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Characterization and correlation analysis of pharmaceutical gelatin

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Characterization and correlation analysis of pharmaceutical gelatin
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English
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Felix, Pascal Georges
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polymer
stress test
statistical analysis
rheology
molecular weight
Dissertations, Academic -- Biomedical Engineering -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: The properties of the aged gel and subsequent softgels were examined using mechanical and chemical testing methods. Our hypothesis was that a negligible variation will exist between the aged gel of the same type. The greater difference is expected to be seen between the types of gels described as 150 Bloom (alkaline treated collagen) and 195 Bloom (acid treated collagen). The types of gelatin used were the acid processed (195 Acid Bone) and alkaline processed (150 Lime Bone). Because of the differences expressed as the result of their manufacture sequence (namely their molecular weights), it follows that physical attributes will further contribute to their distinction. In addition to observing different characteristics between the types of gels, we aged the gelatin and produced softgel capsules to qualify and quantify the changes that occur as a function of time.Two production lots of over 1 million softgel capsules were executed to produce a population that lends itself to statistical analysis. Softgel capsules were manufactured with gelatin which was aged at intervals of 0-8 hrs, 32-40 hrs, 66-72 hrs and 88-96 hrs. The manufacturing process made use of this strategy for the acid and alkaline treated gelatin where a total of eight lots were made (4 acid and 4 alkaline). One hundred thousand softgels were manufactured for the acid processed gelatin, per lot. Additionally, one hundred and fifty thousand softgels were manufactured for the alkaline processed gelatin per lot. The results of the different tests provided trends that were not solely a function of time. Gel extensibility for both gel types showed a decrease in the amount of force needed to rupture the gelatin ribbon, as a function of time. The resilience of the tested ribbon remained constant throughout the aging process.The burst strength was the only test showing an inverse relationship between the two gel types. The amount of force needed to rupture the 150 Bloom softgels decrease in time whereas the amount of force needed to rupture the 195 Bloom softgels increase with time. The rheological testing was described in the literature as being associated with the molecular weight distribution. Such association was seen in our research and both the results of the rheological and the molecular weight tests decreased with the aging process.
Thesis:
Thesis (M.S.B.E.)--University of South Florida, 2003.
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Includes bibliographical references.
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by Pascal Georges Felix.
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Title from PDF of title page.
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Characterization and Correlation Analysis of Pharmaceutical Gelatin by Pascal Georges Felix A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Engineering Department of Chemical Engineering College of Engineering University of South Florida Major Professor: William E. Lee III, Ph.D. Leo E. Ondrovic, Ph.D. Rickey S. Shelley, Ph.D. Date of Approval: November 18, 2003 Keywords: Polymer, Stress Test, Statistical Analysis, Rheology, Molecular Weight Copyright 2004, Pascal G. Felix

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i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT xi CHAPTER 1. INTRODUCTION 1 Background 1 Research Objective 7 Research Questions 8 Importance of the Study 9 Limitations of the Study 9 CHAPTER 2. LITERATURE REVIEW 11 An Overview of Collagen 11 An Overview of Gelatin 13 Gelatin Manufacturing 15 Comparison Obtaining Gelatin from Collagen 17 CHAPTER 3. RESEARCH DESIGN AND METHODS 20 Gel Extensibility 20 Resilience 23 Wet Burst Strength 26 Rheometer 28 HPLC (Size Exclusion Chromatography) 31 Experimental Design 31

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ii CHAPTER 4. RESULTS AND DISCUSSION 33 Results 33 Discussion 44 CHAPTER 5. CONCLUSIONS AND RECOMMENDATIONS 51 Conclusions 51 Recommendations 53 REFERENCES 54 APPENDICES 55 Appendix 1. Microsoft (MS) Excel Graphical Representation of Test Results 56 Appendix 2. Wet Burst Strength Test Results 68 Appendix 3. Rheology Test Results 86 Appendix 4. Gel Extensibility Test Results 110 Appendix 5. Resilience Test Results133 132 Appendix 6. Molecular Weight Analysis Results 150

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iii LIST OF TABLES Table 1. Differences Between the Major Types of Gelatin. 2 Table 2. Summary of the Experimental Design. 32 Table 3. Summary of Experimental Results for the Various Methods. 35 Table 4. Summary of Experimental Results for the Gel Extensibility Method. 36 Table 5. Summary of Experimental Results for the Resilience Method. 37 Table 6. Summary of Experimental Results for the Wet Burst Strength Method. 38 Table 7. Summary of Experimental Results for the Rheological Method. 39 Table 8. Molecular Summary of Experimental Results for the Molecular Weight Analysis Method. 40 Table 9. Summary of Statistical Analysis of Table 3 Data. 45 Table 10. Pearson Correlation Coefficient for 150 Bloom Gel. 47 Table 11. Pearson Correlation Coefficient for 195 Bloom Gel. 48

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iv LIST OF FIGURES Figure 1. Diagram of a Softgel. 5 Figure 2. Chemical Structures of Main Amino Acids of Collagen. 12 Figure 3.Triple Helix Visualizations of Collagen Constituents. 12 Figure 4. Alkali and Acid Treatment Processes for Gelatin. 16 Figure 5. Products of the Dissociation of the Triple Helix. 19 Figure 6. Encapsulation Machine. 21 Figure 7. TA.XTR Texture Analyzer. 22 Figure 8. An Example of a Gel Extensibility Test Printout. 23 Figure 9. An Example of a Resilience Test Printout. 25 Figure 10. An Example of a Wet Burst Strength Test Printout. 27 Figure 11. The Haake Rheostress Research Grade Rheometer. 29 Figure 12. An Example of Rheometer Testing Printout. 30 Figure 13. Summary Graph of Averaged Gelatin Extensibility. 41 Figure 14. Summary Graph of Averaged Gelatin Wet Burst Strength. 42 Figure 15. Summary Graph of Averaged Gelatin Molecular Weight. 43

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v Figure 16. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (0-8 hrs). 56 Figure 17. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (32-40 hrs). 56 Figure 18. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (64-72 hrs). 57 Figure 19. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (88-96 hrs). 57 Figure 20. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (0-8 hrs). 58 Figure 21. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (32-40 hrs). 58 Figure 22. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (64-72 hrs). 59 Figure 23. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (88-96 hrs). 59 Figure 24. MS Excel Representation of Resilience Test Results for 195 Gelatin (32-40 hrs). 60 Figure 25. MS Excel Representation of Resilience Test Results for 195 Gelatin (64-72 hrs). 60 Figure 26. MS Excel Representation of Resilience Test Results for 195 Gelatin (88-96 hrs). 61 Figure 27. MS Excel Representation of Resilience Test Results for 150 Gelatin (0-8 hrs). 62 Figure 28. MS Excel Representation of Resilience Test Results for 150 Gelatin (32-40 hrs). 62

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vi Figure 29. MS Excel Representation of Resilience Test Results for 150 Gelatin (64-72 hrs). 63 Figure 30. MS Excel Representation of Resilience Test Results for 150 Gelatin (88-96 hrs). 63 Figure 31. MS Excel Representation of Wet Burst Strength Test Results for 195 Gelatin (32-40 hrs). 64 Figure 32. MS Excel Representation of Wet Burst Strength Test Results for 195 Gelatin (64-72 hrs). 64 Figure 33. MS Excel Representation of Wet Burst Strength Test Results for 195 Gelatin (88-96 hrs). 65 Figure 34. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (0-8 hrs). 66 Figure 35. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (32-40 hrs). 66 Figure 36. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (64-72 hrs). 67 Figure 37. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (88-96 hrs). 67 Figure 38. 195 Gelatin Wet Burst Strength Printout at 32 hrs. 68 Figure 39. 195 Gelatin Wet Burst Strength Printout at 64 hrs. 69 Figure 40. 195 Gelatin Wet Burst Strength Printout at 68 hrs. 70 Figure 41. 195 Gelatin Wet Burst Strength Printout at 88 hrs. 71 Figure 42. 195 Gelatin Wet Burst Strength Printout at 92 hrs. 72 Figure 43. 195 Gelatin Wet Burst Strength Printout at 96 hrs. 73 Figure 44. 150 Gelatin Wet Burst Strength Printout at 0 hrs. 74 Figure 45. 150 Gelatin Wet Burst Strength Printout at 4 hrs. 75

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vii Figure 46. 150 Gelatin Wet Burst Strength Printout at 8 hrs. 76 Figure 47. 150 Gelatin Wet Burst Strength Printout at 32 hrs. 77 Figure 48. 150 Gelatin Wet Burst Strength Printout at 36 hrs. 78 Figure 49. 150 Gelatin Wet Burst Strength Printout at 40 hrs. 79 Figure 50. 150 Gelatin Wet Burst Strength Printout at 64 hrs. 80 Figure 51. 150 Gelatin Wet Burst Strength Printout at 68 hrs. 81 Figure 52. 150 Gelatin Wet Burst Strength Printout at 72 hrs. 82 Figure 53. 150 Gelatin Wet Burst Strength Printout at 88 hrs. 83 Figure 54. 150 Gelatin Wet Burst Strength Printout at 92 hrs. 84 Figure 55. 150 Gelatin Wet Burst Strength Printout at 96 hrs. 85 Figure 56. 195 Gelatin Rheological Testing at 0 hrs. 86 Figure 57. 195 Gelatin Rheological Testing at 4 hrs. 87 Figure 58. 195 Gelatin Rheological Testing at 8 hrs. 88 Figure 59. 195 Gelatin Rheological Testing at 32 hrs. 89 Figure 60. 195 Gelatin Rheological Testing at 36 hrs. 90 Figure 61. 195 Gelatin Rheological Testing at 40 hrs. 91 Figure 62. 195 Gelatin Rheological Testing at 64 hrs. 92 Figure 63. 195 Gelatin Rheological Testing at 68 hrs. 93 Figure 64. 195 Gelatin Rheological Testing at 72 hrs. 94

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viii Figure 65. 195 Gelatin Rheological Testing at 88 hrs. 95 Figure 66. 195 Gelatin Rheological Testing at 92 hrs. 96 Figure 67. 195 Gelatin Rheological Testing at 96 hrs. 97 Figure 68. 150 Gelatin Rheological Testing at 0 hrs. 98 Figure 69. 150 Gelatin Rheological Testing at 4 hrs. 99 Figure 70. 150 Gelatin Rheological Testing at 8 hrs. 100 Figure 71. 150 Gelatin Rheological Testing at 32 hrs. 101 Figure 72. 150 Gelatin Rheological Testing at 36 hrs. 102 Figure 73. 150 Gelatin Rheological Testing at 40 hrs. 103 Figure 74. 150 Gelatin Rheological Testing at 64 hrs. 104 Figure 75. 150 Gelatin Rheological Testing at 68 hrs. 105 Figure 76. 150 Gelatin Rheological Testing at 72 hrs. 106 Figure 77. 150 Gelatin Rheological Testing at 88 hrs. 107 Figure 78. 150 Gelatin Rheological Testing at 92 hrs. 108 Figure 79. 150 Gelatin Rheological Testing at 96 hrs. 109 Figure 80. 195 Gelatin Extensibility Test Printout at 0 hrs. 110 Figure 81. 195 Gelatin Extensibility Test Printout at 4 hrs. 111 Figure 82. 195 Gelatin Extensibility Test Printout at 8 hrs. 112 Figure 83. 195 Gelatin Extensibility Test Printout at 32 hrs. 113

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ix Figure 84. 195 Gelatin Extensibility Test Printout at 36 hrs. 114 Figure 85. 195 Gelatin Extensibility Test Printout at 40 hrs. 115 Figure 86. 195 Gelatin Extensibility Test Printout at 64 hrs. 116 Figure 87. 195 Gelatin Extensibility Test Printout at 68 hrs. 117 Figure 88. 195 Gelatin Extensibility Test Printout at 72 hrs. 118 Figure 89. 195 Gelatin Extensibility Test Printout at 88 hrs. 119 Figure 90. 195 Gelatin Extensibility Test Printout at 92 hrs. 120 Figure 91. 195 Gelatin Extensibility Test Printout at 96 hrs. 121 Figure 92. 150 Gelatin Extensibility Test Printout at 8 hrs. 122 Figure 93. 150 Gelatin Extensibility Test Printout at 32 hrs. 123 Figure 94. 150 Gelatin Extensibility Test Printout at 36 hrs. 124 Figure 95. 150 Gelatin Extensibility Test Printout at 40 hrs. 125 Figure 96. 150 Gelatin Extensibility Test Printout at 64 hrs. 126 Figure 97. 150 Gelatin Extensibility Test Printout at 68 hrs. 127 Figure 98. 150 Gelatin Extensibility Test Printout at 72 hrs. 128 Figure 99. 150 Gelatin Extensibility Test Printout at 88 hrs. 129 Figure 100. 150 Gelatin Extensibility Test Printout at 92 hrs. 130 Figure 101. 150 Gelatin Extensibility Test Printout at 96 hrs. 131 Figure 102. 195 Gelatin Resilience Test Printout at 32 hrs. 132

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x Figure 103. 195 Gelatin Resilience Test Printout at 36 hrs. 133 Figure 104. 195 Gelatin Resilience Test Printout at 40 hrs. 134 Figure 105. 195 Gelatin Resilience Test Printout at 64 hrs. 135 Figure 106. 195 Gelatin Resilience Test Printout at 68 hrs. 136 Figure 107. 195 Gelatin Resilience Test Printout at 72 hrs. 137 Figure 108. 195 Gelatin Resilience Test Printout at 88 hrs. 138 Figure 109. 195 Gelatin Resilience Test Printout at 92 hrs. 139 Figure 110. 195 Gelatin Resilience Test Printout at 96 hrs. 140 Figure 111. 150 Gelatin Resilience Test Printout at 8 hrs. 141 Figure 112. 150 Gelatin Resilience Test Printout at 32 hrs. 142 Figure 113. 150 Gelatin Resilience Test Printout at 36 hrs. 143 Figure 114. 150 Gelatin Resilience Test Printout at 40 hrs. 144 Figure 115. 150 Gelatin Resilience Test Printout at 68 hrs. 145 Figure 116. 150 Gelatin Resilience Test Printout at 72 hrs. 146 Figure 117. 150 Gelatin Resilience Test Printout at 88 hrs. 147 Figure 118. 150 Gelatin Resilience Test Printout at 92 hrs. 148 Figure 119. 150 Gelatin Resilience Test Printout at 96 hrs. 149 Figure 120. 195 & 150 Gelatin Molecular Analysis Results. 150

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xi Characterization and Correlation Analysis of Pharmaceutical Gelatin Pascal Georges Felix ABSTRACT The properties of the aged gel and subsequent softgels were examined using mechanical and chemical testing methods. Our hypothesis was that a negligible variation will exist between the aged gel of the same type. The greater difference is expected to be seen between the types of gels described as 150 Bloom (alkaline treated collagen) and 195 Bloom (acid treated collagen). The types of gelatin used were the acid processed (195 Acid Bone ) and alkaline processed (150 Lime Bone). Because of the differences expressed as the result of their manufacture sequence (namely their molecular weights), it follows that physical attributes will further contribute to their distinction. In addition to observing different characteristics between the types of gels, we aged the gelatin and produced softgel capsules to qualify and quantify the changes that occur as a function of time. Two production lots of over 1 million softgel capsules were executed to produce a population that lends itself to statistical analysis. Softgel capsules were manufactured with gelatin which was aged at intervals of 0-8 hrs, 32-40 hrs, 66-72 hrs and 88-96 hrs. The manufacturing process made use of this strategy for the acid and alkaline treated gelatin where a total of eight lots were made (4 acid and 4 alkaline). One hundred thousand softgels were manufactured for the acid processed gelatin, per lot. Additionally, one

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xii hundred and fifty thousand softgels were manufactured for the alkaline processed gelatin per lot. The results of the different tests provided trends that were not solely a function of time. Gel extensibility for both gel types showed a decrease in the amount of force needed to rupture the gelatin ribbon, as a function of time. The resilience of the tested ribbon remained constant throughout the aging process. The burst strength was the only test showing an inverse relationship between the two gel types. The amount of force needed to rupture the 150 Bloom softgels decrease in time whereas the amount of force needed to rupture the 195 Bloom softgels increase with time. The rheological testing was described in the literature as being associated with the molecular weight distribution. Such association was seen in our research and both the results of the rheological and the molecular weight tests decreased with the aging process.

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1 CHAPTER 1. INTRODUCTION Background The understanding of gelatin is important in the formation of capsules because gelatin-related factors are directly related to the absorption and bioavailability of a drug. Gelatin is defined as a mixture of water soluble proteins derived from collagen by hydrolysis. Gelatin is a protein, therefore it will behave like materials that are hydrolyzed by the majority of the proteolytic pathways to ultimately be degraded to amino acid levels (Singh et al., 2002). The protein fractions consist of all the known amino acids. These amino acids are joined by an amide linkage to form a linear polymer varying in molecular weight from 15K to 250K Type A and Type B gelatin are the two most common varieties of gelatin commercially available. Type A, as it relates to the gel used in this study, is manufactured by acidic hydrolysis of bovine bone and pork skin collagen (in this study we only used bone gelatin); the final product (softgels) displays relatively high plasticity and elasticity. Type B gelatin is manufactured by the hydrolysis of bones and bovine hide; the final product displays high gel strength. The qualitative attributes of the gelatin are not their only differentiating modalities. Type A gelatin has a pH between 3.8-6.0 and an isoelectric point between 6-8 whereas, Type B gelatin has a pH between 5.0-7.4 and an isoelectric point between 4.7-5.3. Table 1 summarizes the main differences between the

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2 two types of gelatin used in this study. One must note that the usage of these types of gelatin are not mutually exclusive, for gelatin used in the pharmaceutical manufacture of hardshell capsule blends are sometimes used, but not in the manufacture of softgels. However there are a few exceptions but for the most part, blends tend to be restricted to hardhsell capsules. Table 1. Differences Between the Major Types of Gelatin. G elatin is normally characterized by its jelly strength or bloom strength. Bloom strength is defined as the weight in grams that, when applied with a 12.7 mm diameter plunger, will produce a depression exactly 4mm deep in a matured jelly containing 6.66% of gelatin in water at 10 C for 16-18 hours. Gelatin is insoluble in acetone, chloroform, 95% ethanol, ether and methanol. It is soluble in glycerin, acids and bases. It is susceptible to swelling and softening in water, absorbing 5-10 times its own weight in water. Reactivity is experienced with acids, bases Type A Gelatin (195 Bloom) Type B Gelatin (150 Bloom) Extracted by acidic hydrolysis of bovine bone, bovine hide and pork skin Extracted by alkaline hydrolysis of bovine bones and hide High gel strength and can resist crosslinking better than type B High plasticity and elasticity (compared to acid pork skin) pH ~ 3.8-6.0 pH ~ 5.0-7.4 Isoelectric point ~ 6-8 Isoelectric point ~ 4.7-5.3

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3 aldehydes, aldehydic sugars, anionic and cationic polymers, electrolytes, metal ions, plasticizers, preservatives and surfactants (Singh et al., 2002). Cross-linking of gelatin before or after, the drying aspect of manufacturing of capsules, permits the gradual transmission of pharmaceutical agents throughout the gastrointestinal tract. No major pharmaceutical products use cross linking of gelatin in the United States due to the various difficulties associated with it. One of the difficulties with cross-linking as a formulation tool for softgels is the lack of reproducibility in the application of such technology. Gradual or time release of pharmaceutical agents allow for a more localized delivery along the gastrointestinal tract. The medical usage of gelatin is not limited to the encapsulation of drugs, but also includes applications in tissue engineering, bioadhesives, and plasma substitutes. It is rare that gelatin is not biocompatible. Biocompatibility can be defined as the ability of a material to function within an organism for a specific purpose. This ability will prevent the initiation of a cascade that may manifest a possible immune, toxic and necrotic responses. Under certain conditions, gelatin can be toxic and/or an irritant. The increase in dissolution time (Q value) as a result of aging is one of the biggest problems associated with gelatin-based formulations. This lack of performance is due to the cross-linking of chemically altered gelatin. The cross-linking results in the appearance of a swollen, very thin, though, rubbery, water-insoluble membrane known as a pellicle. The pellicle is an important contributor in the reduction of softgels’ dissolution. Once the pellicle is formed, it is not easily disrupted by gentle agitation and the dissolution values (Q value) have a tendency to drop to the point of rejection. The Q value is a United States Pharmacopeia sanctioned test that ascertains the dissolution and degradation limits of

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4 pharmaceuticals. The dissolution is a Quality Control test, which is used to show reproducibility of the softgels. Dissolution is used to show lot-to-lot reproducibility. Studies can be done to show in-vivo/in-vitro correlation but the results would not be indicative of the normal dissolution test. A softgel may have an acceptance criterion of not less than 80% dissolution after 60 minutes. The ranges for the Q value are from 0100%, where the closer to 100% the test result is, the better the softgel ability to act as a drug delivery system. If the softgel should not meet the requirements of such test, it can be said that the values have dropped to the point of rejection. During the manufacture of softgels, the shell has high water content. This water is removed from the shell by drying the softgels at low humidity conditions. After the manufacture of the softgels for hydrophilic fills, water can migrate from the shell to the fill. During drying, the water is removed from the shell to the atmosphere which reduces the amount of water in the shell and results in water migrating from the fill to the shell where the water continues to be lost to the atmosphere. If there are cross-linking agents in the fill these agents will migrate from the fill into the shell. When the softgel is stored at accelerated conditions during stability or stored at room temperature over a long period of time, the cross-linking agents will cause the softgel shell to become insoluble. During dissolution, the shell will not dissolve and a pellicle will be formed. This will reduce the percentage of drug released and can result in dissolution failure. Moisture can move from the gelatin shell into the fill material during storage when hydrophilic ingredients have hygroscopic properties. If the environment where the capsules are stored is high in humidity, it is possible that the ambient water concentration

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5 increases the water concentration within the softgel. Figure 1 shows a softgel. The entire structure is the capsules and the casing (capsule without the fill material) is the shell. Figure 1. Diagram of a Softgel. Even though dissolution testing can be used as a predictor of the bioavailibility of the drug, failing such test as a result of cross-linking of the gelatin and formation of pellicle does not necessarily mean that the drug will not be absorbed by the body. Because it has been shown that cross-linked gelatin with pellicle formation can be degraded by the gastrointestinal tract enzymes. To the question of what causes the cross-linking of the gelatin, it has been proposed that it is gelatin’s interaction with chemicals, humidity, temperature and light. An increase of these factors above what has been determined as acceptable will create unpredictable products. To prevent cross-linking of gelatin, one could use one or more of the following strategies: 1) usage of Type A gelatin; 2) reducing or preventing aldehydes formation and potential subsequent release; 3) use of inhibitors; 4) humidity control; and 5) photostabilization. The appearance and/or rate of formation of aldehydes are prevented by lysine, phenylamine, glutamine and other compounds. However, manipulation of pH will prevent aldehyde associated cross-linking. When inhibitors are used, it is to prevent changes in the dissolution rate of the softgels. Compounds that are used as inhibitors are semicarbazide hydrochloride, hydroxylamine hydrochloride, piperazide hydrate, pyridine,

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6 and others. An increase in ambient humidity of the softgel in some cases can increase crosslinking. Humidity control is achieved by using waterproof packaging. Photostabilization can be accomplished by adding color to the gelatin shell and selecting packaging to shield the softgel from destructive lighting. The incorporation of titanium oxide, iron oxide and color pigments may offer good protection against cross-linking resulting from light exposure (Singh et al., 2002). It is noteworthy to recognize that some of the strategies previously mentioned by Singh et al are not feasible for the formulation and manufacture of softgels. In conclusion, the incorporation of gelatin in a manufacturing process should take into consideration the selection of the raw material. The introduction of inferior raw material will have a serious effect in the manufacturing operations downhill of such selection. Depending on the need, type A and/or type B gelatin will be the raw material used in the formulation of the softgel. These two types of gelatin not only have different sources but also have different innate characteristics as a result of their origins. It is these innate characteristics that give softgel manufacturing choices in their formulation. The gelatin portion of the softgel can interact with its environment but also with the active ingredient and exipients. The challenge for the manufacturer of softgels is to balance the aforementioned elements to produce a stable product with the ability to deliver a drug and/or vitamin to a desired GI tract location. The manufacture of gelatin is not restricted to the delivery of pharmaceuticals. Gelatin is also involved in less critical time-release applications such as of cosmetics (bath oils) and recreational products (paintballs). Oils packaged in gelatin will be released once placed in water. This release resembles the behavior of gelatin within the human

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7 body whose degradation is also related to an increase in ambient temperature and solution. In the case of paintballs, the gelatin provides a good barrier against the degradation of the paint while yielding to a force that does not injure the player. Research Objectives When dealing with natural materials and their derivatives, one is forced to deal with their intricacies. Gelatin and its constituent collagen are prone to degradation which may be a function of time. From the moment collagen is synthesized, it undergoes crosslinking. The cross-linking which occurs as a result of aging adds a variable in the manufacturing of gelatin and subsequent products. In spite of the variability attributes of raw material and various sources of impurity, the manufacturing process oftentimes yields commodities with lesser variability than its constituents (Eastoe and Leach, 1977). The overall objective of this research were: 1) to explore gel stability during the first 96 hours post-production; and 2) to demonstrate that the novel application of specific analytical methods provide useful information which can be used as a baseline for future research. Ultimately everything is prone to decay. However for the times delineated in this work, it is argued that the visible decay does not affect the finished product where it would be compromised. A secondary objective of this work is to determine if minimal or no changes occur in the finished product. Statistical analysis will be performed on the result of all test results to prove that there is no correlation between time and test results. The manufacturing of the softgels will be made using gel made between the times of 0-96 hours. It is hypothesized that there will be no appreciable difference between softgels produced between time 0 and those produced at 96 hours.

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8 Research Questions To demonstrate that equivalence exists between the types and different aged gelatin, answers to the following questions will be sought: 1) Are any differences seen in the test results statistically significant? Differences are expected in the different aged samples and between the types of gel used. However, it is proposed that these differences are negligible. 2) Are any observed differences satisfactorily discounted by statistical analysis? Again, differences are expected; however, mathematical justification will serve as a tool of the defense that the product made with the aged gelatin are similar. 3) Can the results of this research be generalized? By analyzing at the results of the characterization, a possible correlation between the types of gel and age at manufacture will be explored.

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9 Importance of the Study Many things are best understood when characterized at their basic level. This research will not only merge some of the knowledge of gelatin from the academic and corporate settings but also serve as a baseline for future research. As means to analyze and clarify gelatin arise, it would be a great exercise to use these options to predict and explain the behavior of this material. This research made use of relatively new testing devices such as a rheometer and texture analyzer. These tests not only allowed the characterization of the different types of gelatin but also provided the trending of the results. Limitations of the Study As with most enterprises, constraints were placed upon this study. It was proposed that the investigation would include the use of electron microscopy. The lack of funding and time constraints did not allow for such ventures. The usage of an electron microscope would allow one to visualize changes at a level which could potentially explain or at least strengthen the theories associated with this research. In the event that this test was a possibility, it would have been impossible to test the samples due to the lack of people to prepare and execute microscopic determinations. These visualization and other investigative tools could have become a reality if more funding was available. However, the practicality of such investment is challenged by the most important determinant in the business world, profit. It would not be good business to invest too much of one’s resources if there is no appreciable return on that

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10 investment. The accumulation of knowledge is not a tangible commodity and will always be placed as a secondary goal to many. Corporate knowledge is not always communicated due to the laws associated with intellectual properties. The companies must protect themselves by recuperating their Research and Development investments. As a result of corporate policies, some information was excluded from this document. One noticeable exclusion is the compositional information of gel formulations used in this research.

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11 CHAPTER 2. LITERATURE REVIEW An Overview of Collagen Collagen is the predominant body protein found in of the skin, tendon, cartilage, bone and connective tissue. Its appearance within the organism is typically white, opaque, nonbranching fibrils integrated in mucopolysaccharide and other proteins. Collagen content will differ from organism to organism and consequently between species’ populations. It is noteworthy to point out that the type of tissue and the age of the animal will also be a contributing factor in the collagen content. The amino acid composition of collagen is surprisingly constant in the mammalian tissues. Glycine, the simplest amino acid accounts for approximately twothirds of the collagen composition, proline and hydroxyproline for about one-fifth and alanine for roughly one ninth. Figure 2 gives a visual representation of common amino acids in collagen. The combined occurrence of the aforementioned amino acids are about two-thirds of the collagen composition. The common knowledge of the structure of collagen is mainly a product of X-ray diffraction and electron microscopy investigation (Balian and Bowes, 1977).

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12 Figure 2. Chemical Structures of Main Amino Acids of Collagen. The visualization of gelatin suggests that it is a rod-like molecule with the following dimensions; length of 2800 , diameter of 15 with a molecular weight of approximately 300,000 Daltons. The constituents of the helix are roughly the same length with a molecular weight of approximately 95,000 Daltons each (hence 3 chains make up the helix) (Balian and Bowes, 1977). Figure 3 gives a representation of the collagen helix. Figure 3. Triple Helix Visualizations of Collagen Constituents. a) Two polypeptide chains each helically wound with a left-handed three-fold screw axis. b) Simplified version. c) Third chain added behind. d) Th ird chain added in front. e) Twisting of the minor axis into the collagen super helix. (Balian and Bowes, 1977)

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13 The stability seen in collagen is directly related to the native collagen’s ability to resist the degradation of enzymes and chemicals. The attempt to degrade collagen at low pH values are negligible as long as the temperature is below 20 C. Furthermore, the swelling and dispersing action of alkali is mitigated by the addition of a salt to stabilize the helix, by protecting the peptide bonds. A decrease in the viscous properties of collagen suggests denaturation which is the collapse of the rod like structure. The loss of physical properties is only one of the manifestation of the denaturation of collagen. The temperature where collagen losses its properties or is denatured varies between 37 C and 40 C. There exists an inverse relationship between the viscous and elastic properties of collagen. Viscosity decreases as collagen approximated denaturation temperature (~40 C) and elasticity increases as collagen approaches the denaturation temperature. Elasticity is defined as the ability of a material to regain its shape after undergoing deformation due to a strain (Balian and Bowes, 1977). An Overview of Gelatin The gelatin manufacturing goal is to facilitate the conversion of collagen to gelatin and the removal of “impurities” (Johns, 1977). These impurities may be organic or inorganic in nature. Examples of organic impurities are proteins from blood, keratins, glycoproteins, mucopolysaccharides such as hyaluronic acid, keratosulphate and chondroitin sulphate, lipids, nucleic acids and other cell components which more than likely contribute to the soluble degradation of the gelatin. Inorganic contribution to degradation is made possible by the following ions; sodium, potassium, calcium,

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14 magnesium, iron, chloride and phosphate. Even though the mentioned impurities are associated with the organism before the extraction process, one should note that contamination may be an addition of the extraction process. Furthermore, one should pay heed to known changes in composition during the collagen-gelatin conversion (Eastoe and Leach, 1977). The majority of collagen is located in the corium layer. The corium layer is the preferred source of pure skin collagen therefore mechanical extraction concentrates on that area. The corium is freed of soluble components by mincing and prolonged interaction with various solutions followed by organic compound extraction to isolate fat and dehydrate the residue. The result of gelatin extraction is highly reproducible and the variance would be a function of the crosslinking associated with the age of the animal. When dealing with the gelatin process, the term tropocollagen describes acid soluble collagen (Johns, 1977). Factors such as species, age of the animal and extraction techniques affect the quantities of soluble collagen during collagen manufacturing (Johns, 1977). Collagen is insoluble as the result of calcification which explains why mature bones contain little or no extractable collagen. The triple helix conformation is the result of hydrogen bonding. When collagen is insoluble as in the case of bones, it is often associated with the presence of covalent bonds (Johns, 1977). The aforementioned complications in gelatin manufacturing may create variability in the end result (in our case, gelatin used to manufacture softgels). However, the intrinsic properties of gelatin make it a good candidate for the encapsulation of pharmaceutical, cosmetic and recreational products. The preferential usage of gelatin in certain applications is due to its “drug delivery vehicle” properties.

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15 Gelatin Manufacturing Two main manufacturing processes are used in the manufacturing of gelatin: alkaline and acid processes. Figure 4 outlines the gelatin manufacture process. The resulting products can be very different in terms of composition and physical properties. Generally, the alkali-processed gelatins possess higher hydroxyproline and lower tyrosine concentrations than acid-processed gelatins or the raw materials. The reasons for such discrepancies are: a) gelatins do not have the same purity ; and b) acid-processed materials are prone to losing peptides richer in hydroxyproline and maintaining peptides richer in tyrosine (and vise versa for the alkaline treated raw materials). In addition to these two processes, one could also extract gelatin by using the dual soak process (using alkaline and acid processes) and the autoclaving process. Acid processed collagen is soaked in dilute acid and then extracted at about pH 4 for gelatin manufacturing. Non-collagenous proteins and mucoproteins of the tissue are isoelectric at this pH and are therefore less soluble and more readily coagulated under the extraction conditions. Contaminants which are removed in this way depend on the quality and origin of the raw material and the reproducibility of the manufacturers process (Eastoe and Leach, 1977).

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16 Figure 4. Alkali and Acid Treatment Processes for Gelatin. Gelatin Extraction Alkali Treatment (150 Bloom) Acid Treatment (195 Bloom) Filtration and Clarification of Gelatin Liquors Sterilization Drying Evaporation of Gelatin Liquors Grinding and Sifting

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17 When dealing with the alkaline process, the pretreatment of the collagen requires a prolonged soaking in the alkaline solutions (generally, saturated lime-water). A good amount of the impurities (proteins and mucosubstances) are soluble at the subjected pH and are extracted. Gelatins from alkali process tend to be purer than acid produced gelatin, but this variation may be due to the manufacturing process (Eastoe and Leach, 1977). Additionally to the difficulties mentioned previously, the manufacture of gelatin is subject to more complications. For example the total number of carboxyl groups available for ionization depends upon the extraction method. Different gelatins can have different ratios of acidic and basic group therefore different isoelectric points. Charged groups influence the interactions between adjacent gelatin molecules, between each molecule and the solvent and between different parts of the same molecule, as the protein chains are flexible. The extent of these variables vary with pH, and are also dependent on the total ionic composition of the system therefore a detailed description of the solvent as well as that of the gelatin is a necessity (Stainsby, 1977). Comparison Obtaining Gelatin from Collagen Literature review offers no conclusive evidence of the correlation between viscosity and molecular weight for gelatins of different origins even if they have the same isoelectric point. Acid processed gels have lower viscosity than lime processed gels. Highly branched molecules in gelatin contribute to the difficulties in determining the correlation between physical properties to molecular arrangement. The branching also

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18 makes it hard to explain solubility (Stainsby, 1977). However, the relationship between viscosity and molecular weight exists over a wide range (Stainsby, 1977). Variables exist in the industrial gelatin processes which impact the uniformity of the end product. A source for the variations is the impossibility to extract gelatin from collagen without incorporating “impurities”. The impurities may contribute to the accelerated decay of the gelatin. However, one should note that many advances and improvements have been made in gelatin manufacture, making impurities less of an issue than during the early days of gelatin manufacturing. Amidst the contributions to the variances, one should note that the way to manufacture gelatin is to denature soluble collagen. The process destroys the triple helix to produce one, two or three random chain gelatin molecules. The molecular change is catalyzed by mild conditions either by heating at neutral pH to about 40 C or by adding hydrolysis promoters at room temperature or lower. The process has the objective of breaking the hydrogen bonds and hydrophobic bonds that stabilize the collagen helix. These process conditions prevent destruction of covalent bonds before their time, within the collagen chain (Johns and Courts, 1977). The formation of gelatin is related to the denaturation of soluble collagen. The transition can not simply be explained in terms of the uncoiling of the helixes, and consideration must be given to the molecular association. When the soluble collagen is exposed to denaturation conditions such as elevated temperatures or in an hydrogen bond breaker environment, the molecular substituents are dissociated. This dissociation is represented by random chain molecules. Upon the reversal of the denaturation conditions, the molecular chains aggregate to achieve collagen’s native state of a triple-helix (Johns

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19 and Courts, 1977). Figure 5 provides a description of the associated and dissociated aspects of collagen. Figure 5. Products of the Dissociation of the Triple Helix (Balian and Bowes, 1977). Gelatin may be considered as a blend of fine and coarse networks whose proportions depend largely on the thermal history of the gel. To achieve the formation of gel there must be two bonding sites per gelatin molecule and this necessity can be assessed by molecular weight. The approximation of molecules by their sites of attachment increase molecular weight. It has been suggested that roughly 1/6 of the chain is needed for such bonding. If values greater than the mentioned numbers are achieved, the quality of the gelatin will increase and plateau around the molecular weight of around 90 K. However, one should note that the correlation between molecular weight and gelatin quality is the determinant for the quality of the product. The inherent characteristics of gelatin and its constituent collagen make them choice materials in pharmaceutical preparations. In our application of gelatin, this biomaterial is used to contain pharmaceutical ingredients and serve as a drug delivery vehicle. However, gelatin can also be used as a binding agent in tablets where its properties are also exploited.

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20 CHAPTER 3. RESEARCH DESIGN AND METHODS Four analytical methods were used in this investigation to measure mechanical properties. Two of the methods measured compression forces, the third method measured tension force, and the fourth recorded viscoelastic properties. Two instruments were principally used in the testing of the gelatin during this exercise. The first instrument used to test mechanical attributes was a texture analyzer whose function was to apply compressive and tension forces on the samples of interest. The second instrument was a rheometer which principal use was the determination of the viscoelastic properties of the samples. Gel Extensibility Gel Extensibility is a destructive test that punches a hole into a gelatin ribbon. Sample of the material of interest is cut in a rectangular shape with an area of approximately 2.5 X 5 inches with a typical thickness approximating 0.010 inches. The gelatin ribbon was extracted from an encapsulation machine similar to that illustrated in Figure 6. The ribbon was consistently gathered from the “pre-wedge” area, meaning that the ribbon was collected before it was to be shaped and made to contain fill material.

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21 Figure 6. Encapsulation Machine. The texture analyzer was manufactured by Stable Micro Systems in Surrey, England. The model used was the TA.XT2 (HR) and was attached to a computer terminal to allow real time data collection (see Figure 7). A robotic arm is activated and a plunger proceeds towards the sample. When testing the gelatin ribbon, an attachment resembling a softgel was used for the extensibility test, with a length and width of 2 cm and 1 cm, respectively. During the extensibility test, the softgel attachment will pierce the ribbon and the compressive force necessary to achieve the destruction was recorded. The results of the Gel Extensibility are culminated in a printout as seen in Figure 6. The printout is a combination of the analysis of ten (10) trials for the same sample, which quantifies the amount of force (in grams) needed to punch a hole through the ribbon. The other types of data that can be extracted from the printout are the time needed to punch the hole and the distance traveled before the puncture (in millimeter). The variability seen in the results can be due to the raw material variability, the intricacies associated with preparing a

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22 highly viscous liquid, and the variability of the different samples subjected to destructive testing. Figure 7. TA.XTR Texture Analyzer. When dealing with gelatin of a particular molecular weight, the ascribed value is an average and as a result of variability in the main component of gelatin, variability will also be seen in the final product. Even though great care was taken to avoid degradation of the gelatin ribbon between testing, it is possible that the ribbon lost water and in turn plasticity since water is a natural plasticizer. Another precautionary step taken to prevent unnecessary variability in the testing methods was to calibrate the texture analyzer using a ten pound weight as per manufacturer’s recommended calibration procedure. The weight was placed where the triangle points on the mechanical arm of the Texture Analyzer in Figure 7. Once the weight was in place, the calibration mode of the software was selected. The calibration was done for everyday of testing and was applicable for the other tests using the texture analyzer (i.e. resilience and wet burst strength).

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23 Resilience The same sample setup for Gel Extensibility was used for the resilience test but the softgel attachment was not allowed to destroy the sample and the test used tension forces. The goal of the resilience test was to ascertain the absorbed energy required to displace the ribbon at a predetermined distance of 5 mm. Both tests pressed through a ribbon, which is harnessed to a stage to insure stability and uniformity. Figure 8. An Example of a Gel Extensibility Test Printout

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24 Resilience testing also makes use of ten (10) trials per sample. The similarity of these two tests does not stop with the resemblance of the printouts, but also include the fact that they use the exact setup but different applications. The Gel Extensibility test is made using compression forces whereas the Resilience test is made using tension forces. Figure 8 shows the printout for the Gel Extensibility test and Figure 9 shows the printout for the Resilience test. The resilience printout provides more information than what was needed for this study. We were interested in the resilience aspect of the printout as the title of our test suggested. Similar to the printout of the gel extensibility test, the force is calculated in grams. Also, the force used can be correlated to time and distance traveled in mm. The calibration was done for everyday of testing following the method mentioned in the Gel Extensibility section (page 22).

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25 Figure 9. An Example of a Resilience Test Printout.

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26 Wet Burst Strength Unlike the previously mentioned tests, the Wet Burst Strength test measures a parameter of the final product. The wet burst strength test involved taking a newly formed softgel and applying a compressive test until rupture of that softgel. Unlike the previously cited texture analyzer test procedures, the attachment was a disk with a 25 mm diameter which allowed for a uniform distribution of force. The softgel is placed with its seam parallel to the floor, to assure uniformity in testing. Figure 10 shows a printout of such test. The wet burst strength printout gives the distance traveled before the softgel ruptured and the amount of force needed to achieve such failure. As with the gel extensibility and resilience tests, ten trials for the same sample location (beginning, middle and end of the encapsulation runs) were used to provide statistically significant results. The calibration was done for everyday of testing following the method mentioned in the Gel Extensibility section (page 22).

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27 Figure 10. An Example of a Wet Burst Strength Test Printout.

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28 Rheometer The Haake Rheostress rheometer (model RS150) was a German made instrument whose function was to melt the gel mass samples and during that process determine the viscous and elastic regions of the melting gelatin (see figure 11). The sample was situated between sensors and a heat transfer system will bring the temperature to 60 C and then drop the temperature to 40 C. The temperature range was established in consideration of the manufacturing parameters and gelatin tolerance. As with the texture analyzer, the probe was attached to a computer to allow the capture of real time data. Unlike the previous tests, we used one sample from the different points of interest and looked at the viscoelastic behavior as a function of temperature. The sample was tested once. Figure 12 shows the printout of the rheometer test where four of the columns were of interest which were: time in seconds; G’ which represents the elastic aspect of the material; G’’ which represent the viscous aspect of the material; and the complex viscosity of the material which is measured in Pascal were its G’ and G’’ constituents. An outside firm calibrates the instrument annually. However, before the beginning of the experiment the instrument was verified using the Brookfield Viscosity standard 5040 cps (lot# 051401). The verification sequence involves placing the standard on the instrument’s plates and taking the reading. In the event the reading does not correlate to the standard (5040 cps), the instrument would have been recalibrated.

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29 Figure 11. The Haake Rheostress Research Grade Rheometer. All tests in this research made use of the plate to plate combination.

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30 Figure 12. An Example of Rheometer Testing Printout.

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31 HPLC (Size Exclusion Chromatography) The HPLC method was also employed to provide additional information. This method is based on size exclusion chromatography where a column was filled with a material having fixed filtration capacity. The sample is allowed to go through the column and is ultimately segregated by molecular size. Large molecules go through the column relatively fast and the smaller molecules adhere to the material in the column and are dissociated later. Such technique is also known as gel filtration or gel permeation chromatography. Three samples were taken from the points of interest (beginning, middle and end of encapsulation run) and subjected to the HPLC method. This testing portion of the study was performed by the gel supplier who also calibrated the instrument. The HPLC method used in this experiment was qualitative. The goal of this method was to obtain a fingerprint of the gel. The molecular weight data are relative and should only be compared if samples are run in sequence. It is not possible to use such method quantitatively due to the heterogeneity of the gelatin, variables within the method itself such as column-to-column variability, and column variability due to age. Due to the qualitative nature of the HPLC test, accuracy values can not be given. Experimental Design The goal of this research was to test two types of gelatin (150 Bloom Lime Processed and 195 Bloom Acid Processed) using the five methods previously mentioned. Table 1 summarizes the experimental design.

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32 Table 2. Summary of the Experimental Design. Gel Types Test Methods 150 Bloom 195 Bloom Gel Extensibility Samples were tested at four time intervals. Ten times per sample points. Samples were tested at four time intervals. Ten times per sample points. Resilience Samples were tested at four time intervals. Ten times per sample points. Samples were tested at four time intervals. Ten times per sample points.. Wet Burst Strength Samples were tested at four time intervals. Ten times per sample points. Samples were tested at four time intervals. Ten times per sample points. Rheology Samples were tested at four time intervals. One time per sample points. Samples were tested at four time intervals. One time per sample points. HPLC Samples were tested at four time intervals. Three times per sample points. Samples were tested at four time intervals. Three times per sample points. Time intervals: 0,4,8; 32,36,40; 64,68,72; 88, 92, 96 hours.

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33 CHAPTER 4. RESULTS AND DISCUSSION Results The results of the different tests are summarized in Table 3, where the standard deviation and sample size are also given. The reason for the variability in sample size was due to the omission of data. This omission was due to occasional technical difficulties with the testing apparatus. On many occasions, the piston refused to progress and simply came back to its initial position when it sensed the sample. The raw data relating to the test methods is presented in Appendices 1 through 5. Appendix 1 contains Microsoft Excel graphical representation of test results. Appendix 2 contains printouts of the Wet Burst Strength test results. Appendix 3 has the printouts of the Rheology test results. Appendix 4 is the location of the Gel Extensibility test results. Appendix 5 contains the Resilience test results and Appendix 6 contains the Molecular Weight analysis When looking at the data for all test parameters, significant differences are observed between the 150 gelatin and 195 gelatin mean values for a given time interval. When regressed as a function of time, significant differences between gel types are also observed. The 195 Acid Processed gelatin ribbon had stronger mechanical properties that the 150 Lime Processed gelatin ribbon based on the Gel Extensibility test. The

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34 deformation seen in the Resilience testing of the 150 Lime Processed gelatin type hovered around the value of 54%, meaning that the gelatin was deformed on an average value of 54% from time 0 to time 96 hrs. The deformation of the 195 Acid Processed gelatin was statistically greater than its counterpart, displaying an average value of about 75%. The Burst Strength results resembled the Gel Extensibility trends, where the 150 Lime Processed gelatin had smaller values than the 195 Acid Processed gelatin. The viscoelastic aspect of the rheological testing shows that the 150 Lime Processed gelatin has lower values than the 195 Acid Processed gelatin. However, the 150 gelatin type had a greater molecular weight distribution than the 195 gelatin. When looking at the test results as a function of time, the following were observed, 1) There was a decline in the amount of force needed to break the 150 and 195 gelatin ribbons in the Gel Extensibility test; 2) The Resilience for the two gelatin types were constant as a function of time; 3) The trend seen in the Gel Extensibility were also seen in the Burst Strength results, in that it took less force to rupture the softgels as a function of time; 4) The viscoelastic test shows decrease in values as a function of time; 5) The molecular weight distribution showed a steady decrease for the two types of gelatin. Table 4 shows a general decrease as a function of time for Gel Extensibility, however some increases can be seen. The first two values (times 0 and 4) for the 150 Gelatin type were omitted due to instrument malfunction. Table 5 shows uniformity of the test results for Resilience. The first two results were omitted for the two gelatin types and the third result was omitted for the 195 gelatin types. Again, instrument malfunction was the cause of such absence.

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35 Table 3. Summary of Experimental Results for the Various Methods. Type of Test And Corresponding Times of Execution 150 Gelatin 195 Gelatin Are the means significantlydifferent ? Gel Extensibility 0-8 hours (mm) 32-40 hours 66-72 hours 88-96 hours 2266 18 (n=10) 1829 273 (n=30) 1277 103 (n=30) 1202 161 (n=30) 3310 131 (n=30) 2741 485 (n=30) 2759 123 (n=30) 2582 177 (n=30) Yes Yes Yes Yes Resilience 0-8 hours (mm) 32-40 hours 66-72 hours 88-96 hours 54 3 (n=8) 54 2 (n=29) 55 1 (n=23) 54 1 (n=29) N/A 75 2 (n=22) 75 0.18 (n=24) 76 1 (n=20) Yes Yes Yes Yes Burst Strength 0-8 hours (mm) 32-40 hours 66-72 hours 88-96 hours 12,312 2715 (n=30) 10,330 1687 (n=30) 10,245 432 (n=30) 7195 1323 (n=30) 13,672 8 (n=12) 11,450 7 (n=30) 12,049 562 (n=20) 14,022 2239 (n=30) Yes Yes Yes Yes Rheological Test 0-8 hours (Pa) 32-40 hours 66-72 hours 88-96 hours 41 32 (n=14) 108 80 (n=28) 17 12 (n=22) 53 9 (n=30) 186 148 (n=20) 362 417 (n=19) 256 135 (n=30) 241 101 (n=29) Yes Yes Yes Yes Molecular Weight 0-8 hours (Da) 32-40 hours 66-72 hours 88-96 hours 128,602 2397 (n=9) 119,822 2260 (n=9) 112,595 3394 (n=9) 106,535 3564 (n=9) 98,566 2499 (n=9) 90,943 702 (n=9) 82,046 797 (n=9) 81,262 865 (n=9) No No No No Values shown are mean standard deviation followed by number of observations in parenthesis. t-test using p 0.05

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36 Table 4. Summary of Experimental Results for the Gel Extensibility Method. Gel Extensibility And Corresponding Times of Execution 150 Gelatin (mm) 195 Gelatin (mm) 0-8 hrs Beginning of lot (Beg) Middle of lot (Mid) End of lot (End) N/A N/A 2265 959 (n=10) 3440 1697 (n=10) 3311 615 (n=10) 3179 726 (n=10) 32-40 hrs Beg Mid End 2143 520 (n=10) 1691 294 (n=10) 1654 517 (n=10) 2457 628 (n=10) 2463 719 (n=10) 3301 583 (n=10) 66-72 hrs Beg Mid End 1180 565 (n=10) 1267 359 (n=10) 1384 585 (n=10) 2826 528 (n=10) 2617 380 (n=10) 2834 516 (n=10) 88-96 hrs Beg Mid End 1069 301 (n=10) 1156 216 (n=10) 1381 544 (n=10) 2377 674 (n=10) 2683 354 (n=10) 2685 320 (n=10) Values shown are mean standard deviation, followed by number of observations in parenthesis. Table 6 shows a general decrease in the Wet Burst Strength results for both gelatin types. Unlike the previous tables, the technical difficulties were experienced during the testing as opposed to the beginning. Time 4, 8, 36, 40 and 72 were omitted as the results of the aforementioned difficulties. A divergence can be seen at the end of the test time (96th hour), where the 195 Gelatin value increases and the 150 Gelatin value decreases.

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37 Table 5. Summary of Experimental Results for the Resilience Method. Resilience And Corresponding Times of Execution 150 Gelatin (%) 195 Gelatin (%) 0-8 hrs Beginning of lot (Beg) Middle of lot (Mid) End of lot (End) N/A N/A 54 3 (n=8) N/A N/A N/A 32-40 hrs Beg Mid End 56 2 (n=10) 52 4 (n=10) 54 2 (n=9) 73 5 (n=8) 77 15 (n=8) 77 3 (n=6) 66-72 hrs Beg Mid End 53 3 (n=4) 56 5 (n=9) 54 4 (n=10) 75 3 (n=9) 75 4 (n=8) 75 2 (n=7) 88-96 hrs Beg Mid End 55 4 (n=10) 52 4 (n=9) 53 3 (n=10) 75 3 (n=7) 77 1 (n=6) 76 3 (n=7) Values shown are mean standard deviation followed by number of observations in parenthesis. Test results are a percentage of the sustained deformation.

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38 Table 6. Summary of Experimental Results for the Wet Burst Strength Method. Wet Burst Strength And Corresponding Times of Execution 150 Gelatin (mm) 195 Gelatin (mm) 0-8 hrs Beginning of lot (Beg) Middle of lot (Mid) End of lot (End) 11,368 2760 (n=10) 15,374 3554 (n=10) 10,195 4269 (n=10) 13,672 9551 (n=12) N/A N/A 32-40 hrs Beg Mid End 8460 2992 (n=10) 11,739 3600 (n=10) 10,790 1892 (n=10) 11,450 7763 (n=30) N/A N/A 66-72 hrs Beg Mid End 10,684 3208 (n=10) 9820 4010 (n=10) 10,230 3939 (n=10) 11,651 6887 (n=10) 12,446 9543 (n=10) N/A 88-96 hrs Beg Mid End 8677 2458 (n=10) 12999 9726 (n=10) 6780 2730 (n=10) 16,552 6990 (n=10) 13,220 5159 (n=10) 12,294 3439 (n=10) Values shown are mean standard deviation followed by number of observations in parenthesis. Table 7 does not show a particular trend in the viscoelastic properties of the two gelatin types. The first result for the 150 Gelatin type was omitted due to technical difficulties. Table 8 shows a strong relationship between the molecular weight distribution and time.

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39 Table 7. Summary of Experimental Results for the Rheology Method. Rheological Testing And Corresponding Times of Execution 150 Gelatin (Pa) 195 Gelatin (Pa) 0-8 hrs Beginning of lot (Beg) Middle of lot (Mid) End of lot (End) N/A 76 6 (n=6) 14 2 (n=8) 345 46 (n=9) 63 8 (n=6) 48 3 (n=5) 32-40 hrs Beg Mid End 209 19 (n=10) 77 9 (n=11) 14 2 (n=7) 65 10 (n=7) 1041 73 (n=5) 138 27 (n=7) 66-72 hrs Beg Mid End 7 1 (n=5) 9 1 (n=9) 33 5 (n=8) 57 12 (n=9) 317 59 (n=11) 369 41 (n=10) 88-96 hrs Beg Mid End 62 13 (n=11) 42 7 (n=11) 57 8 (n=8) 414 15 (n=6) 245 40 (n=12) 142 34 (n=11) Values shown are mean standard deviation followed by number of observations in parenthesis. Test results are in Pascals.

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40 Table 8. Summary of Experimental Results for the Molecular Weight Analysis Method. Molecular Weight Analysis And Corresponding Times of Execution 150 Gelatin (Da) 195 Gelatin (Da) 0-8 hrs Beginning of lot (Beg) Middle of lot (Mid) End of lot (End) 130,886 2919 (n=3) 128,814 1123 (n=3) 126,107 2144 (n=3) 100,682 797 (n=3) 99,207 562 (n=3) 95,809 412 (n=3) 32-40 hrs Beg Mid End 121,999 3328 (n=3) 117,487 3178 (n=3) 119,980 998 (n=3) 90,682 1584 (n=3) 91,738 768 (n=3) 90,408 960 (n=3) 66-72 hrs Beg Mid End 108,762 371 (n=3) 113,806 1609 (n=3) 115,217 883 (n=3) 82,966 777 (n=3) 81,576 398 (n=3) 81,595 533 (n=3) 88-96 hrs Beg Mid End 104,670 766 (n=3) 104,291 1948 (n=3) 110,645 810 (n=3) 81,909 1159 (n=3) 80,279 945 (n=3) 81,598 277 (n=3) Values shown are mean standard deviation followed by number of observations in parenthesis. Test results are in Daltons. Figure 13-15 provide graphical representation of the tabulated data of Table 3 where strong associations could be recognized visually. Figure 13 represents the Gel Extensibility data of Table 4. A gradual decrease in the amount of force necessary to destroy the sample can be observed. Figure 14 represents the Wet Burst Strength data of Table 6. Figure 15 represents the Wet Burst Strength data of Table 7. As previously mentioned a divergence can be seen at the end point of the data set hence also in the graph.

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41 Gel Extensibility Summary GraphForce (195) = -219.6(hour) + 3399.5 R2 = 0.7706 Force (150) = -374.4(hour) + 2579.5 R2 = 0.93310 500 1000 1500 2000 2500 3000 3500 4367092Time (hours)Force (grams) 150 Gel Extensibility 195 Gel Extensibility Linear (195 Gel Extensibility) Linear (150 Gel Extensibility) Figure 13. Summary Graph of Averaged Gelatin Extensibility. Error bars represent standard deviation.

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42 Wet Burst Strength Summary GraphForce (195) = 164.9(hour) + 12386 R2 = 0.0293 Force (150) = -856.6(hour) + 12735 R2 = 0.8390 2000 4000 6000 8000 10000 12000 14000 16000 4367092Time (hours)Force (grams) 150 Burst Strength 195 Burst Strength Linear (195 Burst Strength) Linear (150 Burst Strength) Note: For clarity, only half of the standard deviation appears on this graph. Figure 14. Summary Graph of Averaged Gelatin Wet Burst Strength. Error bars represent standard deviation.

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43 Molecular Weight Distribution Summary GraphMWD (150) = -7342.8(hour) + 135246 R2 = 0.9932 MWD (195) = -6080.9(hour) + 103407 R2 = 0.91990 20000 40000 60000 80000 100000 120000 140000 4367092Time (hours)Molecular Weight Distribution (Daltons) 150 Gel M.W. 195 Gel M.W. Linear (150 Gel M.W.) Linear (195 Gel M.W.) Figure 15. Summary Graph of Averaged Gelatin Molecular Weight. Error bars represent standard deviation.

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44 Figure 12 visualizes the Molecular Weight data of Table 8. The association seen in this graph is quasi-linear and is the behavior being sought between the different tests in the interest of interchangeability. Tables 9 and 10 are the matrices relating to the 1st order data analysis. As can be seen, no strong correlation exists in Table 9. Discussion Certain trends have been identified within the Results section. For example, the Gel Extensibility values decrease as the gel ages. It seems that such decline suggests a correlation to time. When looking at the 150 gelatin type, a gradual decrease can be seen for the Gel Extensibility, Burst Strength and Molecular Weight Analysis tests. Since mechanical properties are related to atomic/molecular aggregations, molecular weight degradation suggests mechanical strength decline. The behavior of the 195 Acid Processed gelatin shows a decline in Gel Extensibility and Molecular Weight Analysis test results. However, the Burst Strength test does not show a correlation with the Gel Extensibility and Molecular Weight Analysis as with the 150 Lime Processed gelatin. The difference between the 195 Acid Processed and 150 Lime Processed behavior suggest that the extraction processes impart different characteristics to the raw material and ultimately the end product. This is summarized in Table 9.

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45 Table 9. Summary of Statistical Analysis of Table 3 Data. Test Type Is 150 Gel Type Different from the 195 Gel Type for a given time interval? Are the results a function of time (0-96 hrs)? Are the overall means different for the 150 and 195 Gel Types? Gel Extensibility Yes 150: Yes 195: Yes Yes Resilience Yes 150: No 195: No Yes Burst Strength Yes 150: Yes 195: No Yes Viscoelasticity Yes 150: No 195: No Yes Molecular Weight Yes 150: Yes 195: Yes Yes The differences between the two different gel types can also be seen not only in the different test results, lack of correlation between some of the tests, but also in behaviors as a function of time. When looking at the 195 gelatin type, the burst strength results decreased to ultimately achieve a greater value then the one seen at the start of the test. Such behavior suggest that either: 1) the gelatin becomes stronger as a function of time due to molecular rearrangement or; 2) the softgel becomes brittle as a function of time. The latter of the two possibilities can be disregarded due the resilience test results whose value remains constant as a function of time. When dealing with the resilience of both the 195 Acid Processed and 150 Lime Processed gelatin, it is relatively constant throughout this research. Most of the literature suggest that molecular weight analysis and rheological determination are related. However, the characterization of this research

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46 does not show a correlation between these two tests at first glance therefore, the data collected from the various tests were subjected to statistical analysis using the SAS statistical software. The correlation used in the comparison of the tests is the Pearson Correlation Coefficients. It is understood that any values that come close to one (1) has a correlation to whatever characteristic it is being compared to.

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47 Table 10. Pearson Correlation Coefficient for 150 Bloom Gel. Pearson Correlation Coefficient Time in minutes Wet Burst Strength in mm Gel Extensibility in minutes % Resilience Complex Viscosity HPLC results Time in minutes 1 -0.915 0.085 -0.985 0.0015 0.336 0.663 -0.20 0.8 0.998 0.001 Wet Burst Strength in mm 1 0.835 0.164 -0.071 0.93 0.076 0.92 0.93 0.07 Gel Extensibility in minutes 1 -0.488 0.512 0.275 0.724 0.978 0.4667 % Resilience 1 -0.653 0.34 0.3 0.699 Complex Viscosity 1 0.158 0.84 HPLC results 1

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48 Table 11. Pearson Correlation Coefficient for 195 Bloom Gel. Pearson Correla-tion Coeffi-cient Time in minutes Wet Burst Strength in mm Gel Extensibility in minutes % Resilience Complex Viscosity HPLC results Time in minutes 1 0.1 0.9 -0.877 0.113 -0.797 0.412 -0.13 0.88 0.98 0.02 Wet Burst Strength in mm 1 0.24 0.76 -0.97 0.14 -0.81 0.19 -0.045 0.95 Gel Extensibility in minutes 1 -0.995 0.06 0.565 0.434 -0.88 0.11 % Resilience 1 -0.595 0.594 0.56 0.62 Complex Viscosity 1 -0.17 0.82 HPLC results 1

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49 Table 10 and 11 provide a correlation analysis of the data provided by the various test methods used in this research. The tests in the Table 10 and 11 for the 195 Bloom gel relate to the following tests; time in minutes, Wet Bust Strength in mm, the distance traveled in mm for the Gel Extensibility test. The following were also part of the table, the % Resilience aspect of the Resilience test, the complex viscosity factor of the rheometer test and the results of a HPLC molecular weight distribution analysis. The 150 Bloom gel does not have the high frequency of correlation when compared to the 195 Bloom gel. When l ooking at the 195 Gel, a correlation (0.99) can be seen between % Resilience and HPLC results, a negative correlation (-0.96) can be seen between complex viscosity and HPLC results, a negative correlation can be seen between % Resilience and complex viscosity (-0.97). These strong correlations suggest that either of these test can be a predictor for the other correlated test. However, the tests that do not show strong correlations provide unique independent data and their execution can not be substituted. It can be inferred that the tests represented by % Resilience, complex viscosity, and HPLC results can be used interchangeably to predict an outcome measured by one of them. Something not seen in the first order analysis is a correlation between the individual tests and time. It has been established that molecular weight distribution and rheological properties have a correlation. The correlation seen in the matrix reconciles general knowledge to the data being analyze. As a whole, weak correlations are seen and such behavior may be corrected by transformation of x and y variables. Such transformation is further warranted by the residual plots that show uneven distributions around the residual baseline. Also, the

PAGE 63

50 nonlinear appearance of the regression line suggests that a transformation of the data may provide a better model of the treatments over time. The data of the test results were subjected to the following transformations so that a direct relationship with time, could be found for most of the tests results; 1) taking the square root of the data sets and plotting them, 2) natural logarithm transformation, 3) logarithm transformation to the base 2 and 4) common logarithm transformation. However, when the data was subjected to the four transformations previously mentioned, an inverse correlation was been between molecular weight distribution and time. That correlation coefficient was -0.881 with an error margin of 0.0017. It was deduced that molecular weight distribution decreases with time. The transformation of Table 10 did not provide stronger correlations. However, when the data was subjected to the four transformations previously mentioned, an inverse correlation was been between molecular weight distribution and time. That correlation coefficient was -0.881 with a error margin of 0.0017. It was deduced that molecular weight distribution decreases with time. The transformation of Table 10 did not provide stronger correlations.

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51 CHAPTER 5. CONCLUSIONS AND RECOMMENDATIONS Conclusions When looking at the MS Excel representation of the test data, it is clear that the profiles of the 150 and 195 Bloom gels differ substantially. This divergence may be related to the obvious (molecular weight distribution) and may be compounded by process related additions. Because of the intrinsic behavior of the two types of gelatin, it is expected that their end products also differ. The change of behavior was a function of time and that determination was made possible by multiple regression analysis. It can be said that differences were seen as a function of aging the gel. Eventhough there were differences in the gel due to aging, the intrinsic properties of the gelatin allowed for the production of good products (softgels). Thus the variability betweeen the two gelatins used and their properties over time was negligible. Mathematical justification was the validation tool for the study. It can be said with a great amount of certainty that the products made with the aged gelatin are similar to the ones not being aged. There were no appreciable changes in the mechanical properties of the gelatin. The mechanical properties were mainly ascertained by using the Wet Burst and Gel Extensibility tests. However, changes were observed on the molecular level and it is these changes that are the contributors in the decay of gelatin. The determinants of

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52 decay are the rheological, resilience and molecular weight tests. It was observed that the 195 Bloom Gelatin data has strong correlation between test methods and that the 150 Bloom Gelatin has an inverse correlation between time and molecular weight. The dynamic between the two gelatins should be the same and the observed divergence may be due to human error or a skewness due to sample size. Therefore if the gels should show the same dynamics, the correlations seen in the two types should apply to each other. As a result of these assumptions, it can be said that rheological, resilience, and moleculaer weight analysis have a correlation with time. When production unit operation uses gelatin between time 0-96 hours of its manufacture, one can extrapolate the results of this study. Softgels undergo certain dynamics which should be mentioned before addressing the findings of this study. Once the manufacturing process is completed, the softgels undergo a drying period where they stabilize and are readily manipulated. In this period of stabilization, the softgel hardens and its content loose moisture to the environment. By manipulating the parameters that were the subject of this document, one could possibly affect the final product and possibly increase its yield. When looking at the dynamics of the results provided by the Extensibility, Resilience, Wet Burst Strength, Rheological and Molecular Weight analysis testing, one can generally observe that the properties of gelatin are subject to change as a function of time. The literature review presented many theories for such behavior and these prove themselves to point to the unavoidable degradation which affects whatever has an organic base.

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53 In this study, we were able to quantify the changes experienced by the gelatin as a function of time and provide visual representations of such changes. Our assumption that degredation is the primary force in this work has been explained as the chemical reaction known as hydrolysis. The presence of hydrolysis can be ascertained by the molecular weight analysis of the gelatin which shows that the number decreases as a result of time. Recommendations The incorporation of more tests during the manufacturing process could have provided more information about the molecular dynamics of gelatin. In future research, it would be advisable to perform molecular weight analysis and rheology on samples as they are gathered from the encapsulation machine. In this study it was necessary to keep the gel mass samples days before the pertinent tests were performed. This waiting period was due to the lack of resources, mainly manpower. By performing the tests as the samples come out of the machine, the results are more representative of the sample at the time of collection. The gel mass samples were stored in glass jars that provided a good barrier to degradation due to air. The sampling technique introduced air and the ramifications however minute, could be enough to affect the results. When preparing the samples for the various tests, molds would prove themselves beneficial in reducing human error. Great care was taken to minimize human error but variations were unavoidable oftentimes. As the sample size increases, the parameters of interest resemble that of the population. It can be said that great care was taken to provide data with minimal interference however, few ventures follow the charted course.

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54 REFERENCES Balian, G. and Bowes, J. H. 1977. The Structure and Properties of Collagen. In: The Science and Technology of Gelatin, A.G. Wards and A. Courts (eds.), London: Academic Press. Eastoe, J.E. 1977. Chemical Constitution of Gelatin. In: The Science and Technology of Gelatin, A.G. Wards and A. Courts (eds.), London: Academic Press. Gilsenan, Paula M. and Ross-Murphy, Simon B. 2001. Shear creep of gelatin gels from mammalian and piscine collagens. International Journal of Biological Macromolecules. 53,61. Johns, P. 1977. The Structure and Composition of Collagen Containing Tissues. In: The Science and Technology of Gelatin, A.G. Wards and A. Courts (eds.), London: Academic Press. Johns, P. and Courts, A. 1977. Relationship Between Collagen and Gelatin. In: The Science and Technology of Gelatin, A.G. Wards and A. Courts (eds.), London: Academic Press. Marcotte, M., Teherian Hoshahili, Ali R., Ramaswamy, H.S. 2001. Rheological properties of selected hydrocolloids as a function of concentration and temperature. Food Research International 34. 695,703. Nazzal, S. and Wang, Y. 2001. Characterization of soft gelatin capsules by thermal analysis. International Journal of Pharmaceutics 230. 35,45. Singh, S., Rama Rao, K.V., Venugopal, K., Manikandan, R. 2002. Alteration in Dissolution Characteristics of Gelatin-Containing Formulations. Pharmaceutical Technology. 26(4):36,54. Sobral, P.J.A. and Habitante A.M.Q.E. 2001. Phase transitions of pigskin gelatin. Food Hydrocolloids 15. 377,382. Stainsby, G. 1977. The Gelatin and the Sol-Gel Transformation. In: The Science and Technology of Gelatin, A.G. Wards and A. Courts (eds.), London: Academic Press. Stainsby, G. 1977. The Physical Chemistry of Gelatin in Solution. In: The Science and Technology of Gelatin, A.G. Wards and A. Courts (eds.), London: Academic Press. Taylor, David. C.R. Biologies 325. 2002. Inactivation of the BSE agent. 75,76. Usta, M., Piech, D.L., MacCrone R.K., Hillig, W.B. 2003. Behavior and properties of neat and filled gelatins. Biomaterials 24. 165,172.

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55 APPENDICES

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56 Appendix 1. Microsoft (MS) Excel Graphical Representation of Test Results Figure 16. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (0-8 hrs). Figure 17. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (32-40 hrs). E21995 Extensibility 0 1000 2000 3000 4000 5000 6000 0510152025 Distance (mm)Force (g) ~ 0 hrs ~ 4 hrs ~ 8 hrs E21996 Extensibility 0 1000 2000 3000 4000 5000 6000 0510152025 Distance (mm)Force (g) ~ 32 hrs ~ 36 hrs ~ 40 hrs

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57 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 18. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (64-72 hrs). Figure 19. MS Excel Representation of Gel Extensibility Test Results for 195 Gelatin (88-96 hrs). E21997 Extensibility 0 1000 2000 3000 4000 5000 6000 0510152025 Distance (mm)Force (g) ~ 64 hrs ~ 68 hrs ~ 72 hrs E21998 Extensibility0 1000 2000 3000 4000 5000 6000 0510152025 Distance (mm)Force (g) ~ 88 hrs ~ 92 hrs ~ 96 hrs

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58 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 20. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (0-8 hrs). Figure 21. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (32-40 hrs). E21999 Extensibility0 500 1000 1500 2000 2500 3000 3500 4000 010203040 Distance (mm)Force (grams) ~ 8 hrs E22000 Extensibility0 500 1000 1500 2000 2500 3000 3500 4000 010203040 Distance (mm)Force (grams) ~ 32 hrs ~36 hrs ~ 40 hrs

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59 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 22. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (64-72 hrs). Figure 23. MS Excel Representation of Gel Extensibility Test Results for 150 Gelatin (88-96 hrs). E22001 Extensibility0 500 1000 1500 2000 2500 3000 3500 4000 010203040 Distance (mm)Force (grams) ~ 64 hrs ~ 68 hrs ~ 72 hrs E22002 Extensibility0 500 1000 1500 2000 2500 3000 3500 4000 010203040 Distance (mm)Force (grams) ~ 88 hrs ~ 92 hrs ~ 96 hrs

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60 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 24. MS Excel Representation of Resilience Test Results for 195 Gelatin (32-40 hrs). Figure 25. MS Excel Representation of Resilience Test Results for 195 Gelatin (64-72 hrs). E21996 Resilience0 20 40 60 80 100 120 050010001500 Force (g)% Resilienc e ~ 32 hrs ~ 36 hrs ~ 40 hrs E21997 Resilience0 20 40 60 80 100 120 050010001500 Force (g)% Resilienc e ~ 64 hrs ~ 68 hrs ~ 72 hrs

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61 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 26. MS Excel Representation of Resilience Test Results for 195 Gelatin (88-96 hrs). E21998 Resilience0 20 40 60 80 100 120 050010001500 Force (g)% Resilienc e ~ 88 hrs ~ 92 hrs ~ 96 hrs

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62 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 27. MS Excel Representation of Resilience Test Results for 150 Gelatin (0-8 hrs). Figure 28. MS Excel Representation of Resilience Test Results for 150 Gelatin (32-40 hrs). E21999 Resilience0 50 100 150 200 250 020040060080010001200 Force (g)% Resilienc e ~ 8 hrs E22000 Resilience0 50 100 150 200 250 020040060080010001200 Force (g)% Resilience ~ 32 hrs ~ 36 hrs ~ 40 hrs

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63 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 29. MS Excel Representation of Resilience Test Results for 150 Gelatin (64-72 hrs). Figure 30. MS Excel Representation of Resilience Test Results for 150 Gelatin (88-96 hrs). E22001 Resilience0 50 100 150 200 250 020040060080010001200 Force (g)% Resilienc e ~ 64 hrs ~ 68 hrs ~ 72 hrs E22002 Resilience0 50 100 150 200 250 020040060080010001200 Force (g)% Resilienc e ~ 88 hrs ~ 92 hrs ~ 96 hrs

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64 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 31. MS Excel Representation of Wet Burst Strength Test Results for 195 Gelatin (32-40 hrs). Figure 32. MS Excel Representation of Wet Burst Strength Test Results for 195 Gelatin (64-72 hrs). E21996 Wet Burst Strength 0 2 4 6 8 10 0500010000150002000025000300003500040000 Force (g)Distance (mm) ~ 32 hrs E21997 Wet Burst Strength 0 2 4 6 8 10 0500010000150002000025000300003500040000 Force (g)Distance (mm) ~ 64 hrs ~ 68 hrs

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65 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 33. MS Excel Representation of Wet Burst Strength Test Results for 195 Gelatin (88-96 hrs). E21998 Wet Burst Strength 0 2 4 6 8 10 0500010000150002000025000300003500040000 Force (g)Distance (mm) ~ 88 hrs ~ 92 hrs ~ 96 hrs

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66 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 34. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (0-8 hrs). Figure 35. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (32-40 hrs). E21999 Wet Burst Strength0 1 2 3 4 5 6 7 050001000015000200002500030000 Force in gramsDistance in mm ~ 0 hrs ~ 4 hrs ~ 8 hrs E22000 Wet Burst Strength0 1 2 3 4 5 6 7 050001000015000200002500030000 Force in gramsDistance in mm ~ 32 hrs ~ 36 hrs ~ 40 hrs

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67 APPENDIX 1. Microsoft (MS) Excel Graphical Representation of Test Results (Continued) Figure 36. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (64-72 hrs). Figure 37. MS Excel Representation of Wet Burst Strength Test Results for 150 Gelatin (88-96 hrs). E22001 Wet Burst Strength0 1 2 3 4 5 6 7 050001000015000200002500030000 Force in gramsDistance in mm ~ 64 hrs ~ 68 hrs ~ 72 hrs E22002 Wet Burst Strength0 1 2 3 4 5 6 7 050001000015000200002500030000 Force in gramsDistance in mm ~ 88 hrs ~ 92 hrs ~ 96 hrs

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68 Appendix 2. Wet Burst Strength Test Results Figure 38. 195 Gelatin Wet Burst Strength Printout at 32 hrs.

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69 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 39. 195 Gelatin Wet Burst Strength Printout at 64 hrs.

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70 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 40. 195 Gelatin Wet Burst Strength Printout at 68 hrs.

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71 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 41. 195 Gelatin Wet Burst Strength Printout at 88 hrs.

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72 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 42. 195 Gelatin Wet Burst Strength Printout at 92 hrs.

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73 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 43. 195 Gelatin Wet Burst Strength Printout at 96 hrs.

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74 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 44. 150 Gelatin Wet Burst Strength Printout at 0 hrs.

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75 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 45. 150 Gelatin Wet Burst Strength Printout at 4 hrs.

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76 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 46. 150 Gelatin Wet Burst Strength Printout at 8 hrs.

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77 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 47. 150 Gelatin Wet Burst Strength Printout at 32 hrs.

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78 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 48. 150 Gelatin Wet Burst Strength Printout at 36 hrs.

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79 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 49. 150 Gelatin Wet Burst Strength Printout at 40 hrs.

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80 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 50. 150 Gelatin Wet Burst Strength Printout at 64 hrs.

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81 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 51. 150 Gelatin Wet Burst Strength Printout for at 68 hrs.

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82 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 52. 150 Gelatin Wet Burst Strength Printout at 72 hrs.

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83 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 53. 150 Gelatin Wet Burst Strength Printout at 88 hrs.

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84 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 54. 150 Gelatin Wet Burst Strength Printout at 92 hrs.

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85 APPENDIX 2. Wet Burst Strength Test Results (Continued) Figure 55. 150 Gelatin Wet Burst Strength Printout at 96 hrs.

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86 Appendix 3. Rheology Test Results Figure 56. 195 Gelatin Rheological Testing at 0 hrs.

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87 APPENDIX 3. Rheology Test Results (Continued). Figure 57. 195 Gelatin Rheological Testing at 4 hrs.

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88 APPENDIX 3. Rheology Test Results (Continued). Figure 58. 195 Gelatin Rheological Testing at 8 hrs.

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89 APPENDIX 3. Rheology Test Results (Continued). Figure 59. 195 Gelatin Rheological Testing at 32 hrs.

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90 APPENDIX 3. Rheology Test Results (Continued). Figure 60. 195 Gelatin Rheological Testing at 36 hrs.

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91 APPENDIX 3. Rheology Test Results (Continued). Figure 61. 195 Gelatin Rheological Testing at 40 hrs.

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92 APPENDIX 3. Rheology Test Results (Continued). Figure 62. 195 Gelatin Rheological Testing at 64 hrs.

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93 APPENDIX 3. Rheology Test Results (Continued). Figure 63. 195 Gelatin Rheological Testing at 68 hrs.

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94 APPENDIX 3. Rheology Test Results (Continued). Figure 64. 195 Gelatin Rheological Testing at 72 hrs.

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95 APPENDIX 3. Rheology Test Results (Continued). Figure 65. 195 Gelatin Rheological Testing at 88 hrs.

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96 APPENDIX 3. Rheology Test Results (Continued). Figure 66. 195 Gelatin Rheological Testing at 92 hrs.

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97 APPENDIX 3. Rheology Test Results (Continued). Figure 67. 195 Gelatin Rheological Testing at 96 hrs.

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98 APPENDIX 3. Rheology Test Results (Continued). Figure 68. 150 Gelatin Rheological Testing at 0 hrs.

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99 APPENDIX 3. Rheology Test Results (Continued). Figure 69. 150 Gelatin Rheological Testing at 4 hrs.

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100 APPENDIX 3. Rheology Test Results (Continued). Figure 70. 150 Gelatin Rheological Testing at 8 hrs.

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101 APPENDIX 3. Rheology Test Results (Continued). Figure 71. 150 Gelatin Rheological Testing at 32 hrs.

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102 APPENDIX 3. Rheology Test Results (Continued). Figure 72. 150 Gelatin Rheological Testing at 36 hrs.

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103 APPENDIX 3. Rheology Test Results (Continued). Figure 73. 150 Gelatin Rheological Testing at 40hrs.

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104 APPENDIX 3. Rheology Test Results (Continued). Figure 74. 150 Gelatin Rheological Testing at 64 hrs.

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105 APPENDIX 3. Rheology Test Results (Continued). Figure 75. 150 Gelatin Rheological Testing at 68 hrs.

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106 APPENDIX 3. Rheology Test Results (Continued). Figure 76. 150 Gelatin Rheological Testing at 72 hrs.

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107 APPENDIX 3. Rheology Test Results (Continued). Figure 77. 150 Gelatin Rheological Testing at 88 hrs.

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108 APPENDIX 3. Rheology Test Results (Continued). Figure 78. 150 Gelatin Rheological Testing at 92 hrs.

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109 APPENDIX 3. Rheology Test Results (Continued). Figure 79. 150 Gelatin Rheological Testing at 96 hrs.

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110 Appendix 4. Gel Extensibility Test Results Figure 80. 195 Gelatin Extensibility Test Printout at 0 hrs.

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111 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 81. 195 Gelatin Extensibility Test Printout at 4 hrs.

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112 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 82. 195 Gelatin Extensibility Test Printout at 8 hrs.

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113 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 83. 195 Gelatin Extensibility Test Printout at 32 hrs.

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114 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 84. 195 Gelatin Extensibility Test Printout at 36 hrs.

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115 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 85. 195 Gelatin Extensibility Test Printout at 40 hrs.

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116 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 86. 195 Gelatin Extensibility Test Printout at 64 hrs.

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117 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 87. 195 Gelatin Extensibility Test Printout at 68 hrs.

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118 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 88. 195 Gelatin Extensibility Test Printout at 72 hrs.

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119 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 89. 195 Gelatin Extensibility Test Printout at 88 hrs.

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120 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 90. 195 Gelatin Extensibility Test Printout at 92 hrs.

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121 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 91. 195 Gelatin Extensibility Test Printout at 96 hrs.

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122 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 92. 150 Gelatin Extensibility Test Printout at 8 hrs.

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123 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 93. 150 Gelatin Extensibility Test Printout at 32 hrs.

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124 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 94. 150 Gelatin Extensibility Test Printout at 36 hrs.

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125 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 95. 150 Gelatin Extensibility Test Printout at 40 hrs.

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126 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 96. 150 Gelatin Extensibility Test Printout at 64 hrs.

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127 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 97. 150 Gelatin Extensibility Test Printout at 68 hrs.

PAGE 141

128 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 98. 150 Gelatin Extensibility Test Printout at 72 hrs.

PAGE 142

129 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 99. 150 Gelatin Extensibility Test Printout at 88 hrs.

PAGE 143

130 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 100. 150 Gelatin Extensibility Test Printout at 92 hrs.

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131 APPENDIX 4. Gel Extensibility Test Results (Continued). Figure 101. 150 Gelatin Extensibility Test Printout at 96 hrs.

PAGE 145

132 Appendix 5. Resilience Test Results Figure 102. 195 Gelatin Resilience Test Printout at 32 hrs.

PAGE 146

133 APPENDIX 5. Resilience Test Results (Continued). Figure 103. 195 Gelatin Resilience Test Printout at 36 hrs.

PAGE 147

134 APPENDIX 5. Resilience Test Results (Continued). Figure 104. 195 Gelatin Resilience Test Printout at 40 hrs.

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135 APPENDIX 5. Resilience Test Results (Continued). Figure 105. 195 Gelatin Resilience Test Printout at 64 hrs.

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136 APPENDIX 5. Resilience Test Results (Continued). Figure 106. 195 Gelatin Resilience Test Printout at 68 hrs.

PAGE 150

137 APPENDIX 5. Resilience Test Results (Continued). Figure 107. 195 Gelatin Resilience Test Printout at 72 hrs.

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138 APPENDIX 5. Resilience Test Results (Continued). Figure 108. 195 Gelatin Resilience Test Printout at 88 hrs.

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139 APPENDIX 5. Resilience Test Results (Continued). Figure 109. 195 Gelatin Resilience Test Printout at 92 hrs.

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140 APPENDIX 5. Resilience Test Results (Continued). Figure 110. 195 Gelatin Resilience Test Printout at 96 hrs.

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141 APPENDIX 5. Resilience Test Results (Continued). Figure 111. 150 Gelatin Resilience Test Printout at 8 hrs.

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142 APPENDIX 5. Resilience Test Results (Continued). Figure 112. 150 Gelatin Resilience Test Printout at 32 hrs.

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143 APPENDIX 5. Resilience Test Results (Continued). Figure 113. 150 Gelatin Resilience Test Printout at 36 hrs.

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144 APPENDIX 5. Resilience Test Results (Continued). Figure 114. 150 Gelatin Resilience Test Printout at 40 hrs.

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145 APPENDIX 5. Resilience Test Results (Continued). Figure 115. 150 Gelatin Resilience Test Printout at 68 hrs.

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146 APPENDIX 5. Resilience Test Results (Continued). Figure 116. 150 Gelatin Resilience Test Printout at 72 hrs.

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147 APPENDIX 5. Resilience Test Results (Continued). Figure 117. 150 Gelatin Resilience Test Printout at 88 hrs.

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148 APPENDIX 5. Resilience Test Results (Continued). Figure 118. 150 Gelatin Resilience Test Printout at 92 hrs.

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149 APPENDIX 5. Resilience Test Results (Continued). Figure 119. 150 Gelatin Resilience Test Printout at 96 hrs.

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150 Appendix 6. Molecular Weight Analysis Results Figure 120. 195 & 150 Gelatin Molecular Weight Analysis Results.


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Characterization and correlation analysis of pharmaceutical gelatin
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ABSTRACT: The properties of the aged gel and subsequent softgels were examined using mechanical and chemical testing methods. Our hypothesis was that a negligible variation will exist between the aged gel of the same type. The greater difference is expected to be seen between the types of gels described as 150 Bloom (alkaline treated collagen) and 195 Bloom (acid treated collagen). The types of gelatin used were the acid processed (195 Acid Bone) and alkaline processed (150 Lime Bone). Because of the differences expressed as the result of their manufacture sequence (namely their molecular weights), it follows that physical attributes will further contribute to their distinction. In addition to observing different characteristics between the types of gels, we aged the gelatin and produced softgel capsules to qualify and quantify the changes that occur as a function of time.Two production lots of over 1 million softgel capsules were executed to produce a population that lends itself to statistical analysis. Softgel capsules were manufactured with gelatin which was aged at intervals of 0-8 hrs, 32-40 hrs, 66-72 hrs and 88-96 hrs. The manufacturing process made use of this strategy for the acid and alkaline treated gelatin where a total of eight lots were made (4 acid and 4 alkaline). One hundred thousand softgels were manufactured for the acid processed gelatin, per lot. Additionally, one hundred and fifty thousand softgels were manufactured for the alkaline processed gelatin per lot. The results of the different tests provided trends that were not solely a function of time. Gel extensibility for both gel types showed a decrease in the amount of force needed to rupture the gelatin ribbon, as a function of time. The resilience of the tested ribbon remained constant throughout the aging process.The burst strength was the only test showing an inverse relationship between the two gel types. The amount of force needed to rupture the 150 Bloom softgels decrease in time whereas the amount of force needed to rupture the 195 Bloom softgels increase with time. The rheological testing was described in the literature as being associated with the molecular weight distribution. Such association was seen in our research and both the results of the rheological and the molecular weight tests decreased with the aging process.
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