurity profiling

Regulatory guidelines does not provide details of forced degradation testing, the practical procedures

of these tests are not discriminated. The ICH guidelines defines that the fundamental purpose of per-

forming forced degradation testing is to provide information regarding the identification of degradation

products to assist in determining the stability of the API.

Forced degradation tests are performed to generate an amount of data that provides relevant information

about the degradation products, degradation pathways and intrinsic stability of the API. These studies

are carried out by exposing the drug product to different stress scenarios e.g. hydrolytic, oxidation, pho-

tolytic and thermal conditions [19]. These tests are conducted for the formation of degradation products

that do not form in normal stability studies. Forced degradation is generally assessed with no more than

10-20% degradation of although more may be acceptable depending on the particular properties of the

API.

Forced degradation studies are carried out for the following reasons [20]:

• To develop and validate a stability indicating method;

• To determine degradation pathways of API and FPP;

23

• To identify related substances related to API or excipients;

• To understand the API molecule chemistry;

• To define more stable formulations;

• To create a degradation profile that mimics what would be obtained in a formal stability study under

ICH conditions.

As previously mentioned, monitoring the stability of the FPP is a priority to ensure its safety and effec-

tiveness. To control possible product changes over time, an analytical method should be applied, in most

cases, a chromatographic technique which should be stability-indicating. The best way to understand if

a technique is suitable is to use LT stability solutions containing all relevant degradation products. If it is

necessary to form degradation products, stress conditions may vary due to the chemical structure of the

API, FPP, and storage conditions [20].

Therefore, solutions will be stressed in a solid form and/or in solution as shown in figure 3.3. Typically,

stress tests are carried out on one batch of FPP and/or API. For FPP the placebo should be stressed

in a similar way in order to exclude those related substances which are not degradation products (e.g.

related substances arising from excipients).

Figure 3.3: Forced Degradation Studies

Figure 3.4 shows typical stress conditions of API and FPP.

As mentioned, there are different conditions that define forced degradation tests, these tests must

24

Figure 3.4: Stress Conditions

be done taking into account the characteristics of the API/FPP to be analyzed. These tests can be

conducted in various ways depending on the type of sample to be analyzed, i.e. FPP or API, with the

conditions being different for each. Table 3.1 shows the different forced degradation tests and their con-

ditions for the type of sample [12].

Stress conditions Examples Drug substance Drug productAcid/Base 0.01 to 0.1N 1-7 days 24 to 48h

Oxidative 0.3% H2O2few hours to7 days 24 to 48h

Light 1200 Lux h >48h >48hTemperature 10°C to 85°C up to 2 weeks up to 3 weeks

Light/Temperature 10°C to 85°C and60 to 90 RH up to 2 weeks up to 3 weeks

Table 3.1: Typical stress study design

Of all the tests described were chosen two, taking into account the available data history. The

tests which showed a higher degradation rate in the tests already performed in the company were the

hydrochloric acid and hydrogen peroxide, in this sense these were the selected tests.

3.3.1 Hidrolytic study

The hydrolytic study is carried out under acidic conditions by adding a quantity of HCl during 48h.

3.3.2 Oxidation study

Forced oxidation of compounds by hydrogen peroxide is a convenient mean of studying stability. Ox-

idative degradation is a complex process. In this type of study hydrogen peroxide is widely used since

it mimics the action and existence of peroxides in excipients and other oxidizing agents. The oxidative

degradation of drug substances involves an electron transfer mechanism to form reactive anions and

cations. In the case of the product to be studied as already mentioned the main concern is that the API

25

easily undergo oxidation when exposed to atmospheric air. One of the forced degradation tests that has

major relevance given the characteristics of the API is a forced degradation test using hydrogen perox-

ide. Several studies report that subjecting the product to the presence of 0.1% -3% hydrogen peroxide

at neutral pH and room temperature for up to 7 days may lead to a significant percentage of degradation

products [15].

The method consisted of placing an amount of 2% hydrogen peroxide in the regular preparation of

the solutions. The solutions were left for 48 hours in the presence of hydrogen peroxide and were further

analyzed on HPLC as represented in figure 3.5.

Figure 3.5: Forced degradation method

3.3.3 Photostability study

The photostability test was not performed since even though the product was light sensitive, this is not

the most critical parameter to be evaluated. The product is packaged in aluminum opaque tubes to

prevent degradation by light action, so the parameter of photosensitivity is controlled in the product life

cycle.

3.3.4 Thermal study

Thermal degradation is studied by exposing API or FPP to dry and wet heat. This test was not performed

since from the historical data analysis it was not a relevant test considering the degradation obtained in

previous tests.

3.4 Atmospheric air exposure

The purpose of the atmospheric air exposure test is to attempt to reproduce the oxidation of the product

when in contact with oxygen to simulate the oxygen interactions with the API. The main objective is

to test the degradation of the product by oxidation. The study consisted of the use of 10 plastic plates

where the content of a tube was placed. In order to ensure that the product did not react with the plastic,

the plates were lined with aluminum foil. The experiment took place during 30 days in which the plates

26

were in contact with the outside air. The initial idea was to place the contents of each tube in a container

with 100% saturated oxygen, however this was not performed for safety reasons, since the FPP contains

components that may be a source of combustion.

3.5 Pharmaceutical development

The table 3.2 describes the ten proposed formulations, nine containing antioxidants such as Butylated

Hydroxytoluene (BHT), Butylated Hidroxyanisole (BHA), citric acid and Alpha tocopherol and one control

formulation i.e. without inclusion of antioxidants. In order to verify that the quantities determinated by

the HPLC measurement are only from the API and not from the excipients, a placebo should be tested.

Since the placebo had already been studied at the company, it was only necessary to analyze the

chromatogram and identify peaks. Placebo showed no significant peaks, concluding that all peaks are

caused by API degradation. The pharmaceutical development consisted of the production of ten different

batches and an average of 8 tubes per batch was produced to guarantee a sufficient amount for the tests

to be performed.

An important point to note is that all batches were made from the same batch of excipients and API,

with an API with a total percentage of related substances of 0.72%.

Tests Antioxidant(s)I BHTII BHAIII BHT+BHAIV BHT+Citric acidV BHA+Citric acidVI BHT+BHA+Citric acidVII BHT+Alpha TocopherolVIII BHA+Alpha TocopherolIX BHT+BHA+Alpha TocopherolX Control (without antioxidant)

Table 3.2: Pharmaceutical formulations

3.5.1 Sampling procedure

In order to collect HPLC results it was necessary to prepare different solutions represented in table 3.3.

For each sample two replicates were made in order to obtain results more robust and accurate.

Thus, with ten batches the number of solutions to be analyzed is twenty. For each test, 2 tubes were

used per sample in order to have a greater homogeneity of the batch produced. HPLC analysis also

requires the preparation of a standard API solution to determine its retention time.

27

Solutions AmountRT 0 months 20RT 3 months 20AC 3 months 20atmospheric air exposure 20Forced degradation 20

Table 3.3: HPLC solutions

The first sample to be analyzed on the HPLC is the standard solution, then the reference solution and

finally the test solution. Test solutions are the last to run to avoid the carry over phenomena. Between

each 2 sets of solutions a solvent is analysed to clean the injector needle and detector, to eradicate

potential contamination on the next injection. The table 3.4 shows the run order on the HPLC.

Solutions Run time (min)Standard solution 45Solvent 45Reference solution (1,1) 45Reference solution (1,2) 45Solvent 45Test solution (1,1) 45Test solution (1,2) 45Solvent 45

Table 3.4: Running HPLC sequence

3.5.1.A System Suitability Test

The System Suitability Test (SST) is generally performed to assess the suitability and effectiveness

of the entire chromatographic system, this feature is used to verify resolution, column efficiency, and

repeatability of a chromatographic system not only before use, but also during the analysis time. SST is

a key parameter for all HPLC analytical procedures.

3.5.1.B Solvent

Solvent is essential to avoid peaks from previous solutions. The solvent consists of the components in

which the solutions are prepared, in the case of the product, the solvent consists of a n-Hexane/ethanol

mixture.

3.5.1.C Reference sample

Reference solutions is the solution diluted in a mobile phase portion.

28

3.5.1.D Test sample

Test solutions are the solutions that will contain all peaks of interest for the study. Since it is the data

relating to these solutions that enter the calculations of the total percentage of related substances.

Solutions were prepared using two solvents, n-Hexane and ethanol. The solutions were prepared

in the same way and all measurements were uniform from sample to sample, decreasing the variability

between them.

Stirring times were also controlled and equal between all solutions. All sample weights were recorded

and were used for the calculation of related substances.

Sample preparation consisted of the steps illustrated on figure 3.6.

Figure 3.6: Sampling steps

29

30

4Ointment

Contents

4.1 Product characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.2 Manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.3 Product stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

31

32

4.1 Product characteristics

A specific ointment is an antibiotic for topical use containing an API in its composition. This product

is indicated for use in adults in the treatment of localized skin infections, caused by microorganisms

sensitive to API, such as: boils, impetigo, pyoderma, abscesses, folliculitis, acne, infected wounds.

Typically, these infections are caused by staphylococci, streptococci or other organisms sensitive to

API.

4.1.1 Formulation

4.1.1.A API

This ingredient is a bacteriostatic antibiotic derived from a fungus and used as a topical medication

to treat skin infections. It appears as a white or almost white crystalline powder, slightly hygroscopic.

Regarding storage, API should be stored under restricted temperature conditions, between 2°C-8°C.

This API readily oxidizes when it comes in contact with atmospheric O2, so, special precautions should

be taken [21].

4.1.1.B Excipient A

Excipient A is widely used in cosmetics and pharmaceutical formulations such as suppositories, modified-

release solid dosage forms, emulsions, lotions, creams, and ointments. In suppositories is used to raise

the melting point of the base, and in modified-release dosage forms it may be used to form a perme-

able barrier coating. In lotions, creams, and ointments. Excipient A is used because of its emollient,

water-absorptive, and emulsifying properties. It enhances stability, improves texture, and increases con-

sistency. The emollient properties are due to absorption and retention of excipient A in the epidermis,

where it lubricates and softens the skin [22].

4.1.1.C Excipient B

Excipient B is widely used in topical pharmaceutical formulations and cosmetics. Excipient B may be

used as a hydrophobic vehicle and in the preparation of water-in-oil creams and ointments. When mixed

with suitable vegetable oils or with soft paraffin, it produces emollient creams that penetrate the skin and

hence facilitate the absorption of drugs.This excipient mixes with about twice its own weight of water,

without separation, to produce stable emulsions that do not readily become rancid on storage [22].

33

4.1.1.D Excipient C

Excipient C is a soft solid used in topical pharmaceutical formulations and cosmetics as an ointment

base with emollient properties. It is also used in the preparation of creams and lotions [22].

4.1.1.E Excipient D

Excipient D is mainly used in topical pharmaceutical formulations as an emollient-ointment base; it is

poorly absorbed by the skin. This excipient is also used in creams and transdermal formulations and as

an ingredient in lubricant formulations for medicated confectionery together with mineral oil [22].

The product under study is made up of several raw materials, each of which has a specific function

as shown in table 4.1

Raw material FunctionAPI Therapeutic (antibiotic)Excipient A EmollientExcipient B Hydrophobic vehicleExcipient C EmollientExcipient D Ointment base

Table 4.1: Product Formulation

4.2 Manufacturing process

Ointments are semisolid preparations containing either dissolved or suspended functional components.

The ointment base needs to be heated to above its melting temperature prior to the addition of the other

ingredients.

The product manufacturing process has three major steps, and for this process two different equip-

ment are used. In equipment A are added excipients A, B and C where mixing occurs until all com-

ponents are melted. In parallel to the equipment B are added the API and the excipient D, being also

mixed. After these steps, the content of equipment A is transferred to equipment B, and the final mixing

step is performed as described in figure 4.1.

34

Figure 4.1: Unit operations

4.3 Product stability

A problem derived from the fine size of API particles is that it presents a huge surface area for contact

and reaction with molecular O2 during manufacture and handling of the ointment. This has significant

implications to its chemical stability. Degradation due to oxidation is a major cause of instability.

Oxidation stabilization of products containing this API involves observing a number of preventive

measures during manufacturing process and storage. These include:

• replacing O2 in pharmaceutical containers with inert gases such as Nitrogen, Carbon dioxide, Helium

and the like;

• avoid contact of the drug product with heavy metal ions which catalyze oxidation;

• store the API at reduced temperatures through out its shelf life before processing.

Oxygen sensitive semi solid pharmaceuticals are usually protected from degradation by two methods.

One of the methods consist in antioxidants incorporation and/or chelating agents into the formulation,

the other method is maintaining the product in an inert atmosphere. A number of approaches have been

taken during development of pharmaceutical products to improve chemical stability and the shelf-life

of drugs susceptible to oxidation. The obvious approach during processing is to reduce O2 levels and

minimize contact of the API with O2. For example, processing under inert atmosphere and reducing

container headspace may improve the stability of O2 sensitive drugs, although removal of all O2 present

in a container is difficult and trace amounts may be sufficient to initiate oxidation. A simple method for

preventing or decreasing oxidation is to include antioxidants in the formulation. Antioxidants can reduce

localized O2 concentrations, scavenge free radicals, quench singlet O2 bind metal ions, and remove

35

peroxide.

36

5Strategy

37

38

In this chapter will be discussed various strategies aimed at optimizing the stability of the product

under study i.e. the decrease of API degradation. An initial approach to predicting product stability will

also be discussed.

5.1 Prediction Approaches

Stability tests have a disadvantage relative to the time required to prove the behavior of a given product.

An interesting approach is to use the stability data regarding related substances in several batches and

try to predict what the behavior will be at the end of the shelf life. For this, several approaches were

used with the help of the SIMCA software with the Partial Least Squares (PLS) algorithm as shown in

figure 5.1 and the OriginPro software. Different approaches were used, with simultaneous use of the

two software referenced previously.

Figure 5.1: Model predictive approach

5.1.1 Curve fitting

Origin is a software for data analysis and graphical representation of data. For the defined objective the

Curve Fitting function was used in order to adjust the points of the line and to give a line with several

parameters. The line consists of all time points of the nine batches available. The line parameters are

defined in the figures 5.2 and 5.3, this analysis was performed for the different stability conditions.

Figure 5.2: Parameters LT

39

Figure 5.3: Parameters AC

The error associated with the line parameters obtained with the software is very high, not giving

confidence in the predicted values.

5.1.2 Predictive models

PLS modeling is use to identify input and process variables, X, that are “responsible” for the change in

output variables, result variables, Y [23]. In the identified situation there are several variables, the idea

of using PLS is to define some relation between the variables under study.

5.1.2.A Approach I

This approach consists of using the first related substances results in the product to predict the value at

the end of shelf life as represented in figure 5.4. The analysis was done for data under the conditions of

temperature and humidity, i.e., long term, accelerated conditions and intermediate conditions.

Figure 5.4: Approach I

5.1.2.B Approach II

Other approach used takes into account the parameters obtained by the line in the function of curve

fitting in the Origin and the data of raw materials and manufacturing process.

40

Figure 5.5: Approach II

This approach would be interesting as it made it possible to predict the values of related substances

and streamlined all bureaucratic submission processes for the batch and if it was anticipated that the

batch will have a high degradation rate, it would no longer be followed. Trials leading to a saving of

resources and time and anticipate preventive actions. However, for this approach to be successful and

reliable, a large amount of data is required to build a robust model that has a broad predictability based

on numerous batches values already manufactured.

41

5.2 Risk analysis

The project can be divided into two main parts, i) the analysis of the initial formulation and ii) the study

of possible formulations with antioxidant inclusion. For this is important to take a cause-effect approach

and to discriminate which variables most influence product instability.

To be able to identify the problem it is necessary to discriminate the possible causes and all the

variables involved in the process and/or product. The diagram 5.6 is intended to discriminate the possible

causes in order to optimize the process decreasing the degradation of the API.

Figure 5.6: Ishikawa diagram

The quality of pharmaceutical products depends on several factors. To understand what factors have

a greater impact on the product it is necessary to analyze each of the factors.

5.2.1 Raw materials

The starting point for the manufacturing process is the raw materials. It is necessary to evaluate all

possible changes in the raw materials, namely quality attributes that may induce variations in the FPP.

When the there are changes in supplier, it is also necessary to assess its impact.

One of the relevant aspects to evaluate in raw materials, especially in API, is the initial percentage

of related substances and to understand if there is a relation between this value and the final value of

related substances in the FPP. Another important data to evaluate is the storage and handling conditions

of raw materials, namely API.

42

5.2.2 Equipment

In the process two types of equipment are used. It is necessary to evaluate if the equipment can

have any impact on the product quality, namely in the mixture and consequent incorporation of air in

the product that facilitates its oxidation. Whether a manufacturing process uses simple or complex

equipment, changes in the equipment can cause variation on the FPP.

5.2.3 Human Actions

The production operator may introduce process variations that compromise the quality of the FPP. Since

processes need human intervention, they will always have some associated variation. [24] [25].

5.2.4 Environment

The process is performed at controlled, low-light temperature and humidity in the production room. For

oxygen production is carried out without air control, e.g. an inert atmosphere is not used. Changes in

temperature and humidity affect various processes. Also, some manufacturing processes require a clean

room environment, and the introduction of particles from outside the clean room can cause variation.

Changes in the environment can induce variation in raw materials, equipment and human action.

5.2.5 Manufacturing Process

A manufacturing process consists of several unit operations. Variation can be introduced if the order

of unit operations changes, the duration of each unit operation, if one of the operations is suppressed.

When certain parameters are not correctly defined in the process as temperature and stirring speed. [26].

In this particular case, if during mixing, vacuum is not applied, air present into the product might induce

oxidation of product. It is important that the process is uniform across batches so that it does not create

variability that could compromise batch quality.

5.2.5.A Non uniform parameters:

The manufacturing process presents differences in several parameters which includes variability in the

process, this variability may explain stability concerns since parameters of the manufacturing process

differ considerably between batches.

a) Cooling rate;

b) Stirring speed;

c) Product viscosity;

d) Holding time.

43

5.2.5.B Uniform parameters:

The process presents some variables that do not exhibit any variability, being constant among all ana-

lyzed batches.

a) Temperature;

b) Mixing duration of the Mixture (step II);

c) Equipment.

Analyzing all the uniform and non-uniform parameters represented in the figure 5.7, it is noticeable

that the variables that may be responsible for the instability of the product are the ones that vary in the

process, the objective is to identify which variable(s) may contribute to the instability.

Figure 5.7: Process variables

44

The product under study shows no trend towards related substances percentage in the stability

studies. In order to understand the possible root cause of high degradation it is necessary to study what

may be the points where faults can occur that increase the degradation of the product. Analyzing all

the branches of the diagram it is concluded that there are three factors that may have a major influence

on the problem as shown in figure 5.8. In a more general and initial segment include the raw materials

used, with greater attention in the API. The manufacturing process is also a point that can be very critical

since it includes many steps and variables that have to be controlled. Finally, stability tests results may

be included in order to evaluate the precision and accuracy of the analytical methods used in the tests,

in particular in the determination of the related substances on the product and their homogeneity.

Figure 5.8: Possible variables affecting the product quality

5.3 Control

To minimize product quality issues and optimize product stability, tight API control is required, which is

the most problematic raw material in the product. The figure shows the critical points in the raw material

life cycle from its receipt at the end of the manufacturing process.

Figure 5.9: Control points

45

5.4 Formulation with antioxidants

5.4.1 Compatibility

When formulating with antioxidants, compatibility is a major concern. Care must be taken to protect the

antioxidants from neutralizing each other under the conditions of the formulation. In general, antioxidants

are less stable in neutral or alkaline pH, their stability can be greatly enhanced at acidic pH.

5.4.2 Stability

Product stabilization is crucial, antioxidants can be very unstable, they may become oxidized and inactive

before reaching the target.

5.4.3 Penetration

Antioxidants must be absorbed into the skin, reach their target tissue in the active form, and remain there

long enough to exert the desired effects. There are several strategies used by formulators to increase

penetration of topical actives.

5.5 Antioxidants

Many organic compounds undergo appreciable atmospheric oxidation when stored at room temperature

and this is sometimes referred to as ”autoxidation”. Common examples in the pharmaceutical field

include the oxidative rancidity of fats and oils and the fall in activity of several vitamins. It has been found

that several substances have the property of delaying or preventing autoxidation.

Antioxidants are primarily intended to prevent oxidation of pharmaceutical products. Antioxidants can

be divided into three categories. The first category consists of true antioxidants that inhibit oxidation by

reaction with free radicals. Examples of these antioxidants are, BHT, BHA and tocopherols. The second

group includes reducing agents, examples being ascorbic acid, sodium and potassium salts. The third

group is synergistic antioxidants, which have a reduced antioxidant effect but increase the effect of other

antioxidants. Examples are citric acid, lecithin and tartaric acid.

The antioxidants chosen for the formulations were BHT, BHA, alpha tocopherol and citric acid. All

of these antioxidants, especially BHT and BHA, are lipophylic, something important since the product is

made using oily bases.

46

5.5.1 Butylated hydroxytoluene (BHT)

This is 2,6-di-i-butyl-p-cresol, a colourless crystalline powder, melting at 70°C. It is insoluble in water

but soluble in organic solvents, and was originally developed as a preservative for petroleum products

and rubber but was later adopted for foods and drugs. BHT is an alternative antioxidant to tocopherol

for liquid paraffin. BHT is used as an antioxidant in cosmetics, foods, and pharmaceuticals. It is mainly

used to delay or prevent the oxidative rancidity of fats and oils and to prevent loss of activity of oil-soluble

vitamins. The table 5.1 describes the amounts recommended by the Handbook of Pharmaceutical

Excipients [22].

Antioxidant use Concentration (%)Edible vegetable oils 0.01Essential oils and flavoring agents 0.02–0.5Fats and oils 0.02Inhalations 0.01IM injections 0.03IV injections 0.0009–0.002Topical formulations 0.0075–0.1

Table 5.1: Antioxidant uses of butylated hydroxytoluene

5.5.2 Butylated Hydroxyanisole (BHA)

BHA is an antioxidant with some antimicrobial properties. It is used in a wide range of cosmetics, foods,

and pharmaceuticals. BHA is frequently used in combination with other antioxidants, particularly buty-

lated hydroxytoluene and alkyl gallates, and with sequestrants or synergists such as citric acid. It is a

yellow, crystalline substance, with an aromatic odour, melting at about 65°C. BHA is insoluble in water

but soluble in alcohol, fats and other organic solvents.

It is an alternative to BHT in the preservation of wool fat. The table 5.2 describes the amounts recom-

mended by the Handbook of Pharmaceutical Excipients [22].

Antioxidant use Concentration (%)Essential oils and flavoring agents 0.02–0.5IM injections 0.03Fats and oils 0.02IV injections 0.0002–0.0005Topical formulations 0.005–0.02

Table 5.2: Antioxidant uses of butylated hydroxyanisole

47

5.5.3 Alpha Tocopherol

Alpha tocopherol is a source of vitamin E. While alpha tocopherol also exhibits antioxidant properties, the

beta, delta, and gamma tocopherols are considered to be more effective as antioxidants. This compound

is highly lipophilic, and is a solvent for many poorly soluble drugs. The tocopherols are present in several

vegetable oils and fats. Tocopherol is used as an antioxidant, particularly for liquid paraffin. It is effective

at 10 parts per million [22].

5.5.4 Other antioxidants

The addition of another compound in small amount in order to increase the effect of an antioxidant, it

is a possibility. The mechanism of this particular type of synergism is not fully understood, but may be

related, in part, to the chelating of metals, as some metals enables oxidation changes.

One substance in this class is citric acid, which is used as a sequestering agent and antioxidant syner-

gist.

48

6Results and Discussion

Contents

6.1 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.2 Raw Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

6.3 Manufacturing process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

6.4 Stability tests measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6.5 Nitrogen application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

6.6 Pharmaceutical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

6.7 Production scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

6.8 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

49

50

As already stated above, the main objective is to discriminate a cause for the API degradation, for

this, the data from various sources of the history provided by the company was analysed. Subsequently,

a set of formulations was developed and several tests were conducted to study the stability of FPP

batches.

6.1 Data analysis

The history available in the company was investigated, in total were analyzed nine batches. An important

point when analyzing values obtained from measurements by analytical methods is the associated error.

In the case of related substances analysis on FPP, the method was previously validated and it was

demonstrated that the results can have an error equivalent to 0.1%.

By analyzing the figure 6.1 it is possible to discriminate that five batches, B, C, F, H, I have a per-

centage of related substances higher than 4% , in this way these batches are labeled as having a higher

degradation rate when comparing with the other batches, A, D, E, I, which are categorized as having

lower degradation rate. It is relevant to note that all batches have the same formulation and were studied

under the same stability conditions.

Figure 6.1: Long term stability results

51

The analysis of the data was done in several batches, initially the objective was to analyze all avail-

able batches in stability, however, since the analysis will be obtained by crossing stability and manu-

facturing process data, some older batches will not be considered because their manufacturing records

were discarded.

From the history that could be accessed there is a 44% distribution for batches with total related sub-

stances < 4% and the remainder for batches with total related substances > 4% as shown in figure

6.2.

The key is to understand why there is such a sharp discrepancy (2.1%-6.8%) between the results of

the total percentage of related substances in different batches.

Figure 6.2: Batches distribution

6.2 Raw Materials

The raw materials used in the formulation of the product have already been mentioned in Chapter 4. Of

all the raw materials used, API is most prominent in the product. The API is light sensitive, has restricted

storage conditions and is readily oxidizable when in contact with atmospheric oxygen. In this sense it is

important to analyze the conditions in which the API is transported, the reception and the subsequent

storage are made. All these conditions must be controlled in order to prevent the API from degrading

and consequently reducing the stability of the final product.

One question that arises is whether the API shelf life has a direct and negative influence on the FPP.

After the crossing of data from two batches of API with the same shelf life (5 months) the results obtained

are represented in figure ??. Another question was whether the initial value of related substances in the

API was directly proportional to the value of related substances in the FPP upon batch release. From

the analysis of the figure 6.3, it can be seen that there is no relation between the initial percentage of

related substances in the API and the percentage of related substances in the FPP.

52

The API batch I has a value of 0.33% at 5 months and an initial related substances of 1.87% as the

FPP, API batch II has an initial value of related substances of 0.84% at 5 months and at the end of the

manufacturing process has a value corresponding to 0.92%. Thus, there was a 467% of degradation

rate in the batch I and 9.5% in batch II, which indicates that the problem appears to be related to the

manufacturing process.

Figure 6.3: Related substances API vs related substances FPP.

53

6.2.1 Raw materials CoA analysis

In order to understand whether raw materials could have impact on API degradation rate, data was

crossed from five different FPP batches for which there were data from the CoA.

From a cursory analysis of the data illustrated in table 6.5, batches A, B and C for raw materials dif-

fer only in one API batch in a total of three API batches used for each FPP batch. Batches B and C

were batches with total related substances > 4% and both did not differ in any raw material as shown

in figure 6.4, which may indicate that the cause of high related substances of these batches may be the

raw material. For batch A two of the three batches of raw material are the same as batches B and C,

and the batch that is different is one of the batches of API used. One of the factors to take into account

is the shelf life of the raw materials, and the values described in the figure 6.5 are from the CoA, this

may have some influence on the quality of the raw materials and consequently on the product.

Figure 6.4: Differences between API batches

Figure 6.5: Raw materials CoA (A, B and C)

By analyzing batches E and F shown in figures 6.6 and 6.7 an the API batch used was the same,

only the excipients batches differ and there are minor differences which may point to the problem being

in the manufacturing process.

54

Figure 6.6: Differences between raw materials batches

Figure 6.7: Raw materials CoA (E and F)

Since in one situation the API appears to have an impact on the final result of the batch and in

another scenario the opposite result, is not possible to conclude that the raw materials are decisive for

the FPP stability. A major limitation of the study is the number of batches available for analysis.

55

6.3 Manufacturing process

The manufacturing process is composed of several stages of mixing and melting of components. In

these steps there are two important parameters, the stirring speed and the temperature. Within the

process there may be variations between batches such as mixing and cooling times. In this way it is

necessary to investigate if any of these changes can explain the variability between batches with regard

to stability.

The manufacturing process was investigated to identify if any process variable led to the high percent-

age of related substances in some batches.

For this assessment, it was necessary to have access to the company’s data history. Data from batches

that were 2 years beyond the expiry date were discarded, the manufacturing process was investigated

on the basis of available history corresponding of five batches. The objective was to individually analyze

each of the process variables and understand their influence on batches that have high or low degrada-

tion rate. Variables that present more disparate values between batches with low and high degradation

rates may have some influence on the percentage of related substances. Of the five batches analyzed

two of them had high degradation rates after two years.

Each of the variables was analyzed with the aid of radar charts, giving a clear overview of batches that

describe different behavior concerning a given variable, so it is easier to discriminate whether there is

any variable that behaves differently in high and low degradation rates batches.

There were analyzed all variables that showed some variation of values between batches, all constant

variables were ignored in this analysis. The general idea for analyzing the collected data was based on

a simple view of batch variable values. If there is a variable that has a disparate value in a batch with low

degradation rate it can be concluded that this variable will not trigger the problem. In a scenario where

a variable has different values from others batches, only in batches with high degradation rates, it can

already be seen that it is possibly a variable with some influence.

56

The figure 6.8 represents the stirring speed. By the analysis it is possible to observe that the batches

F and H have similar values, these being the two batches with high degradation rates, however the batch

E also presents a similar value, with this data is possible to infer this variable will not be decisive for the

problem.

Figure 6.8: Stirring speed for the five analyzed batches

The figure 6.9 shows the viscosity values of all batches, the values are very similar among all batches.

Since it is not a variable that presents wide variation between low and high degradation rate batches,

it can be concluded that it is not a variable directly related to the problem. The only batch that has a

slightly different value is E , this batch has a low degradation rate which reinforces the non-binding of

this variable with the identified problem.

Figure 6.9: Viscosity for the five analyzed batches

The figure 6.10 shows all values related to the cooling rate, it is possible to identify that the batches

that have a high degradation rate F and H have different values from the other batches.These batches

have a high degradation rate which may indicate a possible relation of this factor to the problem. Batches

F and H have higher cooling rates values than other batches, this variable may have some influence on

the process and consequently on the problem, however, it will hardly be the only cause since the values

57

are higher but not markedly different from others batches to justify that this is a single responsible

variable.

Figure 6.10: Cooling rate for the five analyzed batches

The holding time variable is represented in figure 6.11, as shown, has a heterogeneous behavior

between batches, and the batch that moves farther away from the trend (G) has a low degradation rate

so this variable should not be the cause.

Figure 6.11: Holding time for the five analyzed batches

Of all the analyzed variables, the variable which demonstrated a possible correlation was the cooling

rate, however the number of batches is not significantly large to be able to infer with certainty that this

variable has a negative impact on the development of related substances.

58

6.4 Stability tests measurements

For a stability evaluation, several tests are carried on, some solely dependent on the operator e.g.

appearance of the ointment, others are conditioned by the apparatus used. Like all methods and devices

used there is always an associated margin of error. As previously mentioned the process was studied in

order to understand if there was any variable that had a greater influence on the stability of the product.

Another point studied was the raw materials, particularly the API. These are the two key points that

may indicate the cause of product stability instability. One corrective measure implemented was to

continuously track the related substances of the API from the moment it is shipped at the factory, at the

weighing and in the process utilization.

When evaluating the measurements and stability results another issue that arose was the possibility

of lack of homogeneity of the samples, this being due to an inefficient mixture. To understand if this

hypothesis is factually a problem it has been suggested to collect three samples in the filling container

which is used for the filling in order to ascertain if the three samples have different assay results, which

should not happen. This test was performed and the results showed uniformity between several tubes

originating from different positions of the mixer container indicating that the tubes are homogeneous with

each other.

6.5 Nitrogen application

Regarding the product, there is no trend in the results of the stability tests. The treatment with nitrogen

may have an influence on the stability results, however, since there have been batches that have low

degradation rates and have not had any treatment with nitrogen, this leads to the idea that the solution

does not pass solely by the treatment with nitrogen.

In the currently available ointments, API is used in fine powder form. The small particle size enhances

its dermal contact by providing a large specific surface area and penetration. One way to minimize API

contact with oxygen is to conduct the manufacturing process in an inert atmosphere.

6.5.1 Manufacturing process

As previously mentioned the manufacturing process is an important variable. Since the product is sen-

sitive to contact with atmospheric air, one of the possible approaches is to perform the manufacturing

process in the presence of nitrogen to minimize or eradicate it.

The process was executed at the factory and critical points were pointed out and preventive mea-

sures proposed as illustrated in tables 6.1 and 6.2. One of the most important steps for using nitrogen

is the filling step as it is the last step of the process and it is necessary to ensure that the tube does not

59

contain oxygen. Oxygen trapped in the tubes is responsible for oxidation. One of the pertinent tests to

be performed would be to measure dissolved oxygen in tubes from different batches at time 0 and to

see if this value is directly related to the results of the stability tests.

Unitary operation Critical pointsPlacing of raw materials on equipment A NA

Placing of raw materials on equipment B

API exposed to light and oxygen when removed from the bag;Stainless steel tank has a lid that stays open when the API is added;The room where the product is produced has two large windows with light entry;Nitrogen placement through wide opening relative to the nitrogen tube;Placing adhesive tape on the lid to reduce contact with outside air.

Transfer of the contents of thestainless steel tank to equipment A

Hose placed inside the tank by the lid, which is lifted and the mixture in contact with light and oxygen;Part of the content is manually transferred in the equipment B.

Transfer of equipment A content to filling container Product contact with outside air once filling container lid is tilted;Placing the adhesive tape on the filling container to reduce contact with outside air.

End of transfer processProduct that is retained in the walls in the equipment A is removedmanually by the operators and placed in a container that is in permanent contact with the air,this content is later added to the filling container.

Filling Filling container lid must be removed and a lid fitted with the filling equipment attached;Feeding of the hopper made in an open environment.

Table 6.1: Critical points

Unitary operation Preventive measuresPlacing of raw materials on equipment A NA

Placing of raw materials on equipment B

The API should be removed from the bag in an environment without light with the help of the sodium lamp;The nitrogen must be ejected into the equipment with a system that allows it to be trapped;Lid sealing system with silicone or other material that avoids any external air intake;The placing of the raw materials that are in contact with the API, in the particular case the paraffin must alsobe introduced into the tank by means of a tube that is attached to the tank in order to eradicate contact with air andlight from the outside environment.

Transfer of the contents of thestainless steel tank to equipment A

Hose coupling directly into the tank without the need to lift the lid;System that pushes all content to the everything that transports the product to the equipment A.

Transfer of equipment A to filling container System that allows the coupling of the hose of equipment A to the filling container, making possible the total absence of contactof the product with the outside air.

End of transfer process System that pushes all contents to the hose inlet to prevent manual placement of the remaining product in the filling container.

FillingLid system that allows direct coupling with the filling equipment;Feed the hopper in an isolated system.;Automated system of nitrogen ejection in the tubes.

Table 6.2: Preventive measures

By analyzing the results illustrated in figures 6.12, 6.13 and 6.14 which demonstrate the influence

of the nitrogen on the total percentage of related substances in the FPP over time. It should be noted

that there is only one batch under study and is not sufficient to corroborate the fact that nitrogen is pre-

ponderant for the reduction of total related substances in the FPP. However if the analysis is performed

taking into account the trend it can be seen that the batch line with nitrogen is always below the line

without nitrogen, indicating that the introduction of nitrogen into the manufacturing process reduces the

related substances percentage.

60

Figure 6.12: Comparative study-LT (Batch I)

Figure 6.13: Comparative study-IC (Batch I)

Figure 6.14: Comparative study-AC (Batch I)

61

In order to understand more clearly the influence of the inclusion of nitrogen in the manufacturing

process, an analysis of the addition of related substances between the nitrogen free and nitrogen batch

was performed.

To calculate the degradation increase it is necessary to know the initial value of related substances (x1)

and the final value (x2) represented in equation 6.1. In the case of nitrogen the initial value corresponds

to the value of the batch without nitrogen and the end to the batch with nitrogen. The results are shown

in the figure 6.15.

x2 − x1x1

× 100 (6.1)

Figure 6.15: Increase percentage of related substances

Batch II corresponds to another batch where the results were compared between with and without

nitrogen application. By analyzing the figure 6.16 and making a trend analysis it is possible to analyze

that under most conditions and time points the line corresponding to part of the batch without nitrogen

treatment is below the line with nitrogen indicating that nitrogen has not improved the percentage of

related substances.

62

Figure 6.16: Nitrogen application results (Batch II)

To prove that nitrogen has an influence it will be necessary to study more batches so that the results

are reproducible and reliable.

6.6 Pharmaceutical development

6.6.1 HPLC analysis

As previously described, laboratory batches were analyzed by HPLC to quantify the related substances

and to study the extent of degradation of each sample. In this regard it is important to understand the

usefulness of this technique and the chromatograms obtained for the calculation of the total percentage

of related substances.

63

Typical chromatograms of an HPLC analysis, an SST, solvent, reference and test solutions are shown

in figures 6.17, 6.18, 6.19 and 6.20.

Figure 6.17: SST chromatogram

Figure 6.18: Solvent chromatogram

64

Figure 6.19: Reference solution chromatogram

Figure 6.20: Test solution chromatogram

65

The batches were placed in stability study in order to ascertain the degradation rate of each batch in

different conditions. As already mentioned, stability tests were performed under two conditions, LT and

AC at two time points, 0 (release) and 3 months as shown in figure 6.21.

Figure 6.21: Total related substances percentage

The table 6.3 reflects the difference between the initial value at 0 months and the final value at 3

months in LT and AC conditions, establishing which batches had the highest degradation rate over time.

From the table 6.3, it is possible to conclude that there are two batches (III and Control) which show

a large increase between the value of related substances at 0 months and the value at 3 months in

LT conditions. The batch that has the lowest increase percentage relative to the initial value of related

substances is batch VII.

The table 6.3 shows the values for the percentage increase between 0 and 3 months of batches

placed under AC. In this study the batches with the higher degradation rate were the I and Control. The

batch that presents the smallest difference between the initial and final values was the VII.

Batch I II III IV V VI VII VIII IX ControlLT 0M-3M (%) 188 122 212 37,5 100 100 22 40 110 190AC 0M-3M (%) 275 156 225 137 122 222 100 220 150 230

Table 6.3: Increase percentage of related susbtances

Laboratory batches were subjected to various stress conditions, namely hydrochloric acid and hy-

drogen peroxide, to understand the amount of degradation products resulting in each batches. The

hydrogen peroxide assay can provide an indication of the susceptibility of each batch to oxidative pro-

cesses, thus within the forced degradation tests, this is what demonstrates most useful results in the

66

product under study since the central point of the study is the oxidation of API.

The figure 6.22 shows the results obtained in the hydrogen peroxide forced degradation tests which,

as referred to, test the oxidation of the compounds. It can be seen that the tested batches which have

the smallest value of related substances were the batches I, IV and VII. The batch with the highest value

was the batch Control.

It is also illustrated the results of the forced degradation tests by HCl. Importantly, this test is not as

relevant as the hydrogen peroxide test. As it is possible to observe the values between the batches do

not present very significant differences. The batches with the lowest related substances values were I,

IV and VII. The batches with the highest related substances values were IX and V.

Figure 6.22: Total Related Substances-Forced Degradation

The table 6.4 shows the values for the percentage increase between between 0 months and the

48h period in which the samples were in the presence of degrading agents. In this study the batches

with the higher degradation rate were the III and Control for the H2O2 test. The batch that presents

the smallest difference between the initial and final values is the VII. For the HCl test the batches with

higher degradation rate were III and V. The batch which presents the smallest degradation rate is the

Control.

Batch I II III IV V VI VII VIII IX ControlH2O2(%) 288 366 425 338 333 367 233 330 380 690HCl (%) 175 222 275 175 289 211 144 190 200 130

Table 6.4: Increase percentage of related susbtances (Forced degradation tests)

67

Regarding the results of the atmospheric air exposure tests, it is possible to identify as shown in

figure 6.23, the behavior of the various batches of the product at 30 days exposure to atmospheric air.

From the analysis of the results it can be observed that the batches that batches IX and Control have

the highest values of related substances. The batches with the lowest percentage of related substances

were IV and VII.

Figure 6.23: Total Related Substances-(Atmospheric air exposure)

The table 6.5 represents the increase percentage in the related substances between the LT 0M and

the 30 days atmospheric air exposure test. The batches that stand out for having a low percentage

increase were IV and VII, the batch with the higher increase in the percentage of related substances is

the Control.

Batch I II III IV V VI VII VIII IX ControlAtmospheric air exposure (%) 212 167 215 150 156 178 78 130 200 230

Table 6.5: Increase percentage of related substances (Atmospheric air exposure)

6.7 Production scale

A key question for the entire study is whether the values of stability tests in laboratory batches were

indicative of what is expected on an industrial scale.

One fact that may indicate that the value of related substances in laboratory batches will be similar

to that of industrial batches is by comparing values and understanding if there is any correlation.

There are several industrial batches where LT and AC have values very close to the control batch in the

68

same time points, which may indicate that the other batches may also have close values when produced

on a laboratory scale.

6.8 Discussion

By analyzing all the results obtained with the stability tests, forced degradation tests and atmospheric

air exposure it is possible to highlight batches IV and VII for very positive results regarding the total

percentage of related substances in H2O2, atmospheric air exposure and stability tests.

Nitrogen studies indicate that its application can minimize API degradation but to support this idea a

larger batch repertoire is required. Raw material data were inconclusive since divergent results were

obtained from both scenarios, being limited by the reduced number of batches evaluated. Regarding the

manufacturing process, one of the variables was potentially related to the high degradation rate of the

API since the high degradation batches had similar values but distinct values from the low degradation

batches.

69

7Conclusions

Contents

7.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

7.2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

71

72

7.1 Conclusions

This thesis was divided into two major parts, i) data analysis and ii) pharmaceutical development with

the inclusion of antioxidants.

The first part of the project concerned the analysis of raw material and manufacturing process data in

order to identify the cause of the high percentage of related substances i.e. high degradation of the

API. The raw material analysis included only data from five batches for which stability studies were also

reported. Information from the five batches was not conclusive since part of the batches indicated that

the cause could be the API batches, while others showed no correlation. The conclusions were unclear,

in the raw material data, it was not possible to establish a correlation between API chemical parameters

and API degradation rate. The manufacturing process data were analyzed resourcing to five batches,

and only the variables that presented variation between batches were analyzed. Of the four variables

analyzed, one of them can be considered as a possible cause of high degradation, since it is the only

one with similar values between the two high degradation batches and different from the low degradation

batches. Five batches were analyzed in this part, which is insufficient to obtain robust and representative

results of the actual situation under analysis.

For the second part, because the data collected were insufficient to draw conclusions regarding the

cause of the high degradation of the API, it was proposed to develop several formulations using antiox-

idants compatible with the components of the initial pharmaceutical formulation. The formulations were

subjected to various tests to study their stability over time and under various conditions, including forced

conditions. By analyzing the results it is possible to conclude that there were two formulations which

stood out, IV and VII. These formulations had considerably lower total values of related substances than

the control formulation.

The study of forced degradation showed that some of the formulations are sensitive to degradation but

the control had a much higher degradation, which shows that part of the solution for product stability

involves the inclusion of antioxidants in the formulation.

The oxygen exposure test demonstrated that the product is oxygen sensitive and contact leads to degra-

dation of the API. Of all the formulations, the highest value of related substances percentage was batch

control which supports the idea that the inclusion of antioxidants is essential to improve API protection

against oxidation.

Concerning the application of nitrogen by the two batches analyzed it cannot be inferred that nitrogen

has a positive impact on the degradation of the API, as one of the batches shows that nitrogen tends

to decrease the degradation rate of the API relative to the control batch (without nitrogen), however in

another batch the scenario is the opposite.

The prediction approach was unsuccessful as the data used was not large enough for the model to be

73

robust and to make a prediction with a high level of certainty.

Thus the conclusion with respect to the analysis of raw material data is that there is no correlation, in

the manufacturing process the cooling rate variable can be identified as a variable with potential impact

on the degradation rate. Finally in the pharmaceutical development step it was possible to identify two

potential formulations which showed very positive results regarding API degradation. These results are

systematized in the figure 7.1.

Figure 7.1: Summary of results

To summarize, the most conclusive approach used was pharmaceutical development since two for-

mulations were identified which exhibit much lower API degradation values than the control batch i.e.

the initial formulation.

7.2 Future Work

One of the pertinent tests to be performed would be to quantify trapped oxygen in several tubes of each

batch to evaluate if the value varied widely between tubes and batches.

It is also necessary to control the manufacturing process at various points in the process. It would be

interesting to implement process analytical technologies (PAT) in the factory to optimize the manufactur-

ing process and minimize possible failures. Since a process variable that could have a potential impact

on product stability has been identified, it would be interesting to do studies with different cooling rate

times and assess if there is a correlation.

The study of nitrogen application to be properly sustained should be done with a considerable number

of batches to be able to prove that there is a real impact on nitrogen application with respect to the

degradation rate of API.

74

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