Principles of Chemotherapy

CHEMOTHERAPY
Principles

Cancer is the uncontrolled growth of cells coupled with malignant behavior: invasion and metastasis. Cancer is thought to be caused by the interaction between genetic susceptibility and environmental toxins.
Broadly, most chemotherapeutic drugs work by impairing mitosis (cell division), effectively targeting fast-dividing cells. As these drugs cause damage to cells they are termed cytotoxic. Some drugs cause cells to undergo apoptosis (so-called "programmed cell death").

Unfortunately, scientists have yet to identify specific features of malignant and immune cells that would make them uniquely targetable (barring some recent examples, such as the Philadelphia chromosome as targeted by imatinib). This means that other fast dividing cells such as those responsible for hair growth and for replacement of the intestinal epithelium (lining) are also often affected. However, some drugs have a better side-effect profile than others, enabling doctors to adjust treatment regimens to the advantage of patients in certain situations.

As chemotherapy affects cell division, tumors with high growth fractions (such as acute myelogenous leukemia and the aggressive lymphomas, including Hodgkin's disease) are more sensitive to chemotherapy, as a larger proportion of the targeted cells are undergoing cell division at any time. Malignancies with slower growth rates, such as indolent lymphomas, tend to respond to chemotherapy much more modestly.

Drugs affect "younger" tumors (i.e. more differentiated) more effectively, because mechanisms regulating cell growth are usually still preserved. With succeeding generations of tumor cells, differentiation is typically lost, growth becomes less regulated, and tumors become less responsive to most chemotherapeutic agents. Near the center of some solid tumors, cell division has effectively ceased, making them insensitive to chemotherapy. Another problem with solid tumors is the fact that the chemotherapeutic agent often does not reach the core of the tumor. Solutions to this problem include radiation therapy (both brachytherapy and teletherapy) and surgery.
Over time, cancer cells become more resistant to chemotherapy treatments. Recently, scientists have identified small pumps on the surface of cancer cells that actively move chemotherapy from inside the cell to the outside. Research on p-glycoprotein and other such chemotherapy efflux pumps, is currently ongoing. Medications to inhibit the function of p-glycoprotein are undergoing testing as of June, 2007 to enhance the efficacy of chemotherapy.

When chemotherapy drugs attack reproducing cells, they cannot tell the difference between reproducing cells of normal tissues (that are replacing worn-out normal cells) and cancer cells. The damage to normal cells can cause side effects. Each time chemotherapy is given, it involves trying to find a balance between destroying the cancer cells (in order to cure or control the disease) and sparing the normal cells (to lessen unwanted side effects).

Treatment schemes

There are a number of strategies in the administration of chemotherapeutic drugs used today. Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms.

Combined modality chemotherapy is the use of drugs with other cancer treatments, such as radiation therapy or surgery. Most cancers are now treated in this way.
Combination chemotherapy is a similar practice which involves treating a patient with a number of different drugs simultaneously. The drugs differ in their mechanism and side effects. The biggest advantage is minimizing the chances of resistance developing to any one agent.

In

neoadjuvant chemotherapy

(preoperative treatment) initial chemotherapy is aimed for shrinking the primary tumour, thereby rendering local therapy (surgery or radiotherapy) less destructive or more effective.

Adjuvant chemotherapy

(postoperative treatment) can be used when there is little evidence of cancer present, but there is risk of recurrence. This can help reduce chances of resistance developing if the tumour does develop. It is also useful in killing any cancerous cells which have spread to other parts of the body. This is often effective as the newly growing tumours are fast-dividing, and therefore very susceptible.

Palliative chemotherapy

is given without curative intent, but simply to decrease tumor load and increase life expectancy. For these regimens, a better toxicity profile is generally expected.

All chemotherapy regimens require that the patient be capable of undergoing the treatment. Performance status is often used as a measure to determine whether a patient can receive chemotherapy, or whether dose reduction is required.

Quality audit

Quality Audit
Quality audit is the process of systematic examination of a quality system carried out by an internal or external quality auditor or an audit team [1]. It is an important part of organization's quality management system and is a key element in the ISO quality system standard, ISO 9001.
Quality audits are typically performed at predefined time intervals and ensure that the institution has clearly-defined internal quality monitoring procedures linked to effective action. This can help determine if the organization complies with the defined quality system processes and can involve procedural or results-based assessment criteria.
With the upgrade of the ISO9000 series of standards from the 1994 to 2000 series, the focus of the audits has shifted from purely procedural adherence towards measurement of the actual effectiveness of the Quality Management System (QMS) and the results that have been achieved through the implementation of a QMS.
Audits are an essential management tool to be used for verifying objective evidence of processes, to assess how successfully processes have been implemented, for judging the effectiveness of achieving any defined target levels, to provide evidence concerning reduction and elimination of problem areas. For the benefit of the organisation, quality auditing should not only report non-conformances and corrective actions, but also highlight areas of good practice. In this way other departments may share information and amend their working practices as a result, also contributing to continual improvement.
Quality audits can be an integral part of compliance or regulatory requirements.

Definition

Periodic, independent, and documented examination and verification of activities, records, processes, and other elements of a quality system to determine their conformity with the requirements of a quality standard such as ISO 9000. Any failure in their proper implementation may be published publicly and may lead to a revocation of quality certification. Also called conformity assessment or quality system audit.

Audits can also be used for safety purposes. Evans & Parker (2008) describe auditing as one of the most powerful safety monitoring techniques and 'an effective way to avoid complacency and highlight slowly deteriorating conditions', especially when the auditing focuses not just on compliance but effectiveness. [4]

The processes and tasks that a quality audit involves can be managed using a wide variety of software and self-assessment tools. Some of these relate specifically to quality in terms of fitness for purpose and conformance to standards, while others relate to Quality costs or, more accurately, to the Cost of poor quality. In analyzing quality costs, a cost of quality audit can be applied across any organization rather than just to conventional production or assembly processes

The principle behind Quality Audit

The principles of Quality Audit, in the sense we mean it here, are based on the style of quality standards used in several formal national and international standards such as the ISO-900x international quality standards. These standards do not in themselves create quality. The logic is as follows.

Every organisation should define comprehensive procedures by which their products or services can be delivered consistently to the desired level of quality. As was discussed in the section on Quality Management, maximum quality is rarely the desired objective since it can cost too much and take too long. The average product or service provides a sensible compromise between quality and cost. There is also a legitimate market for products that are low cost and low quality.

Standards authorities do not seek to make that business judgment and enforce it upon businesses, except where certain minimum standards must be met (eg all cars must have seat belts that meet minimum safety standards, but there is no attempt to define how elegant or comfortable they are).

The principle is that each organisation should create thorough, controlled procedures for each of its processes. Those procedures should deliver the quality that is sought. The Quality Audit, therefore, only needs to ensure that procedures have been defined, controlled, communicated and used. Processes will be put in place to deal with corrective actions when deviations occur. This principle can be applied to continuous business process operations or recurring project work. It would not be normal to establish a set of quality controlled procedures for a one-off situation since the emphasis is consistency.

This principle may be applied whether or not the organisation seeks to establish or maintain an externally recognised quality certification such as ISO-900x. To achieve a certification, the procedures will be subjected to internal and external scrutiny.
Preparing for Quality Audit
Thorough procedures need to be defined, controlled, communicated and used.

Thorough Procedures should cover all aspects of work where conformity and standards are required to achieved desired quality levels. For example, one might decide to control formal program testing, but leave the preliminary testing of a prototype to the programmer's discretion.
Procedures Any recurring aspect of work could merit regulation. The style and depth of the description will vary according to needs and preferences, provided it is sufficiently clear to be followed.
Defined A major tenet is that the defined procedures are good and will lead to the desired levels of quality. Considerable thought, consultation and trialing should be applied in order to define appropriate procedures. Procedures will often also require defined forms or software tools.
Controlled As with any good quality management, the procedures should be properly controlled in terms of accessibility, version control, update authorities etc.
Communicated All participants need to know about the defined procedures - that they exist, where to find them, what they cover. Quality reviewers are likely to check that team members understand about the procedures.
Used The defined procedures should be followed. Checks will be made to ensure this is the case. A corrective action procedure will be applied to deal with shortcomings. Typically the corrective action would either be to learn the lesson for next time, or to re-work the item if it is sufficiently important.
There is no reason why these Quality Audit techniques should conflict with the project's Quality Management processes. Where project work is recurring, the aim should be for the Quality Methods and other procedures to be defined once for both purposes.
Problems may occur where the current project has significant differences from earlier ones. Quality standards may have been set in stone as part of a quality certification. In extreme situations this can lead to wholly inappropriate procedures being forced upon the team, for example, using traditional structured analysis and design in a waterfall style approach for what would be handled best using iterative prototyping. The Project Manager may need to re-negotiate quality standards with the organization’s Quality Manager.
Operating Quality Audit
A Quality Audit approach affects the entire work lifecycle:
• Pre-defined standards will impact the way the project is planned
• Quality requirements for specific work packages and deliverables will be identified in advance
• Specific procedures will be followed at all stages
• Quality Methods must be defined and followed
• Completed work and deliverables should be reviewed for compliance.
This should be seen as an underlying framework and set of rules to apply in the project's Quality Management processes.

Intellectual Property Rights

Intellectual Property Rights
Introduction

The protection of intellectual property rights in India continues to be strengthened. There is a well-established statutory, administrative and judicial framework to safeguard rights, whether they relate to patents, trademarks, copyright or industrial designs. Well-known international trademarks have been protected in India even when they were not registered in India. The Indian Trademarks Law has been extended through court decisions to service marks in addition to trade marks for goods. Computer software companies have successfully curtailed piracy through court orders. Computer databases have been protected. The courts, under the doctrine of breach of confidentiality, accorded an extensive protection of trade secrets. Right to privacy, which is not protected even in all developed countries, has been recognized in India. At Annexure 1 is a note on the steps being taken by the Government of India to strengthen and modernize the intellectual property administration system, including patent information services, trademarks registration and patent offices in India.
With specific reference to the obligations under Article 70.8 and 70.9 of the TRIPS Agreement, where India was required to provide a means for receipt of applications for product patents for pharmaceuticals and agricultural chemicals and to grant exclusive marketing rights on fulfillment of certain conditions, the Embassy would like to state the following:

1. India has established a mailbox system through administrative instructions. More than 2200 applications have been filed in this mailbox. About 600 of these applications have been filed by US companies.
2. No Exclusive Marketing Rights (EMR) application has so far been filed.
3. In respect of Article 70.8, the dispute raised by US related only to the legal security of the patent applications filed in the mail box system established through administrative instructions;
4. In respect of Article 70.9, the US argues that India should have had the necessary legislation in place for grant of EMR with effect from 1.1.95. India's stand was that the obligation to grant EMR arises only after an application for EMR is filed after fulfilling all the prescribed conditions and that, therefore, India had flexibility with regard to the timing of legislation;
5. In view of (3) and (4) above, the points raised by the US relate to only how and when India should fulfill these obligations. Even the US has not claimed that India has not accepted these obligations.

1. Comments on the Submission of Pharmaceutical Research and Manufacturers Association
PhRMA has mentioned that India should adopt a patent law which offers immediate product patent protection for pharmaceuticals in line with the highest international standards, and offer protection for all products not yet available in the Indian market. It is submitted that this is a demand that goes beyond India's obligations under the TRIPS Agreement as India is availing the full transition period therein. The ten year transition period available for providing product patents to pharmaceutical products is within WTO rules and a unilateral examination on the part of USA should not be allowed to overrule this multilateral understanding. Regarding the provision of the mailbox and EMR facility, PhRMA is aware that the issue has been taken up in the WTO and India has committed itself to implementation of the recommendations of the panel/appellate body.
Regarding the lack of intellectual property protection in the pharmaceutical sector, it may be mentioned that India does provide for patents in the pharmaceutical sector. However, in terms of Section 5 of the Patents Act, the patents are restricted to the methods of process of manufacture and not to the substances themselves. In terms of the TRIPS Agreement, India has time till January 1, 2005 to extend the product patent protection to this area. Therefore, there is no reason why the US pharmaceutical industry should have reservations on this score.
As regards the drug pricing policy of India, PhRMA has alleged that the pharmaceutical industry is unable to attract investment and the research based pharmaceutical industry is either withdrawing from India or not expanding operations. It is submitted that after the commencement of the liberalization process in India i.e. from August 1991 to November 1997 a total of 249 approvals for foreign direct investment have been obtained in the drug and pharmaceutical sector. These investment approvals envisage a total investment of Rs.6220.27 million. It may be noted that the number of approvals has doubled in 1994-96 as compared to 1991-93. In respect of the PhRMA averment that drug prices are being fixed on outdated cost data, the Department of Chemicals and Petrochemicals, Government of India has stated that the pricing is done based on actual cost data at manufacturing plants. It may be mentioned that the Government of India has not accepted the offer in 'good faith' for abolition of drug price controls. As regards the interests of Indian patients mentioned by PhRMA, under the present socio-economic conditions prevailing in India, drug price controls are required, particularly when no social security or an effective insurance system is yet in place.
PhRMA has mentioned that foreign companies experience arbitrary pricing norms of the Bureau of Industrial Costs & Prices, arbitrary local FDA decisions, high (42%) import duties and complex import procedures. It is submitted that these have no real basis and that specific cases need to be pointed out. The BICP pricing norms are based on accepted principles of costing viz. Actual cost plus a reasonable return of 14% on net worth and 22% on capital employed. 4% is added if the production is undertaken from basic stage. Further, FDA decisions are based on provisions of the Drugs and Cosmetics Act and the rules made thereunder. In fact, the US FDA has stricter norms than those prevailing in India. With regard to import duties, for pharmaceutical products, they have been brought down from 40% (plus 2%) to 30% (plus 5%) in the 1997-98 budget. These tariff rates have declined and are within India's WTO bindings. As regards import procedures, very few pharmaceutical items require import licenses. Only Penicillin and its derivatives, 6APA, Tetracycline, Oxytetracycline, Vitamin B 1, B2, Rifampicin and its intermediates and Streptomycin are placed in the negative list, requiring import licenses. Nevertheless, anomalies, if any, in the price control order and measures for free pricing are continuously under consideration of the Government of India.
PhRMA has stated that Government regulations regarding equity holdings are stringent and non-transparent. This is incorrect. In fact, the thrust of the modifications effected in Drug Policy is on attracting more investment so as to ensure the availability of quality medicines at reasonable prices in the country. The delicensing, the simplified price control mechanism and allowing higher return on investments made for basic stage manufacture would ensure a strong base for indigenous manufacture, which in turn will enhance availability. Automatic approvals to foreign equity participation up to 5 1 % and treating such companies at par with wholly Indian companies would help in bringing in newer technologies and making available newer products. Thus, modifications have made the Drug Policy attractive for domestic as well as foreign investment so as to ensure availability of quality medicines at reasonable prices.
On the question of drug price liability there is now a three-member committee, headed by a retired Judge of High Court, which looks into the Drug Price Equalization Account (DPEA) cases. The committee makes recommendations after hearing all affected parties in the matter. The Government while taking decisions in respect to DPEA considers these recommendations.
Copyright Law and Related Issues
The IIPA has been appreciative of the Copyright Act of 1957 as amended in 1994. It has observed that overall, the new law is Trade Related Aspects of Intellectual Property (TRIPS) compatible from the stand point of substantive rights, except that the term of protection for performers should be increased from 25 to 50 years. IIPA has also observed that the criminal provisions in the Act are among the toughest in the world. It, however, suggested that the level of fines should be increased. At present the minimum fine for the first offence is Rs.50, 000 and the maximum Rs. 200,000. In dollar terms this comes to 1,400 and 5,500. The amount of fines, decided in 1994, on the basis of the per capita income in India are satisfactory, even if they should appear to be low when one considers them in US dollars and in the context of the US per capita income. For making the Copyright Act TRIPS-compatible, India has time till January 1, 2000, as per Article 65.2 of the TRIPS Agreement.
IIPA has also suggested that the lack of presumptions with respect to ownership and subsistence of rights, and the need to prove actual knowledge must be remedied in amendments to the Copyright Act to make the law fully TRIPs-compatible, and bring it into conformity with the WIPO Copyright Treaty and the Performances and Phonograms Treaty.
In regard to the presumption of ownership of copyright issue, it is observed that in 1996 the Supreme Court of India, in a major ruling aimed at curbing piracy in audio and video cassettes in the country, said that ownership evidence of the original work was not required to prove the charge of copyright violation. Thus, even if there is no explicit provision in the Act, the case law makes the presumption of ownership of copyright. In fact the IIPA has also referred to the Supreme Court ruling.
Justifying suggested amendments to the Copyright Act in regard to the need to prove actual knowledge for raising/confiscating equipment for piracy, the IIPA has stated, "while raids on cable systems have been successful to date, it has been necessary to catch the pirate in the act of broadcasting a particular film without a licence in order for the police to be able to seize the pirate's broadcasting equipment." In this connection it may be mentioned that, in Section 64(l) of the Copyright Act, it has been clearly provided as under:

"Any police officer not below the rank of a sub-inspector, may, if he is satisfied that an offence under Section 63 in respect of the infringement of copyright in any work has been, is being, or is likely to be committed, seize without warrant, all copies of the work, and all plates used for the purpose of making infringing copies of the work, wherever found, and all copies and plates so seized shall, as soon as practicable, be produced before a Magistrate"
It will thus be seen that law does not require that the police witness the act of infringement in order to seize the goods involved. The law only requires that the police officer be satisfied that an offence "in respect of the infringement of copyright in any work has been, is being, or is likely to be committed" for the purpose of seizing, without warrant, all copies of the work and the plates used for making the infringing copies. This can further be adduced from the fact that he has the power to seize an infringing work even when he is satisfied that an offence is likely to be committed, thereby ruling out the essentiality of witnessing use of the goods to be seized in an act of infringement. Thus, the powers vested with the police take care of not only the past and present cases of infringement but also future cases where there is a likelihood of infringement of copyright provisions.
The WIPO Copyright Treaty, 1996 and the WIPO Performances and Phonograms Treaty, 1996 have not yet come into force as the requisite number of countries have not yet ratified or acceded to the treaties. India has also not yet acceded to the treaties. Since the two treaties have not yet come into force, it is premature to discuss the compatibility of the Copyright Act with these treaties.
The IIPA has also observed that as cable-showing of pirated products has begun to diminish, there has been an increase in unauthorized showing of US films in student unions in universities. The Copyright Act provides an exception "to the performance, in the course of the activities of an education institution, of a cinematography film,...if the audience is limited to ... staff and students, the parents and guardians of the students and persons directly connected with the activities of the institution..." (Section 52 (1)). IIPA has suggested that his provision should be interpreted narrowly to make such showings an infringement. If not, the provision must be amended and narrowed to be Berne and TRIPS-compatible. It may be observed that the Indian Copyright Act is fully compatible with the Berne and the Berne provisions included in the TRIPS. As such, no amendment is needed in the Act. However, the enforcement agencies may ensure that the exemption provisions for educational purposes are not misused for copyright infringement.
The IIPA in their submissions have also stated, "on the recording side, RIAA is concerned about an apparently over board provision in section 33 which may limit the ability of authors, and composers, as well as producers, to licence their works individually, rather than through a registered collection society." IIPA has suggested that this should be clarified in favor of full rights of all right holders to licence their rights individually. Section 33 of the Copyright Act, which provides for copyright societies, does not take away the rights of individual copyright owners. As per proviso to Section 33 "an owner of copyright shall, in his individual capacity, continue to have the right to grant licences in respect of his own works consistent with his obligations as a member of the registered. copyright society."
IIPA have also demanded a narrower interpretation of provisions relating to educational use of copyrighted work. First of all, no such instance has been brought to the notice of the Department of Education, Ministry of Human Resource Development, Government of India, which is the nodal Ministry for copyright. Further, it is quite likely that instances, if any, do not involve any commercial losses.

Modernization of Intellectual Property Administration System
(Annexure 1)
Government of India has taken several measures to streamline and strengthen the intellectual property administration systems in the country. Two projects were recently implemented with the help of WIPO/UNDP for the modernization of patent information services and trademarks registry, details of which are given below:
Modernization of Patent Information Services

I . The Office of Patent Information System (PIS) was set up in 1980 with a view to provide scientific and technological information contained in patents documents to the users, namely scientists, researchers, universities, Indian Institutes of Technology and others for taking up further research and development. It is estimated that about 80% of the world's knowledge of viable and adaptable technology are contained in about 30 million patent documents. One million documents are being added every year, covering 400,000 inventions. If the information contained in these documents is made available to industry, it can provide information for further research, facilitate evaluation of technology to be purchased under licensing arrangements, and identify worldwide developments in specific fields.
2. It was, therefore, felt necessary to establish a modernized patent information service centre through upgrading and mechanization of procedures relating to collection, retrieval and dissemination of patent information, using state-of-the-art technology and trained personnel.
3. Accordingly, a project for modernization of PIS was taken up with the financial assistance of UPDP and Government of India (at a cost of US$1.22 million of which $0.69 million provided by LTNDP) and technical assistance of World Intellectual Property Organisation (WIPO) in January, 1992. This project has been completed in June 1996.
4. As a part of activities, several awareness/training programs were organized in India for the benefit of scientific and industrial communities. Additional manpower to PIS was provided along with modem office equipment such as computers, CD-ROMS and on-line access to external and internal databases. Computerized Indian patent databases have also been established. All these activities are expected to result in better and improved services to the users.

Modernization of Trade Marks Registry
1. The use and promotion of trademarks helps inform consumers about goods and services available in the market. The use of trademarks facilitates identifying those responsible for putting those goods in the market. An effective trademarks system would help protect consumers against misleading practices. Therefore, it was recognized that in the interest of consumers, the Government of India should promote the use and provide effective protection for trademarks.
2. Accordingly, a project for modernization of trademarks registry was taken up in April 1993 at a cost of US$1.24 million, out of which US$ 0.58 million were provided by the UNDP. The project has been implemented in May 1996 with the technical assistance of WIPO.
3. Under the project, the capability of the Registry has been enhanced by modernizing and streamlining trade marks registration procedures; posts of electronic data processing personnel have been created; awareness programs organized; Registry has been strengthened in terms of modem office equipment; networking established between Head and Branch Offices, etc. It is felt that it would now be possible to issue registration certificate in a shorter period than before.
4. Further strengthening of the capabilities of the trademark registry is also being taken up in the Ninth Plan and a provision has also been incorporated in the Annual Plan 1997-98.

Modernization of Patent Offices
1. A proposal is now under consideration for taking up modernization of Patent Offices.
2. The Patent Offices, apart from grant of patent, perform other functions like public information and guidance, maintenance of public library and search room, production of journals, publication of official gazette, etc.
3. In view of the economic initiatives and liberalization measures taken by the Government since mid-1991, the number of applications for patents are expected to increase significantly. From a level of 3552 in 1991-92, it has increased to 8562 in 1996-97 and is expected to rise further in coming years. Accordingly, a proposal is under consideration for modernization of patent offices incorporating the following elements:-

• Human Resource Development
• Infrastructure Support and Strengthening
• Computerization and Modernization
• Elimination of backlog of patent applications
4. The project will be implemented in a period of 5 years. A detailed project report has already been prepared and the project has been included in Ninth Five-Year Plan and Annual Plan 1998-99.

Types of process validation

Types of process validation
Depending on when it is performed in relation to production, validation can be prospective, concurrent, retrospective or revalidation (repeated validation).

Prospective validation
Itis carried out during the development stage by means of a risk analysis of the production process, which is broken down into individual steps: these are then evaluated on the basis of past experience to determine whether they might lead to critical situations.
Where possible critical situations are identified, the risk is evaluated, the potential causes are investigated and assessed for probability and extent, the trial plans are drawn up, and the priorities set. The trials are then performed and evaluated, and an overall assessment is made. If, at the end, the results are acceptable, the process is satisfactory. Unsatisfactory processes must be modified and improved until a validation exercise proves them to be satisfactory. This form of validation is essential in order to limit the risk of errors occurring on the production scale, e.g. in the preparation of injectable products.

Concurrent validation
is carried out during normal production. This method is effective only if the development stage has resulted in a proper understanding of the fundamentals of the process. The first three production-scale batches must be monitored as comprehensively as possible.1The nature and specifications of subsequent in-process and final tests are based on the evaluation of the results of such monitoring.
1 This careful monitoring of the first three production batches is sometimes regarded as prospective validation.
Concurrent validation together with a trend analysis including stability should be carried out to an appropriate extent throughout the life of the product.

Retrospective validation
involves the examination of past experience of production on the assumption that composition, procedures, and equipment remain unchanged; such experience and the results of in-process and final control tests are then evaluated. Recorded difficulties and failures in production are analysed to determine the limits of process parameters. A trend analysis may be conducted to determine the extent to which the process parameters are within the permissible range.
Retrospective validation is obviously not a quality assurance measure in itself, and should never be applied to new processes or products. It may be considered in special circumstances only, e.g. when validation requirements are first introduced in a company. Retrospective validation may then be useful in establishing the priorities for the validation programme. If the results of a retrospective validation are positive, this indicates that the process is not in need of immediate attention and may be validated in accordance with the normal schedule. For tablets which have been compressed under individual pressure-sensitive cells, and with qualified equipment, retrospective validation is the most comprehensive test of the overall manufacturing process of this dosage form. On the other hand, it should not be applied in the manufacture of sterile products.

Revalidation
is needed to ensure that changes in the process and/or in the process environment, whether intentional or unintentional, do not adversely affect process characteristics and product quality.
Revalidation may be divided into two broad categories:
• Revalidation after any change having a bearing on product quality.
• Periodic revalidation carried out at scheduled intervals.

Revalidation after changes. Revalidation must be performed on introduction of any changes affecting a manufacturing and/or standard procedure having a bearing on the established product performance characteristics. Such changes may include those in starting material, packaging material, manufacturing processes, equipment, in-process controls, manufacturing areas, or support systems (water, steam, etc.). Every such change requested should be reviewed by a qualified validation group, which will decide whether it is significant enough to justify revalidation and, if so, its extent.
Revalidation after changes may be based on the performance of the same tests and activities as those used during the original validation, including tests on subprocesses and on the equipment concerned. Some typical changes which require revalidation include the following:
• Changes in the starting material(s). Changes in the physical properties, such as density, viscosity, particle size distribution, and crystal type and modification, of the active ingredients or excipients may affect the mechanical properties of the material; as a consequence, they may adversely affect the process or the product.
• Changes in the packaging material, e.g. replacing plastics by glass, may require changes in the packaging procedure and therefore affect product stability.
• Changes in the process, e.g. changes in mixing time, drying temperature and cooling regime, may affect subsequent process steps and product quality.
• Changes in equipment, including measuring instruments, may affect both the process and the product; repair and maintenance work, such as the replacement of major equipment components, may affect the process.
• Changes in the production area and support system, e.g. the rearrangement of manufacturing areas and/or support systems, may result in changes in the process. The repair and maintenance of support systems, such as ventilation, may change the environmental conditions and, as a consequence, revalidation/requalification may be necessary, mainly in the manufacture of sterile products.
• Unexpected changes and deviations may be observed during self-inspection or audit, or during the continuous trend analysis of process data.

Periodic revalidation. It is well known that process changes may occur gradually even if experienced operators work correctly according to established methods. Similarly, equipment wear may also cause gradual changes. Consequently, revalidation at scheduled times is advisable even if no changes have been deliberately made.
The decision to introduce periodic revalidation should be based essentially on a review of historical data, i.e. data generated during in-process and finished product testing after the latest validation, aimed at verifying that the process is under control. During the review of such historical data, any trend in the data collected should be evaluated.
In some processes, such as sterilization, additional process testing is required to complement the historical data. The degree of testing required will be apparent from the original validation.
Additionally, the following points should be checked at the time of a scheduled revalidation:
• Have any changes in master formula and methods, batch size, etc., occurred? If so, has their impact on the product been assessed?
• Have calibrations been made in accordance with the established programme and time schedule?
• Has preventive maintenance been performed in accordance with the programme and time schedule?
• Have the standard operating procedures (SOPs) been properly updated?
• Have the SOPs been implemented?
• Have the cleaning and hygiene programmes been carried out?
• Have any changes been made in the analytical control methods?

Electrophoresis

Electrophoresis

Principles of Gel Electrophoresis

Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks:

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic.

Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.

Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders).
Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.
Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.


Cross section diagram of gel apparatus design:
A. Tube
B. Vertical Slab
C. Horizontal submarine
D. Horizontal thin layer with buffer pads



Principle of Separation
• According to charge
• According to size

(1) According to charge
• When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.
Instruments and Reagents
• Buffer
• Power supply
• Supporting media
• Detection and Quantification

I. Buffer
• Function of buffer
• 1. carries the applied current
• 2. established the pH
• 3. determine the electric charge on the solute
• High ionic strength of buffer
• produce sharper band
• produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
II. Power Supply
• Function: To supply electric power
• Constant voltage (increasing of heat over time
• Heat = E x I x t
• Constant current
• according to Ohm’s law E= I x R
• R decrease over time, which inturn decrease the heat effect
E= EMF in volts (V)
I = Current in amperes (A)
t = Time in second (s)



III.Support materials used in electrophoresis
• Paper
• Starch
• Agar/agarose
• Cellulose acetate
• polyacrylamide gel
Support Media (ii)
• Agarose and polyacrylamide gels are across-linked, spongelike structure
• It is important that the support media is electrically neutral. Presence of charge group may cause:
• -Migration retardation
-The flow of water toward one or the other electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of the separation
(1) Agarose
• What is agarose?
• a chain of sugar molecules
• Extracted from seaweed
(1) Agarose gels (ii)
• For the separation of (1) large protein or protein complex (2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4% OH, O, CH2OH,

(2) Polyacrylamide gels
CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CH-
-CH2-CH-CH2-CH-CH2-CH-
CO, NH, CH2
A. Agarose gel B. Polyacrylamide gel
Polymerization of acrylamide gel can be initiated either by a chemical peroxide or by photochemical method
1. The most common method, ammonium persulphate as the initiator peroxide and TEMED as the catalyst
2. For photochemical polymerization, riboflavin and long-wave UV light are initiator and TEMED as the catalyst

Types of Detectors in HPLC

Types of Detectors in HPLC
UV/Visible Detector
The 2489 UV/Visible Detector is a versatile, dual-wavelength absorbance detector for HPLC. This detector offers the high sensitivity required for routine UV-based applications to low-level impurity identification and quantitative analysis.
The detector’s extended linear range of 2.5 AU makes for easy quantitation of both major and minor components in the same run. You can also simultaneously monitor absorbance at two user-selectable wavelengths for more information per run.
Operating as a stand-alone or as an integral component of Waters HPLC Systems, the 2489 will enhance the productivity of your chromatography laboratory. It offers a wide range of flow cell options and integrated features:
• Enhanced sensitivity via patented TaperSlit™ flow cell, which channels light through the cell for better energy throughput while minimizing RI effects
• Low noise performance (<5 µAU)
• Flexible sampling rates, from 1 to 80 Hz, for normal and fast LC separations
• Independent optimization of high-speed data rates and filter time constants allows for the accurate integration of narrow, sharp peaks
• Enhanced thermal wander management reduces baseline wander due to fluctuations caused by ambient conditions
• Accessible detector components for easy operation and maintenance

Photodiode Array (PDA) Detector
The 2998 Photodiode Array (PDA) Detector offers advanced optical detection for Waters analytical HPLC, preparative HPLC, or LC/MS system solutions. Providing unprecedented trace impurity detection and quantitation, the 2998 PDA Detector’s integrated software and optics innovations deliver high chromatographic and spectral sensitivity. Enhanced software control provides flexibility for simultaneous 2D and 3D operation with either Empower™ or MassLynx™ Software.
The 2998 PDA Detector is designed for a range of laboratory applications, including trace impurity detection and quantification, compound identification and method development.
Features:
• Patented TaperSlit™ flow cell ensures high sensitivity while maintaining optimal spectral performance
• Maximum signal-to-noise performance of <10 µAU
• Simultaneous quantification of high- and low-level components within a single chromatographic separation for extended linear range
• Flexible sampling rates for normal and fast LC separations from 1 to 80 Hz
• Definitive compound identification & co-elution detection
• Superior linear range with constant optical bandpass
• Spectral exposure optimization to maximize the signal-to-noise across the entire wavelength range with a single Deuterium lamp
• Thermal wander management for maximum baseline stability
Refractive Index (RI) Detector
The 2414 Refractive Index (RI) Detector is designed for high-performance liquid chromatography (HPLC) applications. It provides sensitivity, stability, and reproducibility for the analysis of components with limited or no UV absorption. The solution-centered design combines with Alliance® or Breeze™ HPLC Systems for RI-based HPLC and GPC applications.
The 2414 RI Detector provides solutions for preparative and analytical applications including alcohol, sugar, saccharide, fatty acid, and polymer analysis.
Features:
• Low dispersion volume for high-speed GPC and narrowbore column chromatography from 0.1 to 10 mL/min
• Thermally isolated optics bench for temperature stability and performance reproducibility
• Save solvent and maintain stability with convenient recycle valve
• Diagnostics and simplified design and control options for easy maintenance and everyday use
• Circuitry and software in the detector’s thermally isolated optics bench provide a sensitive, stable signal response to changes in refractive index independent of ambient temperature changes
• Auto-zero, auto-purge, and LED-displayed diagnostic programming enable unattended operation and to simplify methods development
Electrochemical Detector
The 2465 Electrochemical Detector combines sensitivity, reliability and simplicity for HPLC electrochemical detection. Its versatile detection modes are ideally suited for analyzing a wide range of compounds.
The 2465 Electrochemical Detector’s multiple flow-cell configuration combines working and reference electrode designs to minimize cost and enhance flexibility. The flow cell accommodates several reference electrodes: ISAAC; traditional salt-bridge design, which limits air bubble formation; and Hy-Ref, which is ideal for carbohydrate analysis and is compatible with extreme mobile phases.
The detector is designed for ultra-trace analysis in standard and microbore Alliance® HPLC based, Empower™ Software controlled applications.
Features:
• Multiple detection modes, including direct current, pulsed amperometric, and scanning
• Easier maintenance with front-mounted flow cell for simple cleaning and assembly
• Stabilized temperature for better accuracy with the integral flow cell and column heater compartment
• Faraday-shielded oven with thermal operating stability and reduced noise and drift characteristics
• 21 CFR and Part 11 compliant-ready
Conductivity Detector
The 432 Conductivity Detector provides solutions for chemically-suppressed or single-column ion chromatography. Use it as a stand-alone module or configure it into an HPLC system that links to chromatography data workstations
• High sensitivity and stability due to its multi-electrode flow cell and user-programmable temperature control, which automatically adjusts the oven temperature to minimize baseline drift
• Reduced flow path volume for narrow-bore applications
• Increased operational uptime with self-diagnostics
• Easy, unattended routine analyses with autozero and autoranging capabilities
• Sophisticated circuitry efficiently isolates detection electrodes from sources of transient electronic noise for optimal sensitivity
• Autozero feature automatically sets the baseline to zero at the start of each run eliminating the need for manual adjustments
• Autorange ensures a precise conductivity base range
• Leak sensors alerts user if leak is detected in flow cell
Fluorescence Detector
The 2475 Multi-Wavelength Fluorescence Detector is a multi-channel, tunable, fluorescence detector designed for HPLC applications. The detector delivers the highest sensitivity and selectivity, whether you are monitoring low concentrations of target compounds or low-level impurities.
Use the detector as a stand-alone unit or as an integral part of a Waters chromatography system. It operates from 200 to 900 nm and uses newly designed optics, with an enhanced illumination system, for improved performance. These design features increase the optical throughput and sensitivity resulting in an overall increase in the signal-to-noise ratio.
Features:
• Innovative flow cell designed for less dispersion, less stray light, less volume, and more pathlength for maximum sensitivity
• Three-dimensional spectral scanning for faster method development and optimization, and enhanced peak identification
• Single or multi-channel operation monitors fluorescence at one or more discrete wavelength pairs
• Axially illuminated flow cell – Allows for better light absorption resulting in highest sensitivity.
• Advanced optical design - Maximizes light throughput, and reduces light scatter, allowing for better signal-to-noise performance.
• Low noise performance - Less stray light due to orthogonal optics and the use of mirrors, not lenses, minimize scatter.
• Multiple detection modes - 2D, 3D, and on-the-fly spectral scanning – can greatly reduce the time needed to develop fluorescence detection methods by quickly determining wavelength maxima.
• Integral erbium calibration reference – Ensures wavelength accuracy.

Agarose Electrophoresis

Agarose Electrophoresis

Agarose elctrophoresis is performed to visualize your PCR products. This step allows
you to determine whether your PCR was successful, whether the resulting product is the correct size, whether other products were amplified as well, and whether the
concentration of the resulting product is suitable for cycle sequencing. All gel work
should be performed in the HIGH DNA area.

Precautions
1. Always wear gloves! This is essential to limit the spread of PCR products around
the lab as well as protecting against exposure to Ethidium Bromide.
2. Treat Ethidium Bromide with respect (it is a powerful mutagen) and ensure that it
does not get spread around the lab.
3. Wash hands when finished.

Materials Equipment
1. Agarose 1. Balance
2. 0.5X TBE 2. Erlenmeyer flask
3. weighing paper 3. Microwave
4. Gel box with comb

Step 1: Mixing Gel
1. On the scale, weigh 1 gram of agarose onto a piece of weighing paper.
2. Add agarose to Erlenmeyer flask.
3. Add 100mL of 0.5X TBE to Erlenmeyer flask.
4. Swirl vigorously to thoroughly mix agarose.
Slurry will by opaque.

Step 2: Melting Gel
1. Put agarose and 0.5x TBE slurry into microwave.
2. Heat on HIGH for 30 seconds at a time.
3. After 30 seconds, remove from microwave and swirl. Becareful as the mixtue will be HOT.
4. After 1 minute of heating, repeat heating and swirling procedure every 10 seconds until mixture is clear.
5. If the mixture begins to boil, stop and IMMEDIATELY remove the agarose. Swirl until
mixture is clear.


Step 3: Pouring the Gel
1. Before pouring, gel must be cool enough for you to hold the Erlenmeyer flask in your hand. Pouring boiling hot gel will warp the gel boxes.
2. Place gel tray into casting chamber.
3. Add casting comb(s) into the appropriate slot(s).
4. Pour agarose into gel tray to about 5-7mm.
5. Let sit for at least 30 min, until gel is cool to touch and is opaque in appearance.
6. Once set, place gel and tray into gel rig, with wells on the left (cathode) side. Then, fill gel rig with 0.5X TBE sufficient to cover the entire gel.
7. Carefully remove combs by pulling them upwardsfirmly and smoothly in a continuous motion. The remaining depressions are the wells into which your samples will be loaded.

Step 4: Loading the Gel
1. Cut a piece of parafilm and place it flat on the bench top. You may have to rub or scratch it to stick it down.
2. Using a pipette, place small dots of 6X loading dye (about 1-2uL) onto parafilm, in rows of 8, 1 dot for each PCR sample that will loaded on the gel. It is not necessary to be exact, and it is not necessary to change the tip.
3. Using a pipette take 3 μl of product and pipette it onto its corresponding dye dot, then mix the sample and dye by pipetting up and down. Then, pipette up the dye/sample solution and pipette it into the proper well.
4. After loading the first sample into the well, wash out your pipette tip by pipetting up and down several times in the buffer, then blot tip dry with a Kimwipe. In this way, all of your samples may be loaded using ONE tip.
5. Once all samples have been loaded into the gel, using a fresh tip, pipette 3uL of
the DNA ladder into well number 1 of each row of wells.
6. Put gel box cover into place (this step is essential for your gel to run and to minimize the risk of electric shock). Turn on power supply. Run the gel for 75
minutes at 80 volts.
7. Check for bubbles at the cathode side to ensure that gel rig is running. Clean up, deglove, and wash hands.


Step 5: Visualizing the Gel
1. Turn off power supply. Using gloved hands, remove the cover from the gel box.
2. Remove the gel and casting tray from rig, avoiding dripping buffer all over the
benchtop.
3. Carefully slide the gel off of the casting tray and into ethidium bromide solution.
Caution: ethidium bromide is a mutagen and should be treated with respect. If you
get ethidium bromide on you gloves, immediately change your gloves, placing the
contaminated gloves into the ethidium bromide trash. Do not spread ethidium
bromide around the lab.
4. Soad the gel in the ethidium bromide solution for 10-15 minutes to stain. Using a
spatula, remove the gel from ethidium bromide and rinse for 30 seconds in a tray
0.5x buffer. The gel may be left in the 0.5X buffer for longer to “destain” the gel
and improve contrast of stain, but this step is often unnecessary.
5. Using the spatula, carefully remove the gel from the destaining buffer and place
the gel on UV light box, avoiding creating bubbles underneath the gel.
6. Put on UV protecting face shield and turn on UV light box to see stained sample
bands and DNA ladder. Ensure that all around you are protected for UV exposure.
7. If bands are strong, take a picture using the Polaroid camera (below). If bands are
weak, return gel to ethidium bromide stain bath as the gel has been insufficiently
stained.
Step 6: Taking a picture. Remember that the gel has been soaking in Ethidium
Bromide. All objects and surfaces that the gel comes into contact with will be
contaminated with Ethidium Bromide. Always wear gloves and limit the spread of
Ethidium Bromide to protect yourself and your lab mates.
1. Ensure that you are still wearing your gloves and UV face shield.
2. With the gel flat on the UV light box, position the hood of Polaroid camera over
the gel so that the hood is centered over the gel.
3. Turn on the UV light box.
4. Keeping the camera steady, pull the trigger and HOLD the trigger until there is
silence (i.e. the shutter has opened and closed. This may take 0.5-2.0 seconds
depending on the cameras exposure settings).
5. Turn off the UV light box.
6. Pull WHITE film tab on camera to remove film. Wait 1 minute
7. After one minute has passed, pull backing off of film to check whether the gel
imaged properly.
8. Once picture is developed and you have a good picture of your gel, remove your
gel and put in gel disposal box.
9. Wipe down the UV light box with Kimwipes and dispose of in gel disposal box.
Remember that all of these surfaces and wipes are contaminated with Ethidium
Bromide.
10. Remove your gloves and dispose of in the Ethidium Bromide trash.
11. Wash hands thoroughly.
12. Label your picture.