Incorporating Validation Concepts into the Biotechnology Curriculum or minding your P s and Q s

1. Incorporating Validation Concepts into the Biotechnology Curriculum (or minding your P�s and Q�s) Thomas Burkett, Ph.D. The Community College of Baltimore County tburkett@ccbcmd.edu

2. Aims Introduce the role of validation in biomanufacturing Explain basic validation concepts and terminology Provide an example of how validation is incorporated into the biotech. curriculum at CCBC Provide examples of validation exercises Provide examples of validation resources

3. Validation in Biomanufacturing Biomanufacturing is a complex process involving multiple unit operations many of which are critical to insuring patient safety and product efficacy

4. The block flow is a simple representation of the production process illustrating �unit operations� with simple blocks. Major divisions in the production process, i.e. up-stream, downstream, viral and non-viral are separated with dashed lines. Each of these unit operations may contain numerous pieces of equipment, and proceedures that will require validation. In addition, the unit operation will require validation that the material coming out of that unit operation meets predetermined specifications. The block flow is a simple representation of the production process illustrating �unit operations� with simple blocks. Major divisions in the production process, i.e. up-stream, downstream, viral and non-viral are separated with dashed lines. Each of these unit operations may contain numerous pieces of equipment, and proceedures that will require validation. In addition, the unit operation will require validation that the material coming out of that unit operation meets predetermined specifications.

5. Mammalian Antibody Production � Cell Culture

6. Mammalian Process Flow- Upstream Diagram

7. Mammalian Antibody Production - Downstream Processing

8. Validation in Biomanufacturing A central concept in quality is that quality can not be tested for. Quality must be designed and built into the production process. Requires careful attention to raw material specifications, in process material specifications, and final product specifications.

9. Validation in Biomanufacturing Validating the performance of unit operations, analytical methods, and critical process points (sterilization, viral inactivation, cleaning procedures) is essential in insuring that the process generates a quality product.

10. Validation in Biomanufacturing Validation does not replace testing, but it does reduce the testing burden for raw materials, in-process materials, and final product

11. Validation in Biomanufacturing Validation itself is a process that evolves with the product. Validation requirements for production of pre-clinical material much less stringent then for phase III clinical material. Critical operations: raw materials, analytical methods, viral clearance, sterilization, cleaning.

12. Validation in Biomanufacturing A fully validated process is �locked in� Any change outside of the validated space invalidates process Change must be evaluated for effect on patient safety and product efficacy

13. Process Flow Diagram: More detailed then the block flow diagram, the process flow diagram will include equipment identification and process parameters (flow rates, capacities) at specified points in the production process. Process components, including media, water, gases, and product are tabulated at the bottom of the diagram to indicate material flows. To give you some idea of the validation requirments, just in the first unit operation (innoculum expansion) the BSC, and incubators will have to be validated, the cleaning protocol for the spinner flasks and culture flasks, the methods used for cell counting, cell spliting, etc. Process Flow Diagram: More detailed then the block flow diagram, the process flow diagram will include equipment identification and process parameters (flow rates, capacities) at specified points in the production process. Process components, including media, water, gases, and product are tabulated at the bottom of the diagram to indicate material flows. To give you some idea of the validation requirments, just in the first unit operation (innoculum expansion) the BSC, and incubators will have to be validated, the cleaning protocol for the spinner flasks and culture flasks, the methods used for cell counting, cell spliting, etc.

14. Regulatory requirement for validation 21 CFR 211 Subpart F �Production and Process Controls 211.100 �Written procedures; deviations (a) Requires written procedures for production and process control designed to assure that products possess the quality attributes that they purport or are represented to possess. (b) Requires that any deviations from written production and process control procedures be recorded and justified. 211.101 � Change in of components 211.103 � Calculation of yield 211.105 � Equipment identification 211.110 � Sampling and testing of in-process materials and drug products �Requires that control procedures be established to monitor the output and validate the performance of those manufacturing processes that may be responsible for causing variability of in process material and drug product.� 211.111 � Time limit on production 211.113 � Control of microbiological contamination �Requires that sterilization processes be validated� 211.115 � Reprocessing 21 CFR 211 Subpart H- Holding and Distribution 211.165 � Testing and release for distribution �Requires that the accuracy, sensitivity, specificity, and reproducibility of test methods employed by the firm shall be established and documented. Such validation and documentation may be accomplished in accordance with 21 CFR 211.194 (a)(2)� 21 CFR 211 Subpart I- Laboratory Controls 21 CFR 211 Subpart J � Record and Reports 21 CFR 820 Quality Systems Regulations

15. Regulatory requirement for validation Sec. 211.113 Control of microbiological contamination. (a) Appropriate written procedures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed. (b) Appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed. Such procedures shall include validation of any sterilization process.

16. What does �validation of any sterilization process� mean ? What parameters are critical to sterilization? Temperatures, pressures, time, pore size (filtration), radiation dosage, chemical concentration. Must demonstrate that your autoclave reaches the temperatures, pressures, and times necessary for sterilization. Must demonstrate that items representing real world samples achieve those conditions ( 20 ft of 1 � hose; a 20 L carboy; a 500 ml bottle). Must challenge with worse case scenario (may take place in pilot plant if scalability demonstrated).

17. FDA definition of validation �Validation is a process of demonstrating, through documented evidence, that a process, procedure, method, piece of equipment, or facility will consistently produce a product or result that meets predetermined specifications and quality attributes.�

18. Regulatory guidance on validation Guideline on General Principals of Process Validation http://www.fda.gov/cder/guidance/pv.htm Guidance for Industry: For the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products. CDER CVM November 1994. www.fda.gov/CDER/GUIDANCE/cmc2.pdf Working Party on Control of Medicines and Inspections Final Version of Annex 15 to the EU Guide to Good Manufacturing Practice Title: Qualification and validation http://pharmacos.eudra.org/F2/eudralex/vol-4/pdfs-en/v4an15.pdf ICH Q7a Section 12 on validation http://www.fda.gov/cder/meeting/ICH_Q7A/index.htm A WHO guide to good manufacturing practice (GMP) requirements. Part 2: Validation Chaloner-Larsson, G., Anderson, R., and Egan, A. 1997. World Health Organization, Geneva.

19. Critical Operations in Biomanufacturing Some operations are more critical than others. Viral filtration, sterilization, cleaning, analytical methods. These operations will require greater validation efforts then less critical operations (media blending).

20. Validation in the biotech. curriculum at CCBC Validation introduced as part of �quality systems� section in intro. Course First lecture is on �concepts of quality� and �quality attributes Second lecture introduces validation as part of the production process Lab exercises varies. Past examples include validation protocol for an autoclave; validation of bioreactor sterilization.

21. Learning Objectives Upon completion of this module students should: Be familiar with the various government and third party literature pertaining to validation. Understand how component, process, and methods validation fits into the overall quality system. Be aware of pertinent regulations that apply to validation strategies. Understand concept of criticality and be able to identify points in the production process that are critical to product quality. Be able to distinguish between installation qualification, operation qualification, and performance qualification (IQ, OQ, PQ). Given the function of a piece of equipment used in biomanufacturing, discuss valtidation issues related to that specific piece of equipment. Be aware of the vendor, installation, and maintenance documentation required for initiating the validation process. Follow a validation SOP Be able to design a validation protocol for an individual piece of equipment.

22. Concepts of Quality Biomanufacturing

23. Quality Attributes Identity 21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each component of a drug product. Chemical, biological, Immunological Raw materials, In-process intermediates, final products. Safety 21 CFR 600.3 (p) safety as the relative freedom from harmful effect to persons affected, directly or indirectly, by a product when prudently administered, taking into consideration the character of the product in relationship to the condition of the recipient at the time. Activity of active ingredients Activity of the excipients or additives Activity of process related impurities Efficacy Effectiveness of the product in achieving its medicinal purpose (therapeutic, prophylactic, diagnostic). Gathered at phase II and Phase III trials. Potency 21 CFR 600.3 (s) specific ability or capacity of the product, as indicated by its appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner indicated to effect the given result. Purity 21 CFR 600.3 (r) relative freedom from extraneous matters in the finished product, whether or not harmful to the recipient or deleterious to the product. Cleaning Procedures Stability 21 CFR 211.137 (a) to assure that a drug product meets applicable standards of identity, quality, and purity at the time of use; it shall bear an expiration date determined by stability testing. Drugs may use accelerated time studies, biologics must use real time studies. Consistency The ability of the product and/or process to reliably possess specified quality attributes on an ongoing basis. 3 consecutive batches of product meeting predetermined specifications is accepted as proof that a process is consistent. However, in NDA data from up to twenty batches may be submitted.

24. Designing Quality into the product A central concept is that quality can not be tested for! Testing programs are based on testing a statistically significant number of samples However to be absolutely sure that all of your product meets specifications you would have to test everything. Testing by itself will not insure quality and is inefficient Testing is required under the GMP�s Raw materials In-process samples Final Product Quality (identity, safety, efficacy, potency, purity, stability, consistency) must be designed into the production process Begins with predetermined specifications Raw material specifications In-process material specifications Final Product Specifications

25. Predetermined specifications Identity: Size, amino acid sequence, presence of post translational modifications, 3-D structure.

26. Predetermined specifications

27. Testing For Identity Requires the development of validated analytical methods that can determine identity. Chemical Tests: Is the molecule chemically what it is supposed to be? Biological Activity Tests: Does the molecules have the biologic activity that it is supposed to have? Immunogenic Tests: Is the molecule immunogenic (allergic)?

28. Identity 21 CFR requires testing of raw materials: Raw materials quarantined until identity verified Raw materials must meet predetermined specifications Vendors (and alternates) specified in BLA (NDA)

29. Identity 21 CFR requires testing of in-process materials: Product from bioreactor / fermentor Product from purification steps Waste products from above Must meet specifications, if not - stop the process to investigate take corrective action

30. Testing Usually done by the Quality Control Laboratory CFR requires that quality unit be under independent supervision and report directly to senior management

31. Quality Assurance Reviews records from quality control and production departments Verifies that all specifications and production operations met / performed Investigations necessary for any deviations Root cause Affect on quality Corrective action (CAPA) Approves final release of product

32. Designing Quality into the Product Design of production process and specifications all contribute to a quality product: Absence of contamination Clean rooms, closed systems, use of BSC for critical operations. Purity Separation process (chromatography) designed to remove potential contaminants Viral purification / inactivation

33. Insuring the Production of a Quality Product - II Validation & its role in quality

34. What is Validation Validation � An Essential Part of GMPs! Validation is the scientific study of a system To prove that the facility/system/equipment/method is consistently doing what it is supposed to do (i.e., that the process is under control). We want to make decisions based on good science and not hunches and assumptions! To determine the process variables and acceptable limits for these variables, and to set-up appropriate in-process controls. Is it ok if the wash from a chromatography column is pH 6.8 vs. 7.0 ?

35. Validation The FDA�s definition of validation: �Validation is a process of demonstrating, through documented evidence, that a process, procedure, method, piece of equipment, or facility will consistently produce a product or result that meets predetermined specifications and quality attributes.�

36. Quality Attributes Remember these? Identity 21 CFR 211.84 (d) at least one test shall be conducted to verify the identity of each component of a drug product. Chemical, biological, Immunological Raw materials, In-process intermediates, final products. Safety 21 CFR 600.3 (p) safety as the relative freedom from harmful effect to persons affected, directly or indirectly, by a product when prudently administered, taking into consideration the character of the product in relationship to the condition of the recipient at the time. Activity of active ingredients Activity of the excipients or additives Activity of process related impurities Efficacy Effectiveness of the product in achieving its medicinal purpose (therapeutic, prophylactic, diagnostic). Gathered at phase II and Phase III trials. Potency 21 CFR 600.3 (s) specific ability or capacity of the product, as indicated by its appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner indicated to effect the given result. Purity 21 CFR 600.3 (r) relative freedom from extraneous matters in the finished product, whether or not harmful to the recipient or deleterious to the product. Cleaning Procedures Stability 21 CFR 211.137 (a) to assure that a drug product meets applicable standards of identity, quality, and purity at the time of use; it shall bear an expiration date determined by stability testing. Drugs may use accelerated time studies, biologics must use real time studies. Consistency The ability of the product and/or process to reliably possess specified quality attributes on an ongoing basis. 3 consecutive batches of product meeting predetermined specifications is accepted as proof that a process is consistent. However, in NDA data from up to twenty batches may be submitted.

37. Historical Basis for Validation Assumptions concerning virus inactivation resulted in ten deaths and 200 children becoming paralyzed, from a supposedly �inactivated� polio vaccine. Assumptions about sterilization caused severe infections among burn victims given supposedly sterile solutions. Validation eliminates assumptions and relies on experimental proof!

38. Validation Plan Organizations must define an approach towards validation What is to be validated How is it to be validated Who is to validate it Who is to approve the validation When it must be revalidated

39. Validation Plan Regulatory agencies (FDA, EMEA, WHO, etc) identify minimum components of validation. �Industry standards� (the c in cGMP) can increase validation requirements. New & Novel processes / equipment require greater scrutiny then established processes / equipment. Validation requirements increase as a product moves through development (phase I, phase II, phase III).

40. Validation Plans The Validation Master Plan A high level document that outlines the organizations philosophical approach to validation and revalidation. The master validation plan becomes a guideline by which individual validation protocol are developed and implemented. May contain a flow chart or other diagram of the validation process

41. Validation Protocol Specific protocols (SOP�s) that provide detailed information on what is to be validated. Validation Protocols consist of: A description of the process, equipment, or method to be validated. A description of the validation method. A description of the sampling procedure including the kind and number of samples. Acceptance criteria for test results. Schedule or criteria for revalidation.

43. Validation Protocol Validation Protocols may consist of multiple SOP�s each describing specific steps in the validation process

44. Validation Examples of individual systems subject to validation: HVAC systems Autoclaves pH meters Depyrogenation Ovens Lyopholyzers Centrifuges Steam generators Water systems Compressed air systems Vacuum systems

45. Critical Systems How critical is the system being validated to final product quality? Media blending systems for cell growth vs. final fill & finish operations Demonstrating that the device which fills, labels, and caps the final product will require more extensive validation then the blenders used to prepare media for bioreactors. Validation of complex devices can take years!

46. Validation Proceeds in stages with new facilities / equipment. Planning for validation should start with the design process. Leaving validation to the last minute is asking for trouble.

47. Stages of Validation Starts with Design & Receipt: Does the equipment meet the needs (is the autoclave big enough?) Do you have the manuals, spare parts, can you plug it in? Is it installed properly (drain lines, vents, etc) Does it work? Does the autoclave reach the necessary temp. and pressure? Can the autoclave sterilize your equipment (worse case situation)? How does it work in the manufacturing process? Can it handle production quantities? Will failure compromise product quality?

48. IQ, OQ, PQ ? Installation Qualification (IQ) A process used to document that the piece of equipment was supplied and installed properly and that appropriate utilities, i.e., electrical, steam, gas, etc. are available to operate the equipment according to the manufacturers specifications. Operational Qualification (OQ) A process designed to supply the documented evidence that a piece of equipment operates as it is intended through all anticipated operational ranges. Performance (Process) Qualification (PQ) Verifies that a process / piece of equipment performs as it is intended to in the manufacturing process and produces product (in process or final) meeting predetermined specifications.

50. Typical information in an IQ protocol Name and description of equipment, including model numbers Identification, including model and serial numbers Location of the equipment Any utility requirements, i.e. electrical voltage, steam or water pressure, etc. Any safety features of the equipment, including alarms, interlocks, or relief valves. That all documentation, including manufacturers contact information, spare parts inventory, operational manual, and installation drawings are available on site.

51. OQ Protocol

52. OQ Protocol

53. Typical OQ Protocol Components Objective Responsibility Equipment required (Calibration verification & Traceability) SOP(s) used Equipment Identification Parameters measured (Specifications) Documentation

54. Validation Ideally validation takes place prior to actual production runs, however in some cases validation may take place as product is produced, or past production runs may be used to provide validation data. Prospective Validation Concurrent Validation Retrospective Validation

55. A prospective validation study

56. A concurent / retrospective validation study

57. 57 The V-Model

58. Project Plan Agreed by team members Details phases, activities, and milestones Gantt Chart most commonly used Planning for Validation

59. 59 Putting it all together Compliance pyramid is from Pharm Eng, Magazine, March, April 2000, Page 56Compliance pyramid is from Pharm Eng, Magazine, March, April 2000, Page 56

60. Revalidation Is the initial validation of a piece of equipment the end? No! Periodic revalidation may be necessary depending on the criticality of the equipment Changes need to be evaluated for their impact on validation Deviations from specifications may require revalidation Revalidation spelled out in Master Validation Plan

61. Change Control Must assess impact of changes on FDA compliance and validation state. Change control is a formal process defined in company SOP on how process/equipment changes are evaluated. Any change that takes place outside the change control process can jeopardize product quality (patient safety).

62. An example of a facility / process validation Remicade� (infliximab) is a chimeric mAb* directed against TNF-a. Approved in 1998 (US) and 1999 (EU) to treat Crohn�s disease, and RA. Produced by Centocor, Inc. in Malvern, PA * Contains mouse variable domains and human constant domains (IgG1)

63. Antibodies Proteins 2 heavy Chains 2 Light Chains Disulfide Bonds Variable region Recognizes antigen Constant region Effecter function Classes & subclasses

64. Production of Remicade� BLA approved in August 1998 (FDA), 1999 (EMEA). First site for bulk manufacture was Leiden, The Netherlands. Process was transferred to Malvern, PA in April 2002*. Process changes, including larger bioreactors, external spin filters, and a change in media components were introduced to meet increased demand. Not only did a new facility have to be validated, but also the changes to the manufacturing process had to be validated. Necessary to demonstrate that product produced under these new conditions had same quality attributes as product produced in Leiden. An unanticipated consequence of increased product yield was a change in chromatography conditions due to product breakthrough under old conditions. Minor changes can have unanticipated consequences on product quality! A new facility for production of remicade is being constructed in the Republic of Ireland and should be on line in 2007

65. Changes in Production Process in Malvern, PA

67. Remicade Production

69. Some Questions A valve used to transfer material from a holding tank to the purification suite jam�s closed. You have a spare valve that is an identical model. Can you change this valve with the spare and continue operations? What if the valve is from a different manufacturer? You notice that your autoclave loading plan leaves room for additional material. Realizing that increasing that amount of material in the autoclave will shorten the turn around time for the production line you contemplate increasing the amount of material loaded into the autoclave then specified by the loading plan. What should you do? What will be required to implement this change? An SOP for calibration of a pH meter calls for a two point calibration at pH 4 and pH 7. You notice that a single point calibration at pH 7 produces the same result from pH measurements of your buffer solutions and allows you to take a longer break. Is it Ok to do the one point calibration when the SOP calls for a two point calibration? How would you go about changing the SOP to allow for a one point calibration?

70. What documents would provide information concerning the make and model of a particular valve used to regulate the transfer of material from a holding tank to the purification suite? Your supervisor is concerned that the fermentation vessel is not providing sufficient aeration of the culture to get optimal growth and suggests installing a different kind of baffle in the vessel. How would you demonstrate that this change has no effect on product quality?

71. References Pharmaceutical Manufacturers Association�s (Pharmaceutical Research and Manufacturers of America) Validation Advisory Committee �Process Validation Concepts for Drug Products� Pharmaceutical Technology, September 1985 p 82. Bismuth, G. Cleaning Validation: A Practical Approach. CRC Press, 2000. ISBN 1574911082. Pharmaceutical Process Validation, 3rd Ed. Edited by Robert Nash and Alfred Wachter, Marcel Decker, 2003. ISBN 082470838-5 Validation of Pharmaceutical Processes: Sterile Products. 1998. 2nd Edition. Edited by Frederick J. Carlton and James Agalloco. Marcel Decker, 1998. ISBN 0824793846. Validation Standard Operating Procedures: A step by Step Guide for Achieving Compliance in the Pharmaceutical, Medical Device, and Biotech Industries, Syed Imtiaz Haider, St. Lucie Press, 2002. ISBN 1574443313. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total Quality Control From Manufacturer to Consumer, Sidney J. Willig. Marcel Decker, 2000. ISBN 0824704258. Voss, J. Cleaning and Cleaning Validation: A Biotechnology Perspective. CRC Press, 1995. ISBN 0939459507. LeBlanc, D.A. 2000. Validated Cleaning Technologies for Pharmaceutical Manufacturing. CRC Press. ISBN 1574911163. Cloud, P. 1998. Pharmaceutical Equipment Validation: The Ultimate Qualification Guidebook. CRC Press. ISBN 1574910795. Juran, Quality Control Handbook, 4th Edition., McGraw-Hill, 1988. DeSain C, Sutton C. (1995). Process development that supports process validation. Pharmaceutical Technology 19 (Oct.): 130-136, 1995. Garcia T, Wilkinson S, Scott J. The development of a blend-sampling technique to assess the uniformity of a powder mixture. Drug Development and Industrial Pharmacy 27(4): 297-307, 2001.� Chaloner-Larsson, G., Anderson, R., Egan, A. 1997. A WHO guide to good manufacturing practice (GMP) requirements Part 2: Validation . World Health Organization, Geneva. www.who.int/vaccines-documents/DocsPDF/www9666.pdf Accessed on October 2nd, 2006. Brown, F. 1993. Review of accidents caused by incomplete inactivation of viruses. Dev. Biol. Stand. 81: 103-7 Nathanson, N. and Langmuir, A.D. 1995. The Cutter incident. Poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the United States during the Spring of 1955. II. Relationship of poliomyelitis to Cutter vaccine. 1963. Am. J. Epidemiol. 142:109-40. 

72. Laboratory Activities Students develop and carry out a simplified validation plan / protocol Autoclave validation Bioreactor sterilization Bioreactor cleaning Spectroscopy Chromatography Plasmid construct