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  1. 1. QUALITY CONTROLE OF CHORIONIC GONADOTROPHIN BY S.RAVINDER REDDY ROLL NO:1008-10-885-011 M.PHARMACY 1 ST YEAR 1 ST SEM PHARMACEUTICAL ANALYSIS AND Q.A.
  2. 2. <ul><li>CONTENTS </li></ul><ul><li>ABSTRACT </li></ul><ul><li>INTRODUCTION </li></ul><ul><li>HISTORY </li></ul><ul><li>FUNCTIONS </li></ul><ul><li>SIDE EFFECTS </li></ul><ul><li>PRODUCTION </li></ul><ul><li>PURIFICATION PROCESS </li></ul><ul><li>FORMULATIONS </li></ul><ul><li>HCG PREPARATIONS </li></ul><ul><li>BIOLOGICAL ASSAY INTRODUCTION </li></ul><ul><li>QUALITY CONTROLE OF CHORIONIC GONADOTROPHIN </li></ul>
  3. 3. ABSTRACT Gonadotropins are hormones which target the gonads , structures involved in fertility and reproduction. In women, gonadotropins are directed at the ovaries , where they play in a role in the production of eggs and eventual ovulation , while in men, these hormones are involved in sperm production in the testes . Abnormalities in gonadotropin levels can lead to infertility, which can sometimes be treated with hormone injections . crude preparation of hCG, deriving from a concentrated sample of a culture medium obtained after the recombinant process or from a crude concentrate of urine of pregnant women , can be purified such that the resulting hCG is practically free from proteins or other contaminants contained in the crude hCG preparation.
  4. 4. <ul><li>purified pyrogen-free preparation obtained from the urine of pregnant females. It is standardized by a biological assay procedure. </li></ul><ul><li>INTRODUCTION </li></ul><ul><li>Human chorionic gonadotropin (HCG), a polypeptide hormone produced by the human placenta, is composed of an alpha and a beta sub-unit. The alpha sub-unit is essentially identical to the alpha sub-units of the human pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as to the alpha sub-unit of human thyroid-stimulating hormone (TSH). The beta sub-units of these hormones differ in amino acid sequence . </li></ul>
  5. 5. <ul><li>Early screening for infertility can include blood tests to check hormone levels for the purpose of determining whether or not endocrine disruptions are responsible for the infertility. If they are, a doctor may recommend that patients take shots of FSH and HCG. </li></ul><ul><li>HISTORY </li></ul><ul><li>The discovery of hCG arose from the observation made 80 years ago by Ascheim and Zondek that the urine of pregnant women contained a gonad-stimulating substance that induced both follicular maturation and ovarian stromal luteinization when injected into immature mice . </li></ul>
  6. 6. <ul><li>Subse-quently, hCG was shown to be secreted by placental tissue in vitro , localized to the syncytiotrophoblast , and derived from giant syncytiotrophoblast cells . The hormone first was made commercially available in 1931 under the label Pregnonâ, later changed to Pregnylâ in 1932. Remarkably, Pregnylâ still is available today. </li></ul><ul><li>In 1950s, Dr. A.T.W. Simeon was doing a research on boys with undeveloped gonads. Being treated with HCG, Dr. Simeon noticed that they were losing a lot of weight and as well as their appetite. </li></ul>
  7. 7. <ul><li>FUNCTIONS </li></ul><ul><li>Levels of HCG can be used to determine whether or not a woman is pregnant . </li></ul><ul><li>Human chorionic gonadotropin interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy , causing it to secrete the hormone progesterone . Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus . </li></ul><ul><li>Because of its similarity to LH , hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes . As the most abundant biological source is women who are presently pregnant, some organizations collect urine from pregnant women to extract hCG for use in fertility treatment . </li></ul>
  8. 8. <ul><li>Side effects </li></ul><ul><li>a) Hot Flash caused by high levels of LH </li></ul><ul><li>b) Headache caused by over stimulation of the medication which interfere with the outer brain nervous system </li></ul><ul><li>c) Fluid retention as resulting in interfering with the function of lymphatic and kidney function in regulating fluid in the body </li></ul><ul><li>Others such as bloating and mild nausea. </li></ul>
  9. 9. <ul><li>PRODUCTION </li></ul><ul><li>As the most abundant biological source is women who are presently pregnant, some organizations collect urine from pregnant women to extract hCG for use in fertility treatment . </li></ul><ul><li>Like other gonadotropins , hCG can be extracted from urine or by genetic modification. Pregnyl, Follutein, Profasi, Choragon and Novarel use the former method, derived from the urine of pregnant women. Ovidrel , on the other hand, is a product of recombinant DNA </li></ul>
  10. 10. <ul><li>PURIFICATION PROCESS </li></ul><ul><li>The process comprises the steps of subjecting the sample to ion-exchange chromatography and subjecting the eluate to reverse phase HPLC. A further step of applying the eluate to a size exclusion column may additionally be carriedout. </li></ul><ul><li>The two ion-exchange chromatography steps are preferably performed under different conditions in order to obtain optimum results from the purification process . </li></ul><ul><li>A preferred embodiment of the process of the invention comprises the steps of: </li></ul><ul><li>(a) eluting the sample through a silica chromatography column. </li></ul>
  11. 11. <ul><li>(b) eluting through a DEAE SEPHAROSE (cross-linked agarose matrix with diethylaminoethyl weak anion exchanger) ion-exchange chromatography column. </li></ul><ul><li>(c) eluting through a CM-SEPHAROSE (cross-linked agarose matrix with carboxymethyl weak anion exchanger) ion-exchange chromatography column </li></ul><ul><li>(d) eluting through a Silica C18 reverse phase HPLC column.and </li></ul><ul><li>(e) eluting through a CM-SEPHACRYL (spherical allyl dextran and N,N'-methylenebisacrylamide) sizeexclusion chromatographycolumn . </li></ul>
  12. 12. <ul><li>FORMULATIONS </li></ul><ul><li>Both liquid and freeze dried formulations have been developed with the highly purified recombinant hCG. </li></ul><ul><li>Liquid Formulation Two liquid formulations at 10000 IU/ml were prepared in vials using mannitol or sucrose as excipient and submitted to stability tests at 50, 40, 25 and 4° C. Freeze Dried Formulation A freeze dried formulation at 5000 IU strength was prepared in vials for stability tests at 50, 40, 25 and 4° C. using sucrose as excipient. </li></ul>
  13. 13. <ul><li>hCG Preparations </li></ul><ul><li>Urinary Preparations </li></ul><ul><li>Derived from the urine of pregnant women. </li></ul><ul><li>Generic </li></ul><ul><li>(human) chorionic gonadotropin for injection, USP </li></ul><ul><li>Brands </li></ul><ul><li>Pregnyl (Merck/Schering-Plough) </li></ul><ul><li>Follutein </li></ul><ul><li>Profasi </li></ul><ul><li>Novarel </li></ul><ul><li>Recombinant Preparations </li></ul><ul><li>Generic </li></ul><ul><li>choriogonadotropin alfa for injection (recombinant human Chorionic Gonadotropin, r-hCG). </li></ul><ul><li>Brands </li></ul><ul><li>Ovidrel </li></ul>
  14. 14. <ul><li>new tablet form of hCG, marketed under the brand name   Opti-Lean hCG Tablets . </li></ul><ul><li>When used together with a very low calorie diet, it produces outstanding weight loss results very quickly Weight loss of a pound a day is not uncommon. </li></ul>
  15. 15. <ul><li>BIOLOGICAL ASSAY </li></ul><ul><li>DEFINATION </li></ul><ul><li>&quot;The determination of the relative strength of a substance (as a drug) by comparing its effect on a test organism with that of a standard preparation.“ </li></ul><ul><li>PRINCIPLE </li></ul><ul><li>Bioassays are conducted by determining the amount of preparation of unknown potency required to produce a definite effect on suitable test animals or organs or tissue under standard conditions. This effect is compared with that of a standard. </li></ul><ul><li>Thus the amount of the test substance required to produce the same biological effect as a given quantity the unit of a standard preparation is compared and the potency of the unknown is expressed as a % of that of the standard by employing a simple formula . </li></ul>
  16. 16. <ul><li>USES </li></ul><ul><li>Bioassays are procedures that can determine the concentration of purity or biological activity of a substance such as vitamin, hormone, and plant growth factor. </li></ul><ul><li>While measuring the effect on an organism, tissue cells, enzymes or the receptor is preparing to be compared to a standard precipitation. Bioassays may be qualitative or quantitative . </li></ul><ul><li>Quantitative bioassays involve estimation of the concentration or potency of a substance by measurement of the biological response that it produces </li></ul>
  17. 17. <ul><li>QUALITY CONTROLE OF CHORIONIC GONADOTROPHIN </li></ul><ul><li>Human Chorionic Gonadotrophin is a dry, sterile preparation of placental glycoproteins that has luteinising activity. </li></ul><ul><li>It is extracted from the urine of pregnant women. The material is sterilised by filtration and dried under reduced pressure or freeze-dried. </li></ul><ul><li>Chorionic Gonadotrophin contains not less than 2500 Units per mg. </li></ul><ul><li>Description </li></ul><ul><li>A white or almost white, amorphous powder </li></ul>
  18. 18. <ul><li>IDENTIFICATION </li></ul><ul><li>It causes an increase in the weight of the seminal vesicles or of the prostate glands of immature male rats when administered as directed in the Assay. </li></ul><ul><li>TESTS   </li></ul><ul><li>Appearance of solution . </li></ul><ul><li>A 1.0 per cent w/v solution is clear , and colourless .   </li></ul><ul><li>WATER </li></ul><ul><li>Not more than 5 per cent, determined by the following method. </li></ul><ul><li>Determine by gas chromatography . </li></ul><ul><li>Use throughout dry glassware that may be siliconised. </li></ul>
  19. 19. <ul><li>Internal standard </li></ul><ul><li>Dilute 15 μl of anhydrous methanol with sufficient anhydrous 2-propanol to produce 100 ml. </li></ul><ul><li>  </li></ul><ul><li>Test solution (a) </li></ul><ul><li>Dissolve 4 mg of the substance under examination in 0.5 ml of anhydrous 2-propanol. </li></ul><ul><li>  </li></ul><ul><li>Test solution (b) </li></ul><ul><li>Dissolve 4 mg of the substance under examination in 0.5 ml of test solution (a). </li></ul><ul><li>Reference solution </li></ul><ul><li>Add 10 μl of water to 50 ml of test solution (a). </li></ul><ul><li>  </li></ul>
  20. 20. <ul><li>Chromatographic system </li></ul><ul><li>– a stainless steel column 1m × 2 mm, packed with porous </li></ul><ul><li>polymer beads (60 to 80 mesh) (such as Chromosorb 102), </li></ul><ul><li>– temperature: </li></ul><ul><li>column.120°, </li></ul><ul><li>inlet port and detector. 150°, </li></ul><ul><li>– thermal conductivity detector, </li></ul><ul><li>– flow rate. 30 ml per minute of the carrier gas (helium). </li></ul><ul><li>  </li></ul><ul><li>From the chromatograms obtained, and taking into account </li></ul><ul><li>any water detectable in test solution (a), calculate the </li></ul><ul><li>percentage of water taking 0.9960 g as the weight per ml at 25°. </li></ul>
  21. 21. <ul><li>BIOLOGYCAL ASSAY PROCEDURE </li></ul><ul><li>Standard preparation </li></ul><ul><li>The 3rd International Standard for Chorionic Gonadotrophin, human, established in 1986, consisting of a freeze-dried extract of human chorionic gonadotrophin with human albumin (supplied in ampoules containing 650 Units), or another suitable preparation the potency of which has been determined in relation to the International Standard. </li></ul>
  22. 22. <ul><li>Dissolve a sufficient quantity corresponding to the daily doses to be used in sufficient albumin-phosphate buffer pH7.2 so that the daily dose is about 0.2ml. </li></ul><ul><li>Add a suitable antimicrobial preservative such as 0.4 percent w/v of phenol or 0.002 percent w/v of thiomersal. Store the solution at a temperature of 2° to 8°. </li></ul><ul><li>Test preparation </li></ul><ul><li>Dissolve a sufficient quantity of the preparation under examination corresponding to the daily doses to be used in sufficient albumin-phosphate buffer pH 7.2 so that the daily dose is about 0.2 ml. </li></ul><ul><li>Add a suitable antimicrobial preservative such as 0.4 per cent w/v of phenol or 0.002 per cent w/v of thiomersal. Store the solution at a temperature of 2° to 8°. </li></ul>
  23. 23. <ul><li>Use immature male rats of the same strain, approximately 21 days old and of approximately equal weight within the range 25 to 35 g. </li></ul><ul><li>Assign the rats at random to four equal groups of at least eight animals. If sets of four littermates are available, allot one littermate from each set at random to each group and mark according to the litter. </li></ul>Male rats , 21 days old & 25 – 35 g 4 groups (8 animals in each)
  24. 24. <ul><li>Choose two doses of the standard preparation and two of the test solution such that the smaller dose is sufficient to produce a positive response in some of the rats and the larger dose does not produce a maximum response in all of the rats. </li></ul><ul><li>As an initial approximation, doses of 7.5 and 15 Units may be tried although the dose will depend on the sensitivity of the animals used, which may vary widely </li></ul><ul><li>Inject subcutaneously into each rat the daily dose allocated to its group on 4 consecutive days at the same time each day. </li></ul><ul><li>On the fifth day, about 24 hours after the last injection, kill the rats and remove the seminal vesicles or the prostate glands from each animal. </li></ul>
  25. 25. <ul><li>Remove any extraneous fluid and tissue from the vesicles or glands and weigh them immediately. </li></ul><ul><li>Calculate the result of the assay by standard statistical methods using the weight of the vesicles or prostate glands as the response. </li></ul><ul><li>The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency </li></ul>
  26. 26. <ul><li>Bacterial endotoxins </li></ul><ul><li>Not more than 15 Endotoxin Units per ml of a solution prepared in the following manner. </li></ul><ul><li>Dissolve a quantity in water BET to obtain a solution containing 500 units of chorionic gonadotrophin per ml. </li></ul><ul><li>Carry out the test using Maximum Valid dilution of this solution calculated from the declared sensitivity of the lysate used in the test. </li></ul><ul><li>ABNORMAL TOXICITY </li></ul><ul><li>Complies with the test for abnormal toxicity using a quantity equivalent to 1000 Units dissolved in 0.5 ml of sodium chloride injection and observing the animals for 48 hours . </li></ul>
  27. 27. <ul><li>Sterility </li></ul><ul><li>Complies with the test for sterility . </li></ul><ul><li>Storage </li></ul><ul><li>Store protected from light in a tamper-evident container, which is sealed so as to exclude micro-organisms, in a refrigerator (2° to 8°). </li></ul><ul><li>  </li></ul><ul><li>Labelling </li></ul><ul><li>The label states </li></ul><ul><li>(1) the number of Units contained in the container; </li></ul><ul><li>(2) the number of Units per mg; </li></ul><ul><li>(3) whether or not it is intended for use in the manufacture of parenteral preparations.   </li></ul>
  28. 28. <ul><li>CHORIONIC GONADOTROPHIN INJECTION </li></ul><ul><li>Chorionic Gonadotrophin Injection is a sterile material consisting of Chorionic Gonadotrophin with or without excipients such as buffers, diluents or other inert substances such as Lactose or Sodium Chloride. It may also contain an antimicrobial agent. It is filled in a sealed container. </li></ul><ul><li>The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. </li></ul><ul><li>The constituted solution complies with the requirements for </li></ul><ul><li>Clarity of solution and Particulate matter stated under </li></ul><ul><li>Parenteral Preparations (Injections) . </li></ul>
  29. 29. <ul><li>Storage </li></ul><ul><li>The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. </li></ul><ul><li>ASSAY </li></ul><ul><li>Assay procedure follows same as like gonadotrophin powder </li></ul><ul><li>Chorionic Gonadotrophin Injection contains not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. </li></ul>
  30. 30. <ul><li>Chorionic Gonadotropin, Intact, Human (hCG) BioAssay ELISA Kit </li></ul><ul><li>Principle </li></ul><ul><li>This hCG enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay . </li></ul><ul><li>The microtiter plate provided in this kit has been precoated with a monoclonal antibody specific to hCG . </li></ul><ul><li>Standards or samples are added to the microtiter plate wells and incubated. The plate wells are washed to remove any unbound standard or sample, hCG, if present, will bind to the antibody pre-coated on the wells. </li></ul>
  31. 31. <ul><li>A standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific to hCG is added to each well to “sandwich” the hCG immobilized on the plate. </li></ul><ul><li>The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, a TMB (3,3', 5,5' Tetramethyl-benzidene) substrate solution is added to each well. </li></ul><ul><li>This enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain hCG and enzyme-conjugated antibody will exhibit a change in color. </li></ul>
  32. 32. <ul><li>The enzymesubstrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm . </li></ul><ul><li>In order to measure the concentration of hCG in the sample, this ELISA Kit includes a set of calibration standards (6 standards). </li></ul><ul><li>The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density (O.D.) versus hCG concentration (mIU/ml). </li></ul><ul><li>The concentration of hCG in the samples is then determined by comparing the O.D. of the samples to the standard curve. </li></ul>
  33. 33. <ul><li>Sensitivity </li></ul><ul><li><3.0mIU/ml </li></ul><ul><li>Range </li></ul><ul><li>7.5-240mIU/ml </li></ul>
  34. 34. <ul><li>BIOLOGIC ACTIVITY HCG FOLLOWING REVERSED-PHASE HPLC </li></ul><ul><li>Fractions of hCG collected following reversed-phase HPLC were bioassayed by activation of adenylate cyclase to determine their biological potencies. </li></ul><ul><li>The biologic activity of HPLC fractions containing hCG was determined by their ability to activate adenylate cyclase in plasma membranes prepared from luteinized rat ovaries. </li></ul><ul><li>Addition of 2.5µM forskolin in 1.6% ethanol to the assay buffer for the adenylate cyclase assay. </li></ul><ul><li>Forskolin increases the maximal response of the ovarian enzyme to hCG stimulation resulting in a steeper dose-response curve and increase assay precision. </li></ul>
  35. 35. <ul><li>Standard hCG and HPLC fractions were assayed at 8-10 doses in each assay; each dose was tested in triplicate. </li></ul><ul><li>The 3-6 doses falling on the linear portion of the dose- response curve were used in calculating potencies using standard bioassay statistics. </li></ul><ul><li>The standard for bioassay consisted of hormone which had not been subjected to HPLC. </li></ul><ul><li>Potencies of HPLC fractions were expressed relative to the standard ; a relative potency of 1.00 indicates a potency equal to the standard. </li></ul>
  36. 36. <ul><li>Subsequent experiments focused on finding mobile phases which would elute hCG off the reversed phase columns as a single peak. </li></ul><ul><li>Under the best conditions ,hCG retained about 60% of its biological activity. </li></ul><ul><li>The reduced biological activity of hCG exposed to conditions of low pH can be fully restored following neutralization of the pH to 7. </li></ul>

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