125+ Pharmaceutical Interview Questions and Answers for Sterile Formulations

This page covers most of the interview questions and answers during a technical round in Production of Sterile Formulations. The interview questions cover questions from basic to advance level of technical aspects. These interview questions and answers will help to crack an interview, enhance your knowledge, and also be helpful for the interviewer who is involved in the recruitment process.

The topics covered here are sterilization process, aseptic processing, media fill, area classification, and associated topics. In addition, the interview questions and answers cover various equipment used for the manufacturing process of sterile formulations.

You will find it much more enjoyable while going through these interview questions and answers. So enjoy learning, and best of luck with your interview! Happy Learning.

1. What is sterilization:

A suitably designed, validated and controlled process that inactivates or removes viable microorganisms in a product until sterility is obtained.

2. What is Sterility:

Sterility is the absence of viable microorganisms, as defined by a sterility assurance level equal to or less than 10⁻⁶. The inactivation of microorganisms by physical or chemical means follows an exponential law; thus there is always a finite statistical probability that a micro-organism may survive the sterilizing process. For a given process, the probability of survival is determined by the number, types and resistance of the microorganisms present and by the environment in which the organisms exist during treatment.

3. What is Aseptic processing?

A process performed maintaining the sterility of a product that is assembled from components, each of which has been sterilised by steam, dry heat, ionizing radiation, gas or sterile filtration. This is achieved by using conditions and facilities designed to prevent microbiological contaminants.

4. Bioburden:

The total number of micro-organisms associated with a specific item prior to any sterilisation or bioburden reduction step.

5. Biological indicator:

Biological indicators are test systems containing viable microorganisms (usually spores of bacteria) that provide a defined challenge to verify the required effectiveness of a specified sterilisation process.

6. Colony Forming Unit (CFU):

A microbiological term that describes the formation of a single macroscopic colony after the introduction of one or more micro-organisms to microbiological growth media. One colony forming unit is expressed as 1 CFU.

7. Depyrogenation

A process used to destroy or remove pyrogens (e.g. endotoxins).

8. D-value (decimal reduction value)

The value of a parameter of sterilisation (duration or absorbed dose) required to reduce the number of viable organisms to 10 per cent of the original number. It is only of significance under precisely defined experimental conditions. D121 is the D-value of the relevant spores at 121° C.

9. F₀ value

The F0 value of a saturated steam sterilization process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the load in its container with reference to micro-organisms possessing a theoretical Z-value of 10.

10. Holding time

The time between two process steps.

11. Lethal (process)

A process that kills the microorganisms exponentially.

12. Overkill sterilization

A process with a lethality of F_0BIO> 12 minutes. For example a process that provides at least a 12 log reduction of biological indicator microorganisms having a minimum D value of 1 minute.

13. Ph. Eur. sterilization reference conditions

The reference conditions for sterilisation specified in Ph. Eur. 5.1.1, i.e. terminal steam sterilization at ≥121 °C for 15 min, terminal dry heat sterilisation at ≥160 °C for ≥2 h or terminal ionising radiation of 25 kGy.

14. Post-aseptic processing terminal heat treatment

A terminal moist heat process employed after aseptic processing which has been demonstrated to provide a SAL ≤10^-6, but where the requirements of steam sterilisation (for example, F0≥8 min) are not fulfilled.

15. SAL (Sterility Assurance Level)

The SAL for a given sterilisation process is expressed as the probability of micro-organisms surviving in a product item after exposure to the process. An SAL of 10-6, for example, denotes a probability of not more than 1 non-sterile item in 1 × 10⁶ sterilised items of the final product.

16. TAMC (Total aerobic microbial count)

The total aerobic microbial count (TAMC) is considered to be equal to the number of CFU found using casein soya bean digest agar.

17. z-value

The z-value is the change in temperature required to alter the D-value by a factor of 10.

Reference for Q 1 to 17:: 6 March 2019 EMA/CHMP/CVMP/QWP/850374/2015, Committee for Medicinal Products for Human use (CHMP), Committee for Medicinal Products for Veterinary use (CVMP), Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container

18. Difference Between F0 and Fh Values

At this point of time, it’ll be better to differentiate the two i.e. F0 and Fh values.

F0 Value	Fh Value
Used to evaluate the effectiveness of Steam Sterilization	Used to evaluate the effectiveness of Dry Heat Sterilization
The assumed z-value is 10°C for sterilization range of 100 to 130°C	The assumed z-value is 20°C for sterilization range of 160 to 200°C
Theoretical Requirement121.1°C @ 30 min. of sterile hold time	Theoretical Requirement170°C @ 32 min. of sterile hold time
Targetted to mitigate micro-organisms especially living endospores	Targetted to remove the bacterial endotoxins
Much more complicated because of steam quality requirements	Lethality of the microbes is less than that of F0 at the same temperature

19. What are the sterile manufacturing area grades? What are the operations to be carried out in each area?

For the manufacture of sterile medicinal products 4 grades can be distinguished:

Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open ampoules and vials, making aseptic connections. Normally such conditions are provided by a laminar air flow work station. Laminar air flow systems should provide a homogeneous air speed in a range of 0.36–0.54 m/s (guidance value) at the working position in open clean room applications. The maintenance of laminarity should be demonstrated and validated. A uni-directional air flow and lower velocities may be used in closed isolators and glove boxes.

Grade B: For aseptic preparation and filling, this is the background environment for the grade A zone.

Grade C and D: Clean areas for carrying out less critical stages in the manufacture of sterile products.

20. Explain correlation between Area Grades, Area Class and ISO Classification “At Rest” and “In Operation”.

Grade	ISO Class number (At rest)	Class (At rest)	ISO Class number (At rest)	Class (At rest)
A	4.8	100	4.8	100
B	5	100	7	10,000
C	7	10,000	8	100,000
D	8	100,000	Not defined	Not defined

Note:

For Grade A the airborne particle classification is ISO 4.8 dictated by the limit for particles ≥ 5.0 μm.

For Grade B (at rest) the airborne particle classification is ISO 5 for both considered particle sizes.

For Grade C (at rest and in operation) the airborne particle classification is ISO 7 and ISO 8 respectively.

For Grade D (at rest) the airborne particle classification is ISO 8. (In operation no classification is defined).

Reference – Rules and Guidance for Pharmaceutical Manufacturers and Distributors (MHRA)

21. Provide example of operations to be carried out in the various grades for sterile manufacturing facility.

Examples of operations to be carried out in the various grades are as follows:

Grade	Examples of operations for terminally sterilised products.
A	Filling of products, when unusually at risk.
C	Preparation of solutions, when unusually at risk. Filling of products.
D	Preparation of solutions and components for subsequent filling.

Grade	Examples of operations for aseptic preparations.
A	Aseptic preparation and filling.
C	Preparation of solutions to be filtered.
D	Handling of components after washing.

Reference – Rules and Guidance for Pharmaceutical Manufacturers and Distributors (MHRA)

22. Explain clothing requirements for each grade of manufacturing are for sterile manufacturing facility.

Grade A/B: Headgear should totally enclose hair and, where relevant, beard and moustache; it should be tucked into the neck of the suit; a face mask should be worn to prevent the shedding of droplets. Appropriate sterilised, non-powdered rubber or plastic gloves and sterilised or disinfected footwear should be worn. Trouser-legs should be tucked inside the footwear and garment sleeves into the gloves. The protective clothing should shed virtually no fibres or particulate matter and retain particles shed by the body.

Outdoor clothing should not be brought into changing rooms leading to grade B and C rooms. For every worker in a grade A/B area, clean sterile (sterilised or adequately sanitised) protective garments should be provided at each work session. Gloves should be regularly disinfected during operations. Masks and gloves should be changed at least for every working session.

Grade C. Hair and where relevant beard and moustache should be covered. A single or two-piece trouser suit, gathered at the wrists and with high neck and appropriate shoes or overshoes should be worn. They should shed virtually no fibres or particulate matter.

Grade D. Hair and, where relevant, beard should be covered. A general protective suit and appropriate shoes or overshoes should be worn. Appropriate measures should be taken to avoid any contamination coming from outside the clean area.

Reference – Rules and Guidance for Pharmaceutical Manufacturers and Distributors (MHRA)

23. Explain design consideration in sterile manufacturing area for contamination prevention and clean area separation

Airflow direction shall be from areas of higher cleanliness to adjacent less clean areas.
Higher air cleanliness shall have a substantial positive pressure differential relative to adjacent rooms of lower air cleanliness.
Positive pressure differential of at least 10-15 Pascals (Pa) should be maintained between adjacent rooms of differing classification (with doors closed).
When doors are open, outward airflow should be sufficient to minimize ingress of contamination, and it is critical that the time a door can remain ajar be strictly controlled.
When unclassified room adjacent to the aseptic processing room, a substantial overpressure (e.g., at least 12.5 Pa) from the aseptic processing room should be maintained at all times to prevent contamination.
Continuous monitoring of pressure differentials between cleanrooms with frequently recorded.

24. What is the air changes requirement for Class 100,000 (ISO 8)?

For Class 100,000 (ISO 8) supporting rooms, airflow sufficient to achieve at least 20 air changes per hour is typically acceptable.

Significantly higher air change rates are normally needed for Class 10,000 and Class 100 areas.

25. What is difference between filter leak testing and efficiency testing?

An efficiency test is a general test used to determine the rating of the filter. An intact HEPA filter should be capable of retaining at least 99.97 percent of particulates greater than 0.3 μm in diameter.

The purpose of leak test is to detect leaks from the filter media, filter frame, or seal.

26. What is the limit of HEPA filter leak test?

While performing leak test, a single probe reading equivalent to 0.01 percent of the upstream challenge would be considered as indicative of a significant leak.

27. Why measurement of velocity is important in the aseptic area? What should be measurement location and distance from filter face?

HEPA filter leak testing alone is insufficient to monitor filter performance. It is important to conduct periodic monitoring of filter attributes such as uniformity of velocity across the filter (and relative to adjacent filters). Variations in velocity can cause turbulence that increases the possibility of contamination. Velocities of unidirectional air should be measured 6 inches from the filter face and at a defined distance proximal to the work surface for HEPA filters in the critical area. Velocity monitoring at suitable intervals can provide useful data on the critical area in which aseptic processing is performed. The measurements should correlate to the velocity range established at the time of in situ air pattern analysis studies.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

28. Drains are preferably not allowed in which area classification zones?

Drains are considered inappropriate for classified areas of the aseptic processing facility other than Class 100,000 (ISO 8) areas.

29. Types of training required to be conducted before an individual is permitted to enter the aseptic manufacturing area?

Fundamental training topics should include aseptic technique, cleanroom behavior, microbiology, hygiene, gowning, patient safety hazards posed by a non-sterile drug product, and the specific written procedures covering aseptic manufacturing area operations.

30. What measures required to be followed by personnel in aseptic area to maintain sterility of sterile items and surafaces?

Measures required to be followed by personnel in aseptic area are:
- Contact sterile materials only with sterile instruments.
- After initial gowning, sterile gloves should be regularly sanitized or changed, as appropriate, to minimize the risk of contamination.
- Personnel should not directly contact sterile products, containers, closures, or critical surfaces with any part of their gown or gloves.
- Move slowly and deliberately.
- Keep the entire body out of the path of unidirectional airflow.
- Approach a necessary manipulation in a manner that does not compromise sterility of the product.
- Maintain Proper Gown Control – Prior to and throughout aseptic operations, an operator should not engage in any activity that poses an unreasonable contamination risk to the gown.

31. What is the impact of rapid movements in Aspetic area?

Rapid movements can create unacceptable turbulence in a critical area. Such movements disrupt the unidirectional airflow, presenting a challenge beyond intended cleanroom design and control parameters. The principle of slow, careful movement should be followed throughout the cleanroom.

32. What is the potential impact if body parts interrupt the path of unidirectional airflow?

Disruption of the path of unidirectional flow air in the critical area can pose a risk to product sterility because, the purpose of unidirectional airflow design is to protect sterile equipment surfaces, container-closures, and product.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

33. Explain the type of gowning required for aseptic area?

The gown should provide a barrier between the body and exposed sterilized materials and prevent contamination from particles generated by, and microorganisms shed from, the body.
Gowns should be sterile and non-shedding, and cover the skin and hair (face-masks, hoods, beard/ moustache covers, protective goggles, and elastic gloves are examples of common elements of gowns).
An adequate barrier should be created by the overlapping of gown components (e.g., gloves overlapping sleeves).
If an element of a gown is found to be torn or defective, it should be changed immediately. Gloves should be sanitized frequently.

34. What is the recommended gowning requalification frequency for aseptic area?

Annual requalification is normally sufficient for those automated operations where personnel involvement is minimized and monitoring data indicate environmental control.

For any aseptic processing operation, if adverse conditions occur, additional or more frequent requalification could be indicated.

35. Sanitizing gloves just prior to sampling is acceptable or not acceptable?

Sanitizing gloves just prior to sampling is inappropriate because it can prevent recovery of microorganisms that were present during an aseptic manipulation.

36. What is the recommended solvent for rinse sampling for pre-sterilization preparation of glass containers? What is the acceptance criterion of final rinse water?

High purity water. Final rinse water should meet the specifications of WFI, USP.

37. What is a log reduction criterion for endotoxin after depyrogenation process?

Validation study data should demonstrate that the process reduces the endotoxin content by at least 99.9 percent (3 logs).

38. Is sterilizing-grade filters and moist heat sterilization effective in removing endotoxin?

Sterilizing-grade filters and moist heat sterilization have not been shown to be effective in removing endotoxin.

39. What is an effective way to inactivate endotoxins?

Endotoxin on equipment surfaces can be inactivated by high-temperature dry heat, or removed from equipment surfaces by cleaning procedures.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

40. What should be frequency of periodic media fill?

Semi-annual qualification should be conducted for each processing line to evaluate the state of control of the aseptic process.

41. What permutation and combination factors should be covered during the media fill?

Activities and interventions representative of each shift, and shift changeover, should be incorporated into the design of the semi-annual qualification program. E.g. Production shift should address its unique time-related and operational features.

42. What is the requirement of participation of personnel during media fill? What operations need to be done by participants?

All personnel who are authorized to enter the aseptic processing room during manufacturing, including technicians and maintenance personnel, should participate in a media fill at least once a year. Participation should be consistent with the nature of each operator’s duties during routine production.

43. What should be duration of media fill runs?

The duration of aseptic processing operations is a major consideration in media fill design. Although the most accurate simulation model would be the full batch size and duration because it most closely simulates the actual production operations, other appropriate models can be justified. The duration of the media fill run should be determined by the time it takes to incorporate manipulations and interventions, as well as appropriate consideration of the duration of the actual aseptic processing operation. Interventions that commonly occur should be routinely simulated, while those occurring rarely can be simulated periodically.

44. What should be size of media fill runs?

The simulation run sizes should be adequate to mimic commercial production conditions and accurately assess the potential for commercial batch contamination.

The number of units filled during the process simulation should be based on contamination risk for a given process and sufficient to accurately simulate activities that are representative of the manufacturing process. A generally acceptable starting point for run size is in the range of 5,000 to 10,000 units. For operations with production sizes under 5,000, the number of media filled units should at least equal the maximum batch size made on the processing line

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

45. Why high machine speed and low speed should be challenged during media fill runs?

Hgh line speed is often most appropriate in the evaluation of manufacturing processes characterized by frequent interventions or a significant degree of manual manipulation. Use of slow line speed is generally appropriate for evaluating manufacturing processes with prolonged exposure of the sterile drug product and containers/closures in the aseptic area.

46. What types of growth medium should be used during media fill runs?

A microbiological growth medium, such as soybean casein digest medium, should be used.

Use of anaerobic growth media (e.g., fluid thioglycollate medium) should be considered in special circumstances.

47. Which should be characteristic of microbiological growth medium to be used during media fill?

The media selected should be demonstrated to promote growth of gram-positive and gram-negative bacteria, and yeast and mold (e.g., USP indicator organisms).

Environmental monitoring and sterility test isolates can be substituted (as appropriate) or added to the growth promotion challenge.

48. What should be the concentration of organism while testing of Growth promotion units?

Growth promotion units should be inoculated with a <100 CFU challenge.

49. What action should be done if growth promotion test fails?

If the growth promotion testing fails, the origin of any contamination found during the simulation should nonetheless be investigated and the media fill promptly repeated.

50. What should be the incubation duration and temperature for media filled units?

• Incubation temperature should be suitable for recovery of bioburden and environmental isolates and should at no time be outside the range of 20-35 °C. Incubation temperature should be maintained within +2.5 °C of the target temperature.

• Incubation time should not be less than 14 days. If two temperatures are used for the incubation of the media filled units, the units should be incubated for at least 7 days at each temperature (starting with the lower temperature).

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

51. Who should perform inspection of media filled units?

Each media-filled unit should be examined for contamination by personnel with appropriate education, training, and experience in inspecting media fill units for microbiological contamination.

If QC personnel do not perform the inspection, there should be QC unit oversight throughout any such examination.

All suspect units identified during the examination should be brought to the immediate attention of the QC microbiologist.

52. How to perform inspection of media filled units of amber or other opaque containers?

To allow for visual detection of microbial growth, amber or other opaque containers should be substitute with clear containers.

53. After inspection of media filled units which containers shall be incubated and which containers shall not?

All integral units should proceed to incubation. Units found to have defects not related to integrity (e.g., cosmetic defect) should be incubated; units that lack integrity should be rejected.

54. Whether the media filled units generated during the intervention should be incubated or not?

If written procedures and batch documentation are adequate to describe an associated clearance, the intervention units removed during media fills do not need to be incubated. Where procedures lack specificity, there would be insufficient justification for exclusion of units removed during an intervention from incubation. For example, if a production procedure requires removal of 10 units after an intervention at the stoppering station infeed, batch records (i.e., for production and media fills) should clearly document conformance with this procedure. In no case should more units be removed during a media fill intervention than would be cleared during a production run.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

55. How to interpret test results of media fill?

Recommended criteria for assessing state of aseptic line control are as follows:

i. When filling fewer than 5000 units, no contaminated units should be detected.

One (1) contaminated unit is considered cause for revalidation, following an investigation.

ii. When filling from 5,000 to 10,000 units

One (1) contaminated unit should result in an investigation, including consideration of a repeat media fill.

Two (2) contaminated units are considered cause for revalidation, following investigation.

iii. When filling more than 10,000 units

One (1) contaminated unit should result in an investigation.

Two (2) contaminated units are considered cause for revalidation, following investigation.

56. What should be considered when performing investigation of media fill failure?

The microorganisms should be identified to species level.

The investigation should survey the possible causes of contamination. In addition, any failure investigation should assess the impact on commercial drugs produced on the line since the last media fill.

57. To carryout Filtration Efficacy study, which microorganism should be considered for challenge? Why?

The microorganism, Brevundimonas diminuta (ATCC 19146) when properly grown, harvested and used, is a common challenge microorganism for 0.2 μm rated filters because of its small size (0.3 μm mean diameter).

58. What concentration of organism should be considered for Filtration Efficacy study?

A challenge concentration of at least 10⁷ organisms per cm² of effective filtration area should generally be used, resulting in no passage of the challenge microorganism. The challenge concentration used for validation is intended to provide a margin of safety well beyond what would be expected in production.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

59. What are the factors that can affect filter performance?

Factors that can affect filter performance generally include

(1) Viscosity and surface tension of the material to be filtered,

(2) pH,

(3) Compatibility of the material or formulation components with the filter itself,

(4) Pressures,

(5) Flow rates,

(6) Maximum use time,

(7) Temperature,

(8) Osmolality,

(9) and the effects of hydraulic shock

60. What should be frequency of filter replacement for sterilization filters for product manufacturing?

Sterilizing filters should be routinely discarded after processing of a single lot.

However, in those instances when repeated use can be justified, the sterile filter validation should incorporate the maximum number of lots to be processed.

61. What should be frequency of filter integrity testing?

Integrity testing of the filter(s) can be performed prior to processing, and should be routinely performed post-use. It is important that integrity testing be conducted after filtration to detect any filter leaks or perforations that might have occurred during the filtration.

62. What are the generally used methods for filter integrity testing?

Forward flow and bubble point tests, when appropriately employed, are two integrity tests that can be used.

63. What should be frequency of equipment and accessories sterilization?

Sterility of aseptic processing equipment should normally be maintained by sterilization between each batch.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

64. Why air should be removed from the autoclave chamber while sterilization?

The insulating properties of air interfere with the ability of steam to transfer its energy to the load, achieving lower lethality than associated with saturated steam.

65. Where must to have location for the biological indicator during sterilization validation?

Potentially difficult to reach locations within the sterilizer load or equipment train (for SIP applications) should be evaluated. For example, filter installations in piping can cause a substantial pressure differential across the filter, resulting in a significant temperature drop on the downstream side. We recommend placing biological indicators at appropriate downstream locations of the filter.

66. Why empty chamber mapping studies required during the sterilizer validation?

Empty chamber studies evaluate numerous locations throughout a sterilizing unit (e.g., steam autoclave, dry heat oven) or equipment train (e.g., large tanks, immobile piping) to confirm uniformity of conditions (e.g., temperature, pressure).

67. How heat penetration study shall be done while sterilizer validation?

Heat penetration studies should be performed using the established sterilizer loads. Validation of the sterilization process with a loaded chamber demonstrates the effects of loading on thermal input to the items being sterilized and may identify difficult to heat or penetrate items where there could be insufficient lethality to attain sterility. The placement of biological indicators at numerous positions in the load, including the most difficult to sterilize places, is a direct means of confirming the efficacy of any sterilization procedure. In general, the biological indicator should be placed adjacent to the temperature sensor so as to assess the correlation between microbial lethality and predicted lethality based on thermal input.

68. What types of calibration should be checked which could have impact on sterilization?

i. Temperature and pressure monitoring devices for heat sterilization should be calibrated at suitable intervals. The sensing devices used for validation studies should be calibrated before and after validation runs.

ii. Devices used to monitor dwell time in the sterilizer should be periodically calibrated.

iii. Instruments used to determine the purity of steam (as applicable) should be calibrated. iv. For dry heat depyrogenation tunnels, devices (e.g. sensors and transmitters) used to measure belt speed should be routinely calibrated.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

69. During aseptic area environmental monitoring program, what should be covered?

The environmental monitoring program should cover all production shifts and include air, floors, walls, and equipment surfaces, including the critical surfaces that come in contact with the product, container, and closures.

70. Which location should be covered during environmental monitoring for aseptic area?

It is important that locations posing the most microbiological risk to the product be a key part of the program.

It is especially important to monitor the microbiological quality of the critical area to determine whether or not aseptic conditions are maintained during filling and closing activities. Air and surface samples should be taken at the locations where significant activity or product exposure occurs during production. Critical surfaces that come in contact with the sterile product should remain sterile throughout an operation. When identifying critical sites to be sampled, consideration should be given to the points of contamination risk in a process, including factors such as difficulty of setup, length of processing time, and impact of interventions. Critical surface sampling should be performed at the conclusion of the aseptic processing operation to avoid direct contact with sterile surfaces during processing.

71. How to ensure that environmental monitoring locations are reproducibly monitored?

All environmental monitoring locations should be described in SOPs with sufficient detail to allow for reproducible sampling of a given location surveyed. Written SOPs should also address elements such as (1) frequency of sampling, (2) when the samples are taken (i.e., during or at the conclusion of operations), (3) duration of sampling, (4) sample size (e.g., surface area, air volume), (5) specific sampling equipment and techniques, (6) alert and action levels, and (7) appropriate response to deviations from alert or action levels.

72. Why environmental monitoring results should not be averaged?

Averaging of results can mask unacceptable localized conditions.

73. What should be covered while performing trend analysis of the environmental monitoring data for aseptic area?

Trend reports should include data generated by location, shift, room, operator, or other parameters. Significant changes in microbial flora should be considered in the review of the ongoing environmental monitoring data.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

74. Which are the acceptable methods for monitoring the microbiological quality of the environment in aseptic area?

Acceptable methods for monitoring the microbiological quality of the environment include:

a. Surface Monitoring

b. Active Air Monitoring

c. Passive Air Monitoring (Settling Plates)

75. Which surfaces monitored during Surface Monitoring?

Environmental monitoring involves sampling various surfaces for microbiological quality. For example, product contact surfaces, floors, walls, and equipment should be tested on a regular basis.

76. What types of techniques shall be used for surface monitoring?

Touch plates, swabs, and contact plates can be used for such tests.

77. What types of device shall be used for Active Air Monitoring?

Assessing microbial quality of air should involve the use of active devices including but not limited to impaction, centrifugal, and membrane (or gelatin) samplers.

78. What should be considered during method validation of Passive Air Monitoring (Settling Plates)?

As part of methods validation, the laboratory should evaluate what media exposure conditions optimize recovery of low levels of environmental isolates. Exposure conditions should preclude desiccation (e.g., caused by lengthy sampling periods and/or high airflows), which inhibits recovery of microorganisms.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

79. How microbiological identification is useful in sterile product manufacturing facility environmental monitoring program?

Characterization of recovered microorganisms provides vital information for the environmental monitoring program. Environmental isolates often correlate with the contaminants found in a media fill or product sterility testing failure, and the overall environmental picture provides valuable information for an investigation. Monitoring critical and immediately surrounding clean areas as well as personnel should include routine identification of microorganisms to the species (or, where appropriate, genus) level.

80. How uncontrolled area or lesser controlled areas microbiological identification is useful in sterile product manufacturing facility environmental monitoring program?

In some cases, environmental trending data have revealed migration of microorganisms into the aseptic processing room from either uncontrolled or lesser controlled areas. Establishing an adequate program for differentiating microorganisms in the lesser-controlled environments, such as Class 100,000 (ISO 8), can often be instrumental in detecting such trends.

At minimum, the program should require species (or, where appropriate, genus) identification of microorganisms in these ancillary environments at frequent intervals to establish a valid, current database of contaminants present in the facility during processing (and to demonstrate that cleaning and sanitization procedures continue to be effective).

81. Which method of microbial identification is more accurate?

Genotypic methods have been shown to be more accurate and precise than traditional biochemical and phenotypic techniques. These methods are especially valuable for investigations into failures (e.g., sterility test; media fill contamination). However, appropriate biochemical and phenotypic methods can be used for the routine identification of isolates.

82. What is the goal of microbiological monitoring?

The goal of microbiological monitoring is to reproducibly detect microorganisms for purposes of monitoring the state of environmental control.

83. What should be the capability microbiological culture media used for aseptic area environmental monitoring program?

The microbiological culture media used in environmental monitoring should be validated as capable of detecting fungi (i.e., yeasts and molds) as well as bacteria and incubated at appropriate conditions of time and temperature.

84. What should be incubation condition for environmental monitoring media plates and what should be the duration?

Total aerobic bacterial count can be obtained by incubating at 30 to 35°C for 48 to 72 hours. Total combined yeast and mold count can generally be obtained by incubating at 20 to 25°C for 5 to 7 days.

85. How to ensure that the incoming lots of environmental monitoring media is able to reliably recover microorganisms?

Incoming lots of environmental monitoring media should be tested for their ability to reliably recover microorganisms. Growth promotion testing should be performed on all lots of prepared media. Where appropriate, inactivating agents should be used to prevent inhibition of growth by cleanroom disinfectants or product residuals (e.g., antibiotics).

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

86. Why controlling of Prefiltration Bioburden is important?

Manufacturing process controls should be designed to minimize the bioburden in the unfiltered product. In addition to increasing the challenge to the sterilizing filter, bioburden can contribute impurities (e.g., endotoxin) to, and lead to degradation of, the drug product. A prefiltration bioburden limit should be established.

87. In which scenario alternate Microbiological Test Methods should be used?

Other suitable microbiological test methods (e.g., rapid test methods) can be considered for environmental monitoring, in-process control testing, and finished product release testing after it is demonstrated that the methods are equivalent or better than traditional methods (e.g.,USP).

88. How Particle Monitoring is useful in clean room?

Routine particle monitoring is useful in rapidly detecting significant deviations in air cleanliness from qualified processing norms (e.g., clean area classification).

89. What should be the environmental condition of sterility testing area?

The testing laboratory environment should employ facilities and controls comparable to those used for aseptic filling operations. Poor or deficient sterility test facilities or controls can result in test failure. If production facilities and controls are significantly better than those for sterility testing, the danger exists of mistakenly attributing a positive sterility test result to a faulty laboratory even when the product tested could have, in fact, been nonsterile.

90. Which USP chapter and which part of 21 CFR describe about sterility testing?

Sterility testing methods are required to be accurate and reproducible, in accordance with 211.194 and 211.165.

USP <71> “Sterility Tests” is the principal source used for sterility testing methods, including information on test procedures and media.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

91. What is the probability of detection of contamination during sterility testing?

Sterility tests are limited in their ability to detect contamination because of the small sample size typically used. For example, as described by USP, statistical evaluations indicate that the sterility test sampling plan “only enables the detection of contamination in a lot in which 10% of the units are contaminated about nine times out of ten in making the test” (Ref. 13). To further illustrate, if a 10,000-unit lot with a 0.1 percent contamination level was sterility tested using 20 units, there is a 98 percent chance that the batch would pass the test.

92. How to collect the samples for sterility testing?

It is important that the samples represent the entire batch and processing conditions. Samples should be taken:

• At the beginning, middle, and end of the aseptic processing operation

• In conjunction with processing interventions or excursions

93. When sterility test failure results can be invalidated?

An initial positive test would be invalid only in an instance in which microbial growth can be unequivocally ascribed to laboratory error.

Only if conclusive and documented evidence clearly shows that the contamination occurred as part of testing should a new test be performed.

94. In case of inconclusive investigation, what should be the batch disposition decision?

When available evidence is inconclusive, batches should be rejected as not conforming to sterility requirements.

95. What parameters should be considered while performing sterility failure investigation?

The investigation’s persuasive evidence of the origin of the contamination should be based on at least the following:

1. Identification (speciation) of the organism in the sterility test

2. Record of laboratory tests and deviations

3. Monitoring of production area environment

4. Monitoring Personnel

5. Product Presterilization Bioburden

6. Production record review 7. Manufacturing history

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

96. How isolate identification is useful during investigation of sterility testing failure?

Sterility test isolates should be identified to the species level. Microbiological monitoring data should be reviewed to determine if the organism is also found in laboratory and production environments, personnel, or product bioburden. Advanced identification methods (e.g., nucleic-acid based) are valuable for investigational purposes. When comparing results from environmental monitoring and sterility positives, both identifications should be performed using the same methodology.

97. What is the recommended material of construction for aseptic processing design?

Suitable materials should be chosen based on durability, as well as ease of cleaning and decontamination. For example, rigid wall construction incorporating stainless steel and glass materials is widely used.

98. What is expected classification of isolator and surrounding area?

The interior of the isolator should meet Class 100 (ISO 5) standards. The classification of the environment surrounding the isolator should be based on the design of its interfaces (e.g., transfer ports), as well as the number of transfers into and out of the isolator. A Class 100,000 (ISO 8) background is commonly used based on consideration of isolator design and manufacturing situations. An aseptic processing isolator should not be located in an unclassified room.

Reference: FDA Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice – September 2004

99. What should be the laminar air flow systems air speed for Grade A area? Why?

Laminar air flow systems should provide a homogeneous air speed in a range of 0.36 – 0.54 m/s (guidance value) at the working position in open clean room applications.

100. Why length of tubing and the radii is important for particle monitoring system?

The length of tubing and the radii of any bends in the tubing must be considered in the context of particle losses in the tubing.

101. What should be pressure differential between adjacent rooms of different grades?

Adjacent rooms of different grades should have a pressure differential of 10 – 15 pascals.

102. What is the recommended temperature for Water for injections storage and distribution? Why?

Water for injections should be produced, stored and distributed in a manner which prevents microbial growth, for example by constant circulation at a temperature above 70°C.

103. For what types of areas, fumigation is useful?

Fumigation of clean areas may be useful for reducing microbiological contamination in inaccessible places.

Reference: EudraLex – The Rules Governing Medicinal Products in the European Union Volume 4, EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use, Annex 1 – Manufacture of Sterile Medicinal Products – 01 March 2009

104. What should be the method of choice for sterilization as per EU Annex-1?

Where possible, heat sterilisation is the method of choice.

105. What should be the verification schedule for sterilization process?

The validity of the sterilization process should be verified at scheduled intervals, at least annually, and whenever significant modifications have been made to the equipment.

106. How to differentiate products which have not been sterilised from those which have?

Each basket, tray or other carrier of products or components should be clearly labelled with the material name, its batch number and an indication of whether or not it has been sterilised. Indicators such as autoclave tape may be used, where appropriate, to indicate whether or not a batch (or sub-batch) has passed through a sterilisation process, but they do not give a reliable indication that the lot is, in fact, sterile.

107. How to ensure temperature during sterilization process?

Each heat sterilisation cycle should be recorded on a time/temperature chart with a sufficiently large scale or by other appropriate equipment with suitable accuracy and precision. The position of the temperature probes used for controlling and/or recording should have been determined during the validation, and where applicable also checked against a second independent temperature probe located at the same position.

108. Can chemical or biological indicator be replacement of physical measurements during sterilization cycle?

Chemical or biological indicators should not take the place of physical measurements.

109. How to monitor moist heat sterilization cycle?

Both temperature and pressure should be used to monitor the process. Control instrumentation should normally be independent of monitoring instrumentation and recording charts. Where automated control and monitoring systems are used for these applications they should be validated to ensure that critical process requirements are met. System and cycle faults should be registered by the system and observed by the operator. The reading of the independent temperature indicator should be routinely checked against the chart recorder during the sterilisation period. For sterilisers fitted with a drain at the bottom of the chamber, it may also be necessary to record the temperature at this position, throughout the sterilization period. There should be frequent leak tests on the chamber when a vacuum phase is part of the cycle.

110. Explain the quality of wrapping material used during sterilization process?

The items to be sterilised, other than products in sealed containers, should be wrapped in a material which allows removal of air and penetration of steam but which prevents recontamination after sterilisation.

111. What challenge should be carried out during validation when for dry heat sterilization when it is intended to remove pyrogen?

Challenge tests using endotoxins should be used as part of the validation.

112. In what scenario, sterilisation by radiation can be adopted?

Radiation sterilisation is used mainly for the sterilisation of heat sensitive materials and products. Many medicinal products and some packaging materials are radiation-sensitive, so this method is permissible only when the absence of deleterious effects on the product has been confirmed experimentally.

113. Which irradiation is not an acceptable method of sterilisation.?

Ultraviolet irradiation is not normally an acceptable method of sterilisation.

114. How to measure radiation while doing irradiation serilization?

During the sterilisation procedure the radiation dose should be measured. For this purpose, dosimetry indicators which are independent of dose rate should be used, giving a quantitative measurement of the dose received by the product itself. Dosimeters should be inserted in the load in sufficient number and close enough together to ensure that there is always a dosimeter in the irradiator. Where plastic dosimeters are used they should be used within the time-limit of their calibration. Dosimeter absorbances should be read within a short period after exposure to radiation.

115. How to prevent mix-up between irradiated and nonirradiated materials?

Radiation sensitive colour disks should also be used on each package to differentiate between packages which have been subjected to irradiation and those which have not.

116. When Sterilisation with ethylene oxide should be used?

This method should only be used when no other method is practicable. During process validation it should be shown that there is no damaging effect on the product and that the conditions and time allowed for degassing are such as to reduce any residual gas and reaction products to defined acceptable limits for the type of product or material.

117. What precaution should be taken while sterilization of material using ethylene oxide?

i. Direct contact between gas and microbial cells is essential; precautions should be taken to avoid the presence of organisms likely to be enclosed in material such as crystals or dried protein. The nature and quantity of packaging materials can significantly affect the process.

ii. Before exposure to the gas, materials should be brought into equilibrium with the humidity and temperature required by the process. The time required for this should be balanced against the opposing need to minimize the time before sterilisation.

iii. Each sterilisation cycle should be monitored with suitable biological indicators, using the appropriate number of test pieces distributed throughout the load.

iv. For each sterilisation cycle, records should be made of the time taken to complete the cycle, of the pressure, temperature and humidity within the chamber during the process and of the gas concentration and of the total amount of gas used. The pressure and temperature should be recorded throughout the cycle on a chart.

v. After sterilisation, the load should be stored in a controlled manner under ventilated conditions to allow residual gas and reaction products to reduce to the defined level.

118. What is preferred method of sterilization as per EU Annex 1?

Filtration alone is not considered sufficient when sterilisation in the final container is possible. With regard to methods currently available, steam sterilisation is to be preferred. If the product cannot be sterilised in the final container, solutions or liquids can be filtered through a sterile filter of nominal pore size of 0.22 micron (or less), or with at least equivalent micro-organism retaining properties, into a previously sterilised container. Such filters can remove most bacteria and moulds, but not all viruses or mycoplasmas. Consideration should be given to complementing the filtration process with some degree of heat treatment.

119. Number of sterilization filters recommended by EU Annex 1 when sterilization is done through filtration method? What should be the location of the filer?

Due to the potential additional risks of the filtration method as compared with other sterilization processes, a second filtration via a further sterilised micro-organism retaining filter, immediately prior to filling, may be advisable. The final sterile filtration should be carried out as close as possible to the filling point.

120. When to perform filter integrity testing and why it is important?

The integrity of the sterilised filter should be verified before use and should be confirmed immediately after use by an appropriate method such as a bubble point, diffusive flow or pressure hold test. The time taken to filter a known volume of bulk solution and the pressure difference to be used across the filter should be determined during validation and any significant differences from this during routine manufacturing should be noted and investigated. Results of these checks should be included in the batch record. The integrity of critical gas and air vent filters should be confirmed after use. The integrity of other filters should be confirmed at appropriate intervals.

121. What is the preferred duration of sterilization filter as per EU Annex 1?

The same filter should not be used for more than one working day unless such use has been validated.

122. Partially stoppered freeze drying vials should be maintained under which grade area?

Partially stoppered freeze drying vials should be maintained under Grade A conditions at all times until the stopper is fully inserted.

123. At which stage of operation, aseptically filled vial cap is considered integral?

The container closure system for aseptically filled vials is not fully integral until the aluminium cap has been crimped into place on the stoppered vial. Crimping of the cap should therefore be performed as soon as possible after stopper insertion.

124. What is the major risk of vial crimping operation in aseptic area? How to mitigate that risk?

The equipment used to crimp vial caps can generate large quantities of non-viable particulates, the equipment should be located at a separate station equipped with adequate air extraction.

125. Vial capping should be done which grade area?

Vial capping can be undertaken as an aseptic process using sterilised caps or as a clean process outside the aseptic core. Where this latter approach is adopted, vials should be protected by Grade A conditions up to the point of leaving the aseptic processing area, and thereafter stoppered vials should be protected with a Grade A air supply until the cap has been crimped.

126. How many filled containers should be visually inspected? Why?

Filled containers of parenteral products should be inspected individually for extraneous contamination or other defects.

127. Explain the process of visual inspection injectable products?

When inspection is done visually, it should be done under suitable and controlled conditions of illumination and background. Operators doing the inspection should pass regular eye-sight checks, with spectacles if worn, and be allowed frequent breaks from inspection. Where other methods of inspection are used, the process should be validated and the performance of the equipment checked at intervals. Results should be recorded.

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