PROTOCOL FOR MEMBRANE FILTRATION VALIDATION|0.22 MICRON FILTER VALIDATION

1.0 Filter Validation Test Matrix

Test Name	Rational	References
Adsorption	Adsorption study is conducted on entire device as adsorptive filter materials include filtration media, support materials and hardware	PDA Technical Report No.26 (Revised2008) on Sterilizing Filtration of Liquids.
Extractables	Since the concentration of the test product is much higher than the potential filter extracts expected, the product components may interfere with analytical methods used for performing extractable studies, therefore a model stream approach is proposed and rationale behind selected model solvents is documented. The test product is substituted by using several solvents representing key properties of the test product such as pH, ionic strength or level of organic components of the test product	USP Class VI and Biological Reactivity Test, In Vivo<87> USP <1663>: Assessment of Extractables Associated with Pharmaceutical Packaging and Delivery Systems
Integrity Testing	The method for integrity testing of production filters is chosen to provide reliable results based upon the nature and size of the filter, product characteristics and processing conditions. The test product characteristics and process conditions influence the integrity test specifications of the process filters; hence it is imperative to validate production filters for product wetted integrity test specifications at specified test conditions.	PDA Technical Report No.26 (Revised2008) on Sterilizing Filtration of Liquids.
Viability Testing and Bacterial Retention Challenge Test	The viability of the test organism is determined by direct inoculation method in the carrier fluid (test product or surrogate). The test organism is grown in the same manner as that used for Microbial Retention Efficiency study to preserve its morphological and physiological characteristics. The test time should be equal to or exceed the actual process filtration time. If the total reduction in count of the test organism, Brevundimonas diminuta ATCC 19146 after exposure is not more than 1 log, the carrier fluid/test product is considered as non-bactericidal. The bacterial retention challenge test is conducted to validate the microbial retention efficiency of a 0.22-micron sterilizing-grade filter, ensuring it effectively removes Brevundimonas he Throughput Study is a critical component of Sterile Filtration Process Validation, ensuring that the selected 0.22 µm filter can efficiently process the required batch volume of Artemether Injection 40 mg without clogging, excessive pressure buildup, or loss of filtration efficiency. This study helps establish critical process parameters, including maximum filtration time, filtration volume, operating pressure, and flow rate, ensuring compliance with GMP and regulatory guidelines. By monitoring flow rate decay, the study determines the filter's capacity to maintain sterility and efficiency throughout the process. Proper documentation of throughput data in the Batch Manufacturing Record (BMR) supports regulatory compliance and prevents process failures, ensuring a robust and validated filtration process diminuta ATCC 19146, the smallest known vegetative bacterium and the standard test organism for sterilizing filter validation. This test follows regulatory guidelines, including USP <1229.4> and PDA Technical Report No. 26, requiring a ≥ 107 CFU/mL challenges to confirm sterility assurance. The test conditions, including filtration under simulated process parameters, post-filtration microbial analysis, and positive control recovery (50%-200%), ensure reliability. If no bacterial growth is observed in the filtrate, the filter is deemed effective for sterile filtration. This validation is essential for sterile pharmaceutical products ensuring compliance, sterility, and patient safety	USP Microbiological tests and Sterility Tests<71>. ASTM Designation: F 838-05 (Reapproved 2005), Standard Method for Determining Bacterial Retention Ability of Membrane Filters Utilized for Liquid Filtration. PDA Technical Report 26 (Revised 2008) on Sterilizing Filtration of Liquids
Throughput Study	The Throughput Study is a critical component of Sterile Filtration Process Validation, ensuring that the selected 0.22 µm filter can efficiently process the required batch volume of Artemether Injection 40 mg without clogging, excessive pressure buildup, or loss of filtration efficiency. This study helps establish critical process parameters, including maximum filtration time, filtration volume, operating pressure, and flow rate, ensuring compliance with GMP and regulatory guidelines. By monitoring flow rate decay, the study determines the filter's capacity to maintain sterility and efficiency throughout the process. Proper documentation of throughput data in the Batch Manufacturing Record (BMR) supports regulatory compliance and prevents process failures, ensuring a robust and validated filtration process.	PDA Technical Report No. 26 (Revised 2008) on Sterilizing Filtration of Liquids

1. Microbiological Containment Test for Biosafety Cabinets (BSC)

Objective:

To evaluate the containment efficiency of a Biosafety Cabinet (BSC) by assessing its ability to prevent microbiological contamination from escaping into the environment and to ensure product protection.

Reference Standards:

• ISO 12469: Performance criteria for microbiological safety cabinets
• EN 12469: European standard for BSC validation
• NSF/ANSI 49: Certification of Class II BSCs

---

1.1. Test Materials & Equipment Required

• Microbial Challenge Organism: Bacillus subtilis (ATCC 6633) or Bacillus atrophaeus (spore suspension, 10? CFU/mL)
• Nutrient Agar Plates (or Tryptic Soy Agar – TSA)
• Aerosol Generator (Collison Nebulizer or similar)
• Air Sampler (e.g., Andersen sampler or settle plates)
• Incubator (35-37°C)

---

1.2. Test Procedure

A. Operator Protection Test (External Containment)

Purpose: Ensures the BSC protects the operator from hazardous aerosols.

1. Position the Aerosol Generator inside the center of the cabinet workspace.
2. Generate an aerosol of Bacillus subtilis spores at a concentration of 10? CFU/mL for 5 minutes.
3. Place settle plates and air samplers outside the BSC (near the front opening).
4. Run the BSC at normal operation for 30 minutes after aerosolization.
5. Incubate exposed plates at 35-37°C for 24-48 hours.
6. Examine for bacterial colony growth on external plates.

Acceptance Criteria:

• No growth should be detected outside the BSC (or max 1 CFU per plate).
• If any bacterial growth >1 CFU is observed, the BSC fails the containment test.

---

B. Product Protection Test (Internal Containment)

Purpose: Ensures no contamination occurs inside the BSC workspace.

1. Place sterile nutrient agar plates inside the cabinet workspace.
2. Generate an aerosol of Bacillus subtilis spores for 5 minutes.
3. Keep the BSC running for 30 minutes post-aerosolization.
4. Collect plates from inside the BSC and incubate at 35-37°C for 24-48 hours.
5. Check for microbial contamination on the internal plates.

Acceptance Criteria:

• No colony growth (>1 CFU) should be observed on internal agar plates.
• Growth inside the cabinet indicates airflow failure or contamination risk.

---

C. Cross-Contamination Test

Purpose: Verifies the BSC prevents sample cross-contamination.

1. Place an open TSA plate inside the workspace.
2. Perform aerosol generation at one side of the BSC.
3. Incubate plates on the opposite side of the BSC.
4. Examine for contaminant spread across work areas.

Acceptance Criteria:

• No growth (>1 CFU) on opposite-side plates.
• If CFUs appear, the BSC may have turbulent airflow issues.

---

2. KI-Discus Test (Potassium Iodide Test) for Biosafety Cabinets

Objective:

To evaluate personnel protection efficiency of Class II BSCs by measuring the escape of aerosols from the cabinet’s front opening.

Reference Standards:

• ISO 12469 – KI-Discus test is a recommended method for operator protection evaluation.

---

2.1. Test Materials & Equipment Required

• KI Solution (Potassium Iodide, 1% w/v)
• Discus Plates (KI-reactive test papers)
• Aerosol Generator (Collison Nebulizer or similar)
• Air Samplers (Portable Particle Counter or Settle Plates)

---

2.2. Test Procedure

1. Preparation of KI Solution:

   • Dissolve 10g of KI in 1L of sterile water.
   • Mix thoroughly and filter-sterilize if needed.

2. Placement of KI-Discus Plates:

   • Position KI-Discus plates at the BSC front opening.
   • Place some inside the cabinet and some outside (near the operator’s position).

3. Generation of KI Aerosol:

   • Use an aerosol generator to nebulize KI solution inside the cabinet for 5 minutes.
   • Simulate normal working conditions inside the BSC (e.g., hand movement, pipetting).

4. Exposure & Observation:

   • Allow exposure for 10-15 minutes.
   • Observe color change in KI-Discus plates.
   • KI reacts with starch in the plates, forming a dark blue/black stain if aerosols escape.

---

2.3. Acceptance Criteria

• No staining outside → PASS (BSC is effective in personnel protection).
• Any KI-stain outside → FAIL (BSC leaks aerosols and requires maintenance).

---

3. Summary of Acceptance Limits for Both Tests

Difference Between Biosafety Cabinet Class I and Class II (A1, A2, B1, B2)

Biosafety Cabinets (BSCs) are designed to provide personnel, product, and environmental protection when handling biological materials. They are classified based on containment level, airflow patterns, and application.

 

1. Difference between Class I and Class II Biosafety Cabinets

Feature	Class I BSC	Class II BSC
Purpose	Protects personnel and environment, but does not protect the sample/product.	Protects personnel, environment, and product.
Airflow Pattern	Inward airflow prevents contaminants from escaping. Exhausted air is filtered via HEPA filters.	Recirculates filtered air to protect the product, while HEPA-filtered exhaust protects personnel and environment.
Use Case	Used for low-to-moderate risk biological agents (BSL-1, BSL-2) where sample sterility is not a concern.	Used for sterile processing and working with biohazardous materials (BSL-2, BSL-3, BSL-4).
HEPA Filtration	Single HEPA filter in the exhaust.	Dual HEPA filtration (one for supply, one for exhaust).
Product Protection	No product protection – contaminants from the environment can enter the workspace.	Yes – unidirectional airflow prevents contamination of sterile materials.
Airflow Velocity	Typically 0.38–0.51 m/s (75–100 fpm).	Typically 0.38–0.51 m/s (75–100 fpm) with laminar airflow.
Common Use Cases	- Chemical handling with low biohazard risk

• Cage changing in animal facilities
• Basic microbiology research | - Sterile pharmaceutical preparations
• Handling biological hazards (BSL-2, BSL-3)
• Cell culture work, vaccine production |

 

2. Difference Between Class II Biosafety Cabinets (A1, A2, B1, B2)

Feature	Class II, Type A1	Class II, Type A2	Class II, Type B1	Class II, Type B2
Airflow Recirculation	70% recirculated, 30% exhausted	70% recirculated, 30% exhausted	30% recirculated, 70% exhausted	100% exhaust (no recirculation)
Exhaust Connection	Can be ducted or non-ducted	Can be ducted or non-ducted	Must be hard-ducted to an external exhaust system	Must be hard-ducted to an external exhaust system
Negative Pressure Plenum	No	Yes	Yes	Yes
Safe for Handling Volatile Chemicals?	No	Minimal	Yes (small amounts)	Yes (large amounts)
Best Used For	General microbiology, non-hazardous biological materials	Biosafety levels BSL-1, BSL-2 work, some BSL-3 applications	Toxic chemicals & biohazards	Toxic chemicals & highly hazardous bioagents (BSL-3, BSL-4 work)
Common Applications	- Routine microbiology

• Cell culture, diagnostic testing | - Vaccine production
• Aseptic pharmaceutical processes | - Handling radioactive materials with biohazards | - Handling BSL-3 & BSL-4 infectious agents (e.g., tuberculosis, Ebola, SARS-CoV-2) |

 

3. Qualification Procedure for Biosafety Cabinets (BSC)

Biosafety Cabinets (BSCs) must be qualified as per ISO 14644, ISO 12469, and EN 12469 standards to ensure they meet regulatory requirements.

3.1 Qualification Phases

A. Design Qualification (DQ)

• Verify that the BSC meets user requirements and ISO/EN standards.
• Review technical drawings, materials, and compliance documents.
• Confirm compliance with ISO 12469, EN 12469, and NSF/ANSI 49.

B. Installation Qualification (IQ)

• Verify correct installation location, leveling, and connections.
• Confirm electrical supply and exhaust system.
• Ensure filters and lighting meet manufacturer specifications.
• Check HEPA filter integrity.

C. Operational Qualification (OQ)

• Perform airflow velocity tests as per ISO 14644-3.
• Conduct HEPA filter leak test using DOP (Dispersed Oil Particulate).
• Perform smoke pattern tests to verify unidirectional airflow.
• Measure downflow velocity & exhaust air volume.
• Validate alarm functions and interlocks.

D. Performance Qualification (PQ)

• Conduct microbial contamination tests to confirm sterility.
• Verify operational stability under real-time conditions.
• Check operator protection using KI (potassium iodide) test.
• Perform particle count testing (ISO 14644-1 standards).

 

3.2 Test Methods and Standards for Biosafety Cabinet Qualification

Test Name	Standard / Reference	Purpose
HEPA Filter Integrity Test	ISO 14644-3, EN 12469	Ensures filters are properly sealed and not leaking.
Airflow Velocity Test	NSF/ANSI 49, EN 12469	Confirms proper airflow rates and unidirectional flow.
Smoke Visualization Test	ISO 14644-3	Verifies correct airflow pattern (no backflow of air).
Microbiological Containment Test	EN 12469	Ensures cabinet prevents contamination of the product & operator.
Noise Level Test	ISO 11201	Confirms noise levels are within safe limits.
KI-Discus Test	ISO 12469	Confirms operator protection (BSC containment efficiency).
Lighting Intensity Test	ISO 14644-1	Ensures proper illumination for operator visibility.

4. Recommended Requalification Frequency

Test Name	New Installation	Annual Testing	After Major Maintenance
HEPA Filter Integrity	Yes	Yes	Yes
Airflow Velocity	Yes	Yes	Yes
Smoke Pattern Test	Yes	Yes	Yes
Microbial Containment	Yes	Yes	Yes
KI-Discus Test	Yes	Yes	Yes
Noise Level Test	Yes	No	Yes
Lighting Test	Yes	No	Yes

 

5. Summary of Key Differences

Class	Personnel Protection	Product Protection	Airflow Pattern	Best Use Case
Class I BSC	Yes	No	Inward airflow	Non-sterile microbiology
Class II A1	Yes	Yes	70% recirculated, 30% exhausted	General microbiology
Class II A2	Yes	Yes	70% recirculated, 30% exhausted	Sterile work
Class II B1	Yes	Yes	30% recirculated, 70% exhausted	Hazardous chemicals
Class II B2	Yes	Yes	100% exhausted	Highly toxic/hazardous work

 

Conclusion

• Class I BSC protects only personnel and is used for low-risk work.
• Class II BSC protects both personnel & product and is required for aseptic biological work.
• B2 BSC is essential for highly toxic and BSL-3/BSL-4 work.
• Qualification as per ISO 14644, EN 12469 ensures regulatory compliance and safety validation.

Standard Operating Procedure (SOP) for Handling of Recombinant Proteins

1. Purpose

This SOP outlines the procedures for the safe handling, storage, processing, and disposal of recombinant proteins in compliance with Good Manufacturing Practices (GMP), biosafety regulations, and ALCOA++ data integrity principles.

---

2. Scope

This procedure applies to all personnel involved in the handling, processing, testing, storage, and disposal of recombinant proteins in R&D, production, quality control, and warehousing.

---

3. References

• ICH Q5A(R2): Viral Safety Evaluation of Biotechnology Products
• WHO TRS 987 Annex 3: Guidelines on Recombinant DNA Products
• US FDA 21 CFR Part 600: Biological Products General
• EudraLex Volume 4 Annex 2: Manufacture of Biological Products
• NIH Guidelines: Recombinant DNA Research
• OSHA Standard 29 CFR 1910.1030: Bloodborne Pathogens and Biosafety

---

4. Definitions & Abbreviations

• Recombinant Protein: A protein produced using recombinant DNA technology, typically expressed in microbial, mammalian, or insect cell systems.
• GMP (Good Manufacturing Practice): Regulatory framework ensuring quality, safety, and efficacy of biologics.
• BSC (Biological Safety Cabinet): Enclosed, ventilated workspace for handling biological material safely.
• SDS (Safety Data Sheet): Document providing hazard, handling, and disposal information.
• LAF (Laminar Airflow): Clean air environment ensuring sterility during handling.
• PPE (Personal Protective Equipment): Safety gear including gloves, masks, and lab coats.

---

5. Responsibilities

5.1 Operators (Production & QC)

• Handle recombinant proteins according to biosafety guidelines.
• Maintain proper documentation in batch records and logbooks.
• Follow PPE requirements at all times.
• Report deviations and contamination events immediately.

5.2 Quality Assurance (QA) Team

• Ensure adherence to SOPs and GMP requirements.
• Conduct routine audits and training on biosafety and handling procedures.
• Approve changes to this SOP and oversee compliance.

5.3 R&D & Process Development

• Evaluate protein stability, expression, and purification process parameters.
• Ensure appropriate storage conditions are followed.

5.4 Warehouse Team

• Store recombinant proteins as per defined conditions (e.g., -80°C, 2-8°C, or RT).
• Ensure FIFO (First-In-First-Out) or FEFO (First-Expiry-First-Out) principles.

---

6. Materials & Equipment Required

• PPE: Lab coat, gloves, safety glasses, face shield (if required).
• LAF/BSC (Class II A2 or B2) for aseptic handling.
• Refrigerators/Freezers (-80°C, -20°C, 2-8°C) for storage.
• Glassware & Plasticware (Sterile conical tubes, pipettes, vials).
• Buffer solutions & Reagents for processing.
• Autoclave or Biohazard Disposal Bags for waste.

---

7. Procedure for Handling Recombinant Proteins

7.1 Receiving and Storage

1. Upon receipt, check the SDS and certificate of analysis (CoA).
2. Inspect the packaging for leakage, damage, or temperature excursions.
3. Label storage containers with protein name, lot number, concentration, storage conditions, and expiry date.
4. Store at recommended conditions:
   • -80°C: Long-term storage.
   • -20°C: Intermediate storage.
   • 2-8°C: Short-term use.
   • RT (Room Temperature): Only if validated for stability.
5. Record storage details in the inventory management system.

7.2 Aseptic Handling & Processing

1. Work under a BSC Class II or LAF unit to minimize contamination.
2. Ensure all materials are sterile (use filter-sterilized buffers).
3. Use low-binding plasticware or glassware to minimize protein loss.
4. Prepare aliquots to avoid repeated freeze-thaw cycles.
5. Avoid vigorous mixing or vortexing to prevent denaturation.
6. Record all handling steps in laboratory notebooks/logbooks as per ALCOA++ principles.

7.3 Thawing and Reconstitution

1. Thaw proteins gradually at 4°C or on ice.
2. If lyophilized, reconstitute using validated buffer (as per CoA or protocol).
3. Gently mix by inversion (avoid pipetting up/down forcefully).
4. Filter sterilize (0.22 µm) if required before further use.

7.4 Transport of Recombinant Proteins

1. Transport frozen samples using dry ice (-80°C) or cold packs (2-8°C).
2. Use validated shipping containers for maintaining temperature integrity.
3. Record temperature logs for cold chain traceability.

7.5 Waste Disposal

1. Dispose of used materials (tubes, tips) in biohazard waste bins.
2. Inactivate biohazardous protein solutions using validated chemical treatments (e.g., bleach, autoclaving).
3. Follow local biosafety and environmental regulations for disposal.

---

8. Documentation & Record-Keeping

• Maintain batch records, logbooks, and deviation reports.
• Ensure real-time documentation (Contemporaneous ALCOA++ compliance).
• Store electronic data in validated LIMS (Laboratory Information Management System).

---

9. Safety & Biosafety Considerations

• Category of Risk: Biosafety Level 1 (BSL-1) or 2 (BSL-2) based on recombinant protein source.
• Spill Management:
  1. Isolate spill area, wear PPE.
  2. Disinfect with 10% bleach solution, wait 10 min.
  3. Dispose of contaminated materials in biohazard waste.
• Emergency Procedures: In case of exposure, wash area immediately, inform supervisor, seek medical assistance.

Advanced Chromatography Techniques in Biologics – Q&A 

This section continues with topics on cleaning validation, regulatory trends, future innovations, and comparative analysis of chromatography resins in biologics manufacturing.

---

41. How is cleaning validation performed for chromatography columns in biologics manufacturing?

Answer:

Cleaning validation ensures that chromatography columns are free from contaminants such as residual proteins, buffers, microbial growth, and leachables. The process includes:

1. Defining Acceptance Criteria: Establishing limits for carryover, bioburden, and endotoxins.
2. Selection of Cleaning Agents: Using detergents, NaOH, or buffer rinses based on column resin compatibility.
3. Rinse Recovery Testing: Analyzing wash solutions for residuals via HPLC, TOC (Total Organic Carbon), and conductivity measurements.
4. Swab & Rinse Sampling: Validating surface and liquid-phase cleanliness.
5. Resin Reuse Study: Evaluating column performance post-cleaning over multiple cycles.

???? Reference: PDA Technical Report No. 29, "Points to Consider for Cleaning Validation," 2012, p. 30-45.

---

42. What are the key regulatory trends in chromatography for biologics purification?

Answer:

Regulatory authorities emphasize:

• Process Analytical Technology (PAT): Implementing real-time monitoring to improve process control.
• Continuous Manufacturing Approaches: Encouraging multi-column chromatography (MCC) for efficiency.
• Data Integrity Compliance (21 CFR Part 11): Ensuring robust electronic record-keeping.
• Single-Use Chromatography Columns: Addressing concerns regarding extractables and leachables.

???? Reference: FDA Guidance, "PAT: A Framework for Innovative Pharmaceutical Development," 2004, p. 5-10.

---

43. What are the common challenges faced in ion-exchange chromatography (IEX) for biologics?

Answer:

• Charge Heterogeneity: Variation in protein charge due to post-translational modifications.
• pH Sensitivity: Small pH fluctuations can affect binding efficiency.
• Salt Sensitivity: High salt concentrations required for elution may denature proteins.
• Column Fouling: Adsorption of impurities leading to reduced capacity over time.

Reference: Rathore, A.S., "Process Challenges in Ion Exchange Chromatography," Biotech J., 2017, p. 18-22.

---

44. What are the advantages of using Protein A chromatography for monoclonal antibody (mAb) purification?

Answer:

• High Selectivity: Strong and specific binding to the Fc region of antibodies.
• Efficient Purification: >95% purity in a single step.
• Scalability: Suitable for both clinical and commercial production.
• Compatibility with Automation: Integrated into continuous bioprocessing workflows.

Reference: GE Healthcare, "Protein A Affinity Chromatography for mAbs," 2019, p. 10-15.

---

45. How do single-use chromatography columns compare to reusable columns?

Answer:

???? Reference: EMA Guidance, "Use of Single-Use Technologies in Biologics Manufacturing," 2021, p. 18-25.

---

46. What is the role of AI-driven chromatography control in biologics purification?

Answer:

AI and machine learning improve chromatography by:

• Predicting Process Variability: Identifying deviations from historical batch data.
• Optimizing Gradient Elution: Adjusting salt and pH gradients in real time.
• Enhancing Column Performance: Monitoring resin degradation to optimize replacement cycles.
• Reducing Waste: Predicting buffer consumption and minimizing overuse.

???? Reference: MIT AI Bioprocessing Report, "Machine Learning in Bioprocessing," 2023, p. 22-30.

---

47. What are the main considerations when scaling up chromatography processes for commercial biologics production?

Answer:

• Resin Performance at Scale: Evaluating breakthrough curves and binding capacities.
• Flow Rate Optimization: Ensuring linear scalability of residence times.
• Buffer Volume Requirements: Adjusting buffer preparation to maintain consistency.
• Column Packing Consistency: Ensuring uniform bed height and density across larger columns.

Reference: Bioprocess Engineering Handbook, "Scale-Up Considerations for Chromatography," 2020, p. 50-60.

---

48. How does hydrophobic interaction chromatography (HIC) contribute to biologics purification?

Answer:

• Removes Hydrophobic Contaminants: Such as host cell proteins and lipids.
• Provides Mild Elution Conditions: Using decreasing salt concentrations.
• Works as a Complementary Step: After ion-exchange chromatography for increased purity.

Reference: J. Chromatogr. B, "Advancements in Hydrophobic Interaction Chromatography," 2019, p. 30-35.

---

49. What are the key parameters affecting the efficiency of affinity chromatography?

Answer:

• Ligand Density: Higher ligand density increases binding capacity but may reduce selectivity.
• pH & Ionic Strength: Affect binding kinetics and elution efficiency.
• Flow Rate: Higher flow rates can reduce binding efficiency.

Reference: J. Sep. Sci., "Optimization Strategies for Affinity Chromatography," 2021, p. 18-22.

---

50. How is Process Analytical Technology (PAT) implemented in chromatography?

Answer:

• Real-Time UV Monitoring: Ensures consistent protein concentration during elution.
• Online Conductivity & pH Sensors: Adjusts buffer conditions dynamically.
• Automated Fraction Collection: Based on peak shape analysis to improve yield.

Reference: FDA Guidance, "PAT Framework for Process Control," 2014, p. 12-20.

51. What is mixed-mode chromatography, and how is it applied in biologics purification?

Answer:

Mixed-mode chromatography (MMC) combines two or more interaction mechanisms (e.g., ionic, hydrophobic, and hydrogen bonding) to enhance selectivity.

Applications in biologics:

• Removal of Product-Related Impurities: Separates charge and size variants of monoclonal antibodies.
• High Selectivity for Difficult Proteins: Useful for Fc-fusion proteins and bispecific antibodies.
• Salt-Tolerant Binding: Suitable for direct capture from cell culture supernatant.

Reference: J. Chromatogr. A, "Advances in Mixed-Mode Chromatography," 2022, p. 110-125.

---

52. How do you optimize resin lifetime in chromatography for biologics?

Answer:

Resin longevity is critical for cost-effectiveness and performance in large-scale biologics purification. Key strategies include:

1. Cleaning & Sanitization: Using NaOH and proteolytic enzymes to prevent fouling.
2. Flow Rate Optimization: Avoiding excessive shear forces that degrade resin beads.
3. Monitoring Binding Capacity: Regularly testing breakthrough curves to determine when replacement is needed.
4. Storage Conditions: Maintaining proper temperature and hydration to prevent resin shrinkage or microbial contamination.

Reference: BioProcess Int., "Best Practices for Chromatography Resin Maintenance," 2021, p. 34-42.

---

53. What is continuous chromatography, and why is it gaining importance in biologics manufacturing?

Answer:

Continuous chromatography (e.g., multi-column chromatography, countercurrent chromatography) allows for higher throughput and cost reduction compared to batch chromatography.

Advantages:

• Reduced Resin Usage: Uses smaller columns in a continuous cycle.
• Increased Productivity: Operates with shorter residence times.
• Improved Yield & Consistency: Minimizes product loss between cycles.

Reference: PDA J. Pharm. Sci. Tech., "Continuous Chromatography in Biologics," 2020, p. 18-28.

---

54. What are the regulatory expectations for implementing continuous chromatography in biologics?

Answer:

Regulators expect:

• Robust Process Validation: Demonstrating consistent performance across cycles.
• Real-Time Monitoring (PAT): Implementing sensors for in-line quality control.
• Risk Assessment for Carryover: Ensuring no contamination between cycles.
• Comparability Studies: Demonstrating equivalence to traditional batch processes.

Reference: FDA Draft Guidance, "Continuous Manufacturing of Drug Substances and Biologics," 2022, p. 10-15.

---

55. What are the benefits of using multimodal resins in biologics purification?

Answer:

Multimodal resins enhance purification by combining multiple interactions, such as hydrophobic, ionic, and hydrogen bonding.

Benefits include:

• Higher Selectivity: Targets specific protein conformations.
• Salt-Tolerant Binding: Allows flexibility in buffer conditions.
• Improved Removal of Aggregates & Host Cell Proteins (HCPs): Reduces impurity burden.

Reference: Bioprocess Tech. J., "Innovations in Multimodal Chromatography," 2021, p. 50-55.

---

56. How does high-throughput process development (HTPD) improve chromatography optimization?

Answer:

HTPD utilizes miniaturized chromatography columns and robotic systems to screen multiple parameters simultaneously.

Advantages:

• Faster Process Development: Speeds up resin selection and buffer optimization.
• Data-Driven Decision Making: Uses design of experiments (DoE) to optimize conditions.
• Reduced Material Consumption: Uses micro-scale experiments before large-scale production.

Reference: Curr. Opin. Biotechnol., "HTPD for Biologics Process Development," 2020, p. 60-72.

---

57. How does membrane chromatography differ from traditional packed-bed chromatography?

Answer:

Membrane chromatography replaces resin beads with microporous membranes coated with ligands, offering:

• Faster Processing: Eliminates diffusion limitations.
• Lower Pressure Requirements: Suitable for fragile biologics.
• Disposable & Single-Use Format: Reduces cleaning validation needs.

???? Reference: J. Membrane Sci., "Applications of Membrane Chromatography in Bioprocessing," 2021, p. 85-95.

---

58. What factors influence the choice of chromatography technique in biologics downstream processing?

Answer:

Selection is based on:

1. Target Molecule Properties: Charge, hydrophobicity, size.
2. Purity Requirements: Regulatory and market expectations.
3. Process Throughput Needs: Scale and productivity demands.
4. Cost Considerations: Resin cost, buffer usage, and cycle efficiency.

Reference: Biopharm Int., "Strategic Selection of Chromatography Techniques," 2020, p. 25-30.

---

59. What are the advantages of using hybrid chromatography approaches in biologics purification?

Answer:

Hybrid chromatography combines multiple purification steps in a single unit operation, increasing process efficiency.

Example:

• Affinity + Ion Exchange: Captures and removes charge variants in a single step.
• Size Exclusion + Hydrophobic Interaction: Simultaneously removes aggregates and hydrophobic impurities.

Reference: Trends Biotechnol., "Hybrid Chromatography Strategies," 2022, p. 100-110.

---

60. How does chromatography data integrity compliance align with 21 CFR Part 11 regulations?

Answer:

21 CFR Part 11 requires chromatography data to be:

• Secure & Tamper-Proof: Using audit trails and electronic signatures.
• Complete & Accurate: Ensuring all raw data is retained.
• Easily Retrievable: Implementing validated software systems for data access.

Reference: FDA 21 CFR Part 11 Compliance Guide, 2023, p. 15-25.

Advanced Chromatography Techniques in Biologics – Q&A 

31. How can chromatography methods be optimized for biologics purification?

Answer:

Optimization of chromatography methods for biologics purification involves:

1. Resin Selection: Choosing resins based on specificity, binding capacity, and robustness (e.g., Protein A for mAbs).
2. Buffer Optimization: Adjusting pH, ionic strength, and additives to enhance separation efficiency.
3. Flow Rate & Column Packing: Ensuring optimal flow rates and uniform packing to minimize peak broadening.
4. Step Elution vs. Gradient Elution: Fine-tuning elution conditions to achieve high purity and yield.
5. Multicolumn Chromatography: Implementing continuous or periodic counter-current systems for efficiency.

???? Reference: Shukla, A.A., et al., "Process Optimization in Protein Chromatography," BioProcess International, 2017, p. 45-52.

---

32. What are the regulatory requirements for chromatography in biologics purification?

Answer:

Regulatory bodies like the FDA and EMA require:

• Validation of Chromatography Steps: Demonstrating reproducibility, selectivity, and recovery.
• Cleaning Validation: Ensuring removal of product carryover and column leachables.
• Resin Lifetime Studies: Assessing stability and performance over multiple cycles.
• Process Analytical Technology (PAT): Implementing real-time monitoring of critical process parameters.
• Data Integrity & Traceability: Ensuring compliance with 21 CFR Part 11 for electronic records.

???? Reference: FDA Guidance for Industry, "Process Validation: General Principles and Practices," 2011, p. 20-25.

---

33. What are the common analytical chromatography techniques used in biologics?

Answer:

Key analytical chromatography techniques include:

1. High-Performance Liquid Chromatography (HPLC): For quantification and purity assessment.
2. Size Exclusion Chromatography (SEC): For aggregate and fragment analysis.
3. Ion Exchange Chromatography (IEX): For charge variant analysis.
4. Hydrophobic Interaction Chromatography (HIC): For protein hydrophobicity assessment.
5. Affinity Chromatography (AC): For specific ligand-binding interactions.

???? Reference: Rathore, A.S., "Analytical Techniques for Biopharmaceutical Development," J. Chromatogr. A, 2018, p. 10-15.

---

34. How is HPLC applied in biologics manufacturing?

Answer:

HPLC is used for:

• Purity Analysis: Detecting impurities, aggregates, and degradation products.
• Quantification: Measuring protein concentration using UV or fluorescence detectors.
• Glycosylation Profiling: Using reverse-phase HPLC with fluorescence detection.
• Process Monitoring: Tracking purification performance and consistency.

???? Reference: FDA Analytical Procedures and Methods Validation for Drugs and Biologics, 2015, p. 30.

---

35. What are the advantages of using SEC-MALS in biologics characterization?

Answer:

SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering) provides:

• Molecular Weight Determination: Without column calibration errors.
• Aggregate and Fragment Analysis: In real-time without standards.
• Structural Insights: By analyzing radius of gyration (Rg) and hydrodynamic radius (Rh).

???? Reference: Beckman Coulter, "SEC-MALS Applications in Biopharma," White Paper, 2019, p. 5-10.

---

36. How does automation improve chromatography processes?

Answer:

Automation in chromatography enhances:

• Reproducibility: Reducing variability in resin binding and elution steps.
• Throughput: Enabling high-throughput screening of purification conditions.
• Process Control: Implementing real-time monitoring via PAT tools.
• Data Integration: Ensuring compliance with regulatory data integrity guidelines.

???? Reference: Rathore, A.S., "Automated Chromatography in Biopharmaceuticals," BioPharm Int., 2018, p. 40-50.

---

37. How is AI and machine learning applied in chromatography?

Answer:

AI and ML algorithms optimize:

• Peak Resolution: Predicting optimal gradient and buffer conditions.
• Column Performance: Detecting early signs of resin degradation.
• Real-Time Process Control: Adjusting flow rates and buffer compositions dynamically.
• Data Analysis: Identifying trends in historical batch data for process improvement.

???? Reference: EMA QbD Guidelines, "Use of Artificial Intelligence in Bioprocessing," 2022, p. 12-18.

---

38. What is multicolumn chromatography, and why is it used?

Answer:

Multicolumn chromatography (MCC) is a continuous purification method that:

• Increases Throughput: By reducing column downtime.
• Improves Yield: By minimizing product loss during column loading.
• Enhances Consistency: By allowing real-time adjustments to process conditions.

???? Reference: GE Healthcare, "Continuous Chromatography for Biologics," 2019, p. 25-30.

---

39. How are leachables and extractables controlled in chromatography resins?

Answer:

• Resin Selection: Using low-leaching synthetic polymers.
• Preconditioning Columns: Washing with buffers to remove extractables.
• Regulatory Testing: Conducting extractables and leachables studies per ICH Q3D.

???? Reference: ICH Q3D Guideline, "Elemental Impurities in Drug Products," 2014, p. 8-12.

---

40. What factors affect column lifetime in biologics purification?

Answer:

• Chemical Stability: Compatibility with buffer pH and ionic strength.
• Mechanical Stress: Excessive flow rates causing resin compression.
• Microbial Contamination: Poor sanitation leading to biofilm formation.

???? Reference: PDA Journal of Pharmaceutical Science, "Column Maintenance Strategies," 2021, p. 14-19.

Chromatography Techniques in Biologics – Interview Q&A (Part 3: Advanced Techniques & Case Studies)

Continuing with the next set of Q&A on chromatography used in biologics upstream and downstream processing, focusing on advanced techniques, troubleshooting, and case studies.

---

21. What is simulated moving bed (SMB) chromatography, and how is it applied in biologics purification?

Answer:
Simulated Moving Bed (SMB) chromatography is a continuous chromatography technique that mimics the countercurrent movement of mobile and stationary phases, increasing efficiency in separation.

Applications in Biologics Purification:

• Purification of therapeutic proteins and monoclonal antibodies (mAbs).
• Chiral separation of peptides and nucleotides.
• High-purity product recovery with better resin utilization.
• Reduces solvent consumption and buffer usage.

Example: SMB is used in insulin purification to separate different isoforms.

Reference: "SMB Chromatography for Continuous Bioprocessing," Biotechnol. J., p. 512.

---

22. What is perfusion chromatography, and why is it beneficial for biologics purification?

Answer:
Perfusion chromatography uses large-pore resins to enhance mass transfer, allowing for high flow rates without sacrificing resolution.

Benefits in Biologics Processing:

• High-speed separations improve process throughput.
• Allows for better separation of large biomolecules (mAbs, viral vectors).
• Used for high-productivity purification of recombinant proteins.

Example: Perfusion chromatography is applied in continuous monoclonal antibody production.

Reference: "Perfusion Chromatography in Continuous Bioprocessing," J. Chromatogr. A, p. 269.

---

23. How does radial flow chromatography differ from conventional axial chromatography?

Answer:
Radial flow chromatography (RFC) passes the mobile phase radially through a cylindrical resin bed, rather than axially from top to bottom.

Advantages Over Axial Flow Chromatography:

• Higher flow rates without excessive pressure drop.
• Faster processing time and increased productivity.
• Ideal for large-scale biologics purification, such as plasma-derived products.

Example: RFC is used for immunoglobulin (IgG) purification from plasma.

Reference: "Radial Flow Chromatography in Large-Scale Purification," Bioprocess Int., p. 174.

---

24. What is membrane chromatography, and how is it used in biologics purification?

Answer:
Membrane chromatography uses functionalized membrane materials (instead of resin beads) for high-speed, low-pressure separations.

Advantages Over Traditional Column Chromatography:

• Faster processing times with higher flow rates.
• Low-pressure operation reduces shear stress on sensitive biologics.
• Effective for host cell protein (HCP) and DNA removal.

Example: Sartorius Sartobind® Q membranes are used for viral clearance in mAb purification.

Reference: "Membrane Chromatography for Bioprocessing," J. Chromatogr. B, p. 224.

---

25. What are the major failure modes in chromatography, and how can they be prevented?

Answer:
Common Chromatography Failures & Prevention Strategies:

Reference: "Troubleshooting Chromatographic Separations in Biopharmaceuticals," PDA J. Pharm. Sci., p. 341.

---

26. What are some key strategies for scaling up chromatography processes in biologics manufacturing?

Answer:
Scaling up chromatography requires maintaining separation efficiency while increasing process volume.

Key Strategies:

• Geometric scaling: Maintain column length-to-diameter ratio.
• Flow rate optimization: Maintain linear velocity instead of volumetric flow rate.
• Buffer preparation control: Ensure consistent buffer composition at scale.
• Automation and monitoring: Use Process Analytical Technology (PAT) for real-time control.

Reference: "Scale-Up Strategies in Bioprocess Chromatography," Biotech Prog., p. 288.

---

27. What is counter-current chromatography (CCC), and how does it differ from solid-phase chromatography?

Answer:
Counter-current chromatography (CCC) is a liquid-liquid separation technique, meaning there is no solid stationary phase. Instead, it uses two immiscible liquid phases.

Differences from Solid-Phase Chromatography:

• No resin required → Reduces material costs.
• High recovery rates → Avoids sample loss due to binding to solid supports.
• Used for high-purity separation of complex natural products.

Example: CCC is used for antibiotic and peptide purification.

Reference: "Counter-Current Chromatography in Bioseparations," J. Sep. Sci., p. 165.

---

28. What are the most critical chromatography parameters monitored during process validation?

Answer:

1. Peak shape (asymmetry factor, tailing factor)
2. Column efficiency (plate count, HETP)
3. Binding capacity (dynamic binding capacity, DBC50)
4. Recovery yield (%)
5. Host cell protein (HCP) removal efficiency
6. pH, conductivity, and buffer composition consistency

Reference: FDA Process Validation Guidance (2011), p. 142.

---

29. How is chromatography used in nucleic acid purification for gene therapy applications?

Answer:
Chromatography is critical for purifying plasmid DNA (pDNA), mRNA, and viral vectors used in gene therapies.

Key Techniques Used:

• Anion Exchange Chromatography (AEX): Separates supercoiled vs. linear vs. open circular DNA.
• Size-Exclusion Chromatography (SEC): Removes small fragments and host cell contaminants.
• Affinity Chromatography: Used for mRNA purification (e.g., polyA capture).

Example: AEX chromatography is used in AAV (Adeno-Associated Virus) purification.

Reference: "Chromatography in Gene Therapy Manufacturing," Biotech J., p. 421.

---

30. What is Quality by Design (QbD) in chromatography, and how does it impact process development?

Answer:
Quality by Design (QbD) in chromatography means designing processes with predictability, robustness, and control to ensure consistent product quality.

Key Elements of QbD in Chromatography:

• Critical Quality Attributes (CQAs): Define purity, yield, and impurity levels.
• Critical Process Parameters (CPPs): Optimize pH, temperature, flow rate, and buffer conditions.
• Design of Experiments (DoE): Uses statistical models to optimize conditions.

Example: QbD is used to develop scalable chromatography for monoclonal antibodies.

Reference: "ICH Q8 (R2) - QbD Principles in Biopharmaceuticals," ICH Guidelines, p. 32.

Chromatography Techniques in Biologics – Interview Q&A

(Part 2: Advanced Concepts & Applications)

Continuing with the next set of Q&A on chromatography used in biologics upstream and downstream processing, covering critical aspects for interview preparation.

---

11. What is mixed-mode chromatography, and how does it benefit biologics purification?

Answer:
Mixed-mode chromatography (MMC) utilizes resins with multiple interaction mechanisms, such as ion exchange, hydrophobic, and hydrogen bonding, to improve selectivity and efficiency in purification.

Benefits in biologics purification:

• Can capture or polish biomolecules in a single step.
• Improved removal of host cell proteins (HCPs) and DNA.
• Better yield and purity compared to conventional single-mode chromatography.
• Suitable for monoclonal antibodies (mAbs), fusion proteins, and enzymes.

Example: Capto® adhere resin is used for flow-through polishing of mAbs.

Reference: "Mixed-Mode Chromatography in Biopharmaceutical Processing," J. Chromatogr. A, p. 355.

---

12. What are the advantages of multi-column continuous chromatography over batch chromatography?

Answer:
Multi-column continuous chromatography (MCC) improves purification by using several small columns in sequence instead of a single batch column.

Advantages over batch chromatography:

1. Higher productivity – Continuous operation reduces downtime.
2. Better resin utilization – Uses smaller column volumes efficiently.
3. Improved yield – Reduces product loss in column dead volumes.
4. Lower buffer consumption – More efficient use of mobile phase.
5. Scalability – Easily adaptable to large-scale GMP manufacturing.

Reference: "Advances in Multi-Column Continuous Chromatography," Bioprocess Int., p. 129.

---

13. How does flow-through chromatography (FTC) work in impurity removal?

Answer:
FTC removes process-related impurities while allowing the target protein to pass through without binding to the resin.

Applications in biologics:

• Anion exchange flow-through chromatography (AEX-FTC): Removes host cell proteins (HCPs), DNA, and endotoxins.
• Cation exchange flow-through chromatography (CEX-FTC): Used for charge variant analysis and polishing.
• Hydrophobic interaction flow-through chromatography: Removes aggregates from mAbs.

Reference: "Process Development Strategies for Flow-Through Chromatography," PDA J. Pharm. Sci., p. 211.

---

14. What is Reverse Phase Chromatography (RPC), and why is it not commonly used in biologics purification?

Answer:
Reverse Phase Chromatography (RPC) separates biomolecules based on hydrophobic interactions with a non-polar stationary phase (e.g., C18 silica beads).

Limitations in biologics purification:

• Denaturation risk – Harsh organic solvents (e.g., acetonitrile) can damage protein structures.
• Low protein recovery – Strong hydrophobic interactions may cause protein precipitation.
• Limited scalability – Mostly used for analytical HPLC, not bulk purification.

Common Uses: Peptide mapping, protein characterization, and impurity profiling.

Reference: "Reversed-Phase HPLC of Therapeutic Proteins," J. Chromatogr. B, p. 89.

---

15. What are the key steps in chromatography column packing and qualification?

Answer:

1. Column Packing:

   • Prepare the slurry (resin and buffer).
   • Pump the slurry into the column under controlled flow rates and pressure.
   • Compress and stabilize the bed for uniform packing.

2. Qualification Tests:

   • Theoretical Plates (N): Measures column efficiency (N > 2000 for SEC).
   • Asymmetry Factor (AF): Should be 1.0–1.5 for well-packed columns.
   • HETP (Height Equivalent to Theoretical Plate): Measures bed performance.
   • Flow Distribution Test: Confirms uniformity across the column.

Reference: FDA Process Validation Guidance (2011), p. 138.

---

16. What is high-throughput screening (HTS) in chromatography resin selection?

Answer:
HTS uses robotic automation and micro-scale chromatography systems to rapidly test multiple resins and process conditions.

Benefits:

• Faster resin selection for process development.
• Reduces material and time costs.
• Allows for parallel testing of multiple conditions (pH, salt, flow rate).

Example: Tecan Freedom EVO® platform is used for high-throughput resin screening.

Reference: "High-Throughput Screening Strategies for Biologics Purification," Biotech Prog., p. 267.

---

17. What are the key regulatory guidelines for chromatography validation in biologics manufacturing?

Answer:

1. FDA Process Validation Guidance (2011) – Requires documented evidence of chromatography reproducibility.
2. ICH Q7 (Good Manufacturing Practice for APIs) – Ensures chromatography column lifetime studies and cleaning validation.
3. ICH Q8 (Pharmaceutical Development) – Defines chromatography process parameters as part of Quality by Design (QbD).
4. ICH Q9 (Quality Risk Management) – Requires risk assessment of chromatographic separations.
5. ICH Q10 (Pharmaceutical Quality System) – Ensures continuous process verification for chromatography.

Reference: ICH Q7, Section 12.5 (Chromatographic Purification).

---

18. What is viral clearance validation in chromatography, and why is it critical?

Answer:
Viral clearance validation ensures that chromatographic steps effectively remove or inactivate viruses in biologic drugs.

Common Chromatographic Steps for Viral Clearance:

1. Protein A chromatography – Removes viruses through ligand interaction.
2. Anion Exchange Chromatography (AEX-FTC) – Captures negatively charged viruses.
3. Hydrophobic Interaction Chromatography (HIC) – Removes viral particles based on hydrophobicity.

Regulatory Requirement:

• FDA & EMA guidelines require a ≥4 log reduction of virus load in purification processes.
• ICH Q5A: Viral Safety Evaluation for Biotechnology Products.

Reference: "Viral Clearance in Bioprocessing: Strategies and Methods," PDA Technical Report No. 41, p. 74.

---

19. How is chromatography used in biosimilar purification compared to originator biologics?

Answer:
Biosimilars require highly comparable purification strategies to match the reference biologic’s purity profile.

Key Considerations:

• Protein A affinity chromatography remains the same.
• IEX chromatography may be optimized for charge variants.
• HIC and SEC may require fine-tuning to achieve similar impurity levels.
• Additional purification steps may be needed to remove novel host cell proteins.

Regulatory Requirement:

• Biosimilars must demonstrate analytical similarity to the reference biologic per FDA 351(k) pathway.

Reference: "Chromatography Strategies for Biosimilar Development," J. Pharm. Sci., p. 182.

Interview Questions and Answers (Q&A) for a Biological Product Manufacturing Facility

Section 1: Manufacturing Processes

1. What are the key steps in the manufacturing process of a biological product?

The manufacturing of biological products involves several critical steps, including:

1. Cell Banking and Culture Initiation – Establishment of Master and Working Cell Banks.
2. Upstream Processing – Cultivation of cells using bioreactors, including media preparation, seed train development, and cell expansion.
3. Downstream Processing – Harvesting of the product, filtration, chromatography purification, and viral clearance.
4. Formulation and Fill-Finish – Buffer exchange, stabilization, sterile filtration, and aseptic filling.
5. Packaging and Labeling – Final packaging with compliance to regulatory standards.
6. Quality Control and Release – Testing for purity, potency, sterility, and identity before release.

Reference:
ICH Q5D: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products.

---

2. What are the differences between upstream and downstream processing in biologics manufacturing?

Upstream Processing:

• Focuses on cell culture, fermentation, and biomass production.
• Includes media preparation, inoculation, and bioreactor operation.
• Key objective: Maximize cell growth and product expression.

Downstream Processing:

• Involves cell harvest, filtration, chromatography, and purification.
• Includes viral clearance, buffer exchange, and concentration.
• Key objective: Isolate and purify the biologic product for formulation.

Reference:
FDA Guidance for Industry: Process Validation: General Principles and Practices, 2011 (Page 9-11).

---

3. What are the common expression systems used in biologics manufacturing?

Biologics can be produced using different expression systems, each with unique advantages:

1. Mammalian Cells (CHO, HEK293) – Used for monoclonal antibodies and complex proteins due to post-translational modifications.
2. Bacterial Cells (E. coli, Bacillus) – Used for recombinant proteins, insulin, and growth factors.
3. Yeast Cells (Pichia pastoris, Saccharomyces cerevisiae) – Used for vaccines and therapeutic enzymes.
4. Insect Cells (Sf9, Sf21) – Used for viral vectors and some vaccine production.

Reference:
WHO Guidelines on the Quality, Safety, and Efficacy of Biotherapeutic Protein Products, 2022 (Page 45).

---

4. What is the significance of viral clearance in biologics manufacturing?

Viral clearance is crucial to eliminate or reduce potential viral contaminants that may be introduced during cell culture. The key viral clearance methods include:

• Chromatography (e.g., ion-exchange and affinity chromatography)
• Nanofiltration (virus removal filters)
• Low pH treatment (inactivation of enveloped viruses)
• Solvent-Detergent Treatment (used for inactivating lipid-enveloped viruses)

Reference:
ICH Q5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin (Page 18-19).

---

5. What are the major factors influencing cell culture performance?

1. Cell Line Selection – Stability and productivity of the cell line.
2. Media Composition – Availability of essential nutrients, growth factors, and serum components.
3. Bioreactor Conditions – pH, temperature, oxygen levels, and agitation rate.
4. Feeding Strategies – Batch, fed-batch, or perfusion culture modes.
5. Contamination Control – Prevention of microbial and viral contamination.

Reference:
FDA Guidance on Cell-Based Products, 2021 (Page 22-24).

---

Section 2: Quality Control & Testing

6. What analytical methods are used for biologics testing?

1. Potency Assays – ELISA, cell-based assays, receptor binding assays.
2. Purity & Impurity Testing – SDS-PAGE, capillary electrophoresis, HPLC.
3. Identity Testing – Mass spectrometry, peptide mapping, sequencing.
4. Sterility & Endotoxin Testing – USP <71> Sterility Test, LAL test.
5. Viral Safety Testing – PCR-based assays, viral infectivity assays.

Reference:
USP <85> Bacterial Endotoxins Test, USP <71> Sterility Tests.

---

7. How is stability testing performed for biologics?

Stability testing ensures that biologics maintain potency, purity, and safety over time. Common studies include:

• Real-time Stability (storage at recommended conditions for shelf-life determination).
• Accelerated Stability (higher temperature stress testing).
• Forced Degradation Studies (pH, light, oxidation stress).
• Freeze-Thaw Stability (evaluates product integrity during freezing and thawing).

Reference:
ICH Q5C: Quality of Biotechnological Products – Stability Testing of Biotechnological/Biological Products (Page 8-10).

---

8. What are the key regulatory guidelines for biologics manufacturing?

1. FDA 21 CFR Part 600-680 – Biologics regulations.
2. ICH Q5A-Q5E – Biotechnology product guidelines.
3. WHO TRS 999 – Guidelines on biotherapeutics.
4. USP <1046> Biologics Testing – Analytical methods and quality requirements.

Reference:
ICH Q5E: Comparability of Biotechnological/Biological Products, 2004 (Page 6-7).

---

Section 3: Validation & Engineering

9. What is process validation in biologics manufacturing?

Process validation ensures that manufacturing processes consistently produce biologics that meet predetermined quality standards. It consists of:

1. Stage 1: Process Design – Development of process parameters and control strategy.
2. Stage 2: Process Qualification – Demonstration of reproducibility in commercial-scale production.
3. Stage 3: Continued Process Verification – Ongoing monitoring of critical parameters.

Reference:
FDA Guidance for Industry: Process Validation: General Principles and Practices, 2011 (Page 12-14).

---

10. What is the role of a cleanroom in biologics manufacturing?

Cleanrooms control environmental factors like particulate count, microbial contamination, and airflow. The key classifications are:

• Grade A (ISO 5) – Aseptic filling areas.
• Grade B (ISO 7) – Background for aseptic operations.
• Grade C & D (ISO 8-9) – Non-aseptic preparation areas.

Reference:
EU GMP Annex 1: Manufacture of Sterile Medicinal Products, 2022 (Page 18).

 

Section 4: Quality Assurance & Compliance

11. What are the key Good Manufacturing Practice (GMP) requirements for biologics manufacturing?

GMP requirements ensure that biologics are consistently produced and controlled to meet quality standards. The key elements include:

1. Personnel & Training – Qualified staff with GMP training.
2. Facility & Equipment – Cleanrooms, HVAC systems, validated equipment.
3. Documentation & Record Keeping – Batch records, deviation reports, CAPA.
4. Process Validation & Change Control – Consistency of manufacturing and controlled modifications.
5. Quality Control & Testing – Raw materials, in-process, and final product testing.
6. Sterility & Contamination Control – Aseptic processing, environmental monitoring.
7. Product Release & Stability Testing – Ensuring efficacy throughout shelf life.

Reference:
WHO GMP for Biological Products, TRS 999 (Page 34).

---

12. How is risk management applied in biologics manufacturing?

Risk management in biologics manufacturing follows the ICH Q9 Quality Risk Management (QRM) approach:

1. Risk Assessment – Identify potential hazards in materials, processes, or equipment.
2. Risk Control – Implement mitigation measures (e.g., redundancy in critical equipment).
3. Risk Communication – Ensure stakeholders are informed about identified risks.
4. Risk Review – Continuous monitoring and re-evaluation of risks.

Examples:

• Cross-contamination risk: Managed through facility segregation and dedicated equipment.
• Microbial contamination risk: Controlled via validated aseptic processing.

Reference:
ICH Q9: Quality Risk Management, 2005 (Page 7-9).

---

13. What is the difference between Corrective Action and Preventive Action (CAPA)?

Corrective Action (CA): Action taken to eliminate the cause of a detected nonconformity. Example: Identifying and addressing a sterility failure.

Preventive Action (PA): Proactive measures to eliminate the cause of a potential nonconformity. Example: Conducting periodic equipment requalification to prevent failures.

A CAPA system should include:

• Root cause analysis (RCA).
• Investigation reports.
• Effectiveness verification.

Reference:
FDA 21 CFR Part 820.100: CAPA Requirements (Page 56).

---

14. How is environmental monitoring performed in biologics manufacturing?

Environmental monitoring (EM) assesses contamination risks in classified areas. The key components include:

1. Airborne Particulate Monitoring – Particle counters in Grade A/B areas.
2. Viable Air Sampling – Active air sampling for microbial contamination.
3. Surface Monitoring – Contact plates and swabs.
4. Personnel Monitoring – Finger dabs and gowning assessments.

Frequency:

• Grade A/B: Per batch.
• Grade C/D: Periodically.

Reference:
EU GMP Annex 1: Manufacture of Sterile Medicinal Products, 2022 (Page 21).

---

15. What is the significance of Data Integrity in biologics manufacturing?

Data integrity ensures that all recorded information is Attributable, Legible, Contemporaneous, Original, and Accurate (ALCOA+ principles).

Key Requirements:

• Electronic data security (audit trails, access controls).
• Documentation practices (no backdating, corrections with justification).
• Validation of computerized systems (CFR Part 11 compliance).

Reference:
FDA Guidance on Data Integrity and Compliance, 2018 (Page 12-14).

---

Section 5: Validation & Engineering

16. What is cleaning validation, and why is it important?

Cleaning validation demonstrates that equipment cleaning effectively removes residues to prevent cross-contamination.

Key Elements:

1. Selection of Cleaning Agents – Compatibility with residues and equipment.
2. Worst-Case Scenario Studies – Evaluating the most difficult-to-clean areas.
3. Acceptance Criteria – Based on toxicity, therapeutic dose, and analytical limits.
4. Analytical Methods – Swab testing, rinse water analysis.

Reference:
FDA Guide to Inspections: Validation of Cleaning Processes, 1993 (Page 6-7).

---

17. What is HVAC validation, and why is it critical in biologics facilities?

HVAC validation ensures controlled temperature, humidity, and air quality in cleanrooms.

Key Parameters:

• Airflow Patterns (Unidirectional flow in aseptic zones).
• HEPA Filter Integrity Testing (Leak test using particle generators).
• Differential Pressure Monitoring (Maintains proper segregation).

Reference:
EU GMP Annex 1: HVAC Systems in Sterile Manufacturing, 2022 (Page 29).

---

18. What is the significance of media fills in aseptic processing?

Media fills (process simulations) confirm the sterility assurance level of aseptic processes.

Key Aspects:

1. Simulated Process – Uses sterile growth media instead of product.
2. Duration & Line Speed – Mimics routine production.
3. Acceptance Criteria – No microbial growth in filled units.
4. Frequency – At least twice per year per shift.

Reference:
FDA Aseptic Processing Guidance, 2004 (Page 19).

---

19. How is equipment qualification performed?

Equipment qualification (EQ) ensures that equipment functions consistently.

Phases of EQ:

1. Design Qualification (DQ) – Verifies design meets user requirements.
2. Installation Qualification (IQ) – Confirms proper installation and utilities.
3. Operational Qualification (OQ) – Tests equipment functionality.
4. Performance Qualification (PQ) – Ensures reproducible performance under actual conditions.

Reference:
EU GMP Annex 15: Qualification and Validation, 2015 (Page 10).

---

20. What are the differences between sterilization methods used in biologics manufacturing?

Reference:
USP <1229>: Sterilization of Compendial Articles (Page 15).

Section 6: Manufacturing Processes

21. What are the key steps in biologic drug substance manufacturing?

Biologic drug substance (DS) manufacturing involves upstream and downstream processing.

1. Upstream Processing

   • Cell line development (CHO, HEK, bacterial, etc.).
   • Cell culture expansion in bioreactors (fed-batch, perfusion).
   • Media optimization and monitoring.

2. Downstream Processing

   • Cell harvest (centrifugation, depth filtration).
   • Purification (chromatography, ultrafiltration).
   • Formulation (buffer exchange, stabilization).

3. Sterile Filtration & Storage

   • Final filtration through 0.2 µm membrane filters.
   • Bulk storage under controlled conditions.

Reference:
ICH Q5A: Viral Safety Evaluation of Biotechnology Products, 1998 (Page 12).

---

22. How is fed-batch culture different from perfusion culture in biologics production?

Perfusion is preferred for monoclonal antibody (mAb) production due to higher productivity and better product quality.

Reference:
WHO TRS 987: Guidelines on Biopharmaceuticals Manufacturing, 2014 (Page 30).

---

23. What are the common chromatography techniques used in biologics purification?

1. Affinity Chromatography – Uses ligand binding (e.g., Protein A for mAbs).
2. Ion Exchange Chromatography (IEX) – Separates based on charge differences.
3. Size Exclusion Chromatography (SEC) – Purifies based on molecular size.
4. Hydrophobic Interaction Chromatography (HIC) – Uses hydrophobicity for separation.

Example:

• mAb purification: Protein A → IEX → SEC.

Reference:
USP <1052>: Biotechnology-Derived Products (Page 22).

---

24. What are the critical process parameters (CPPs) in biologics manufacturing?

CPPs are parameters that affect product quality and consistency. Examples:

• Upstream CPPs

  • pH, dissolved oxygen, temperature.
  • Agitation speed, glucose concentration.

• Downstream CPPs

  • Column flow rate in chromatography.
  • Filtration pressure for sterile filtration.

Reference:
ICH Q8: Pharmaceutical Development, 2009 (Page 14).

---

25. How is viral clearance achieved in biologics purification?

Since biologics are produced in mammalian cell cultures, viral safety is critical.

Viral clearance methods:

1. Inactivation – Low pH treatment, solvent-detergent method.
2. Removal – 0.1 µm nanofiltration, chromatography steps.
3. Testing – PCR-based viral detection, infectivity assays.

Reference:
ICH Q5A: Viral Clearance & Testing, 1998 (Page 18).

---

Section 7: Analytical & Testing Methods

26. What are the key quality attributes tested for biologics?

Biologics undergo extensive testing to ensure safety, potency, and purity.

Reference:
USP <1043>: Analytical Testing of Biologics (Page 8).

---

27. What is Host Cell Protein (HCP) testing?

HCP testing detects residual cellular proteins from host expression systems (CHO, E. coli).

Methods:

• ELISA – Quantifies total HCP content.
• LC-MS/MS – Identifies specific HCP profiles.

Regulatory guidelines require HCP levels to be <100 ppm in final products.

Reference:
FDA Guidance on Residual Impurities in Biologics, 2016 (Page 22).

---

28. What is the importance of charge heterogeneity testing for biologics?

Charge variants can impact stability and bioactivity.

Methods:

• Capillary Isoelectric Focusing (cIEF) – Separates charge variants.
• Ion Exchange Chromatography (IEX) – Detects acidic or basic isoforms.

Example:

• Acidic variants in monoclonal antibodies may indicate deamidation or glycation.

Reference:
USP <129>: Charge Heterogeneity Analysis of Biologics (Page 5).

---

29. How is sterility testing performed for biologics?

Sterility testing ensures biologics are free from viable microorganisms.

Methods:

1. Membrane Filtration Method (Preferred for injectable biologics).
2. Direct Inoculation Method (For non-filterable products).

Incubation Conditions:

• Aerobic (TSA, 30-35°C, 14 days).
• Anaerobic (FTM, 20-25°C, 14 days).

Reference:
USP <71>: Sterility Tests (Page 10).

---

30. What are the differences between real-time and accelerated stability studies?

Reference:
ICH Q5C: Stability Testing of Biotechnological/Biological Products, 1995 (Page 7).

Section 8: Quality Assurance & Regulatory Compliance

31. What are the key regulatory guidelines for biologics manufacturing?

Biologic products are highly regulated to ensure safety, efficacy, and quality.

Reference:
ICH Q5A: Viral Safety Evaluation, 1998 (Page 15).

---

32. What is the role of Quality Assurance (QA) in biologics manufacturing?

QA ensures compliance with GMP regulations through:

1. Document Control – SOPs, batch records, change control.
2. Training & Qualification – Operator competency checks.
3. Deviation & CAPA Management – Root cause analysis, corrective actions.
4. Product Release – Review of test results, batch certification.

Reference:
WHO TRS 961: Good Manufacturing Practices for Biologics, 2011 (Page 45).

---

33. What is a comparability study in biologics?

Comparability studies demonstrate that manufacturing changes do not impact product quality.

When Required?

• Process changes (e.g., new purification method).
• Facility changes (e.g., new site or equipment).
• Formulation changes (e.g., buffer modification).

Testing Requirements:

• Analytical characterization (HPLC, MS, bioassays).
• Stability & impurity profiling.

Reference:
ICH Q5E: Comparability of Biotechnological/Biological Products, 2004 (Page 12).

---

34. What is the difference between biosimilars and biologics?

Example:

• Biologic: Humira (Adalimumab).
• Biosimilar: Amjevita (Adalimumab biosimilar).

Reference:
FDA Guidance on Biosimilars, 2015 (Page 18).

---

35. What are the major types of deviations in biologics manufacturing?

Deviations are classified based on impact on product quality and compliance.

Critical deviations require immediate investigation and CAPA implementation.

Reference:
EU GMP Annex 16: Certification by a QP, 2016 (Page 9).

---

Section 9: Validation & Engineering in Biologics Manufacturing

36. What are the key validation requirements for biologics manufacturing?

Validation ensures that processes consistently produce high-quality products.

Reference:
ICH Q11: Development and Manufacture of Drug Substances, 2012 (Page 21).

---

37. What are the phases of process validation in biologics?

1. Stage 1 – Process Design

   • Define Critical Process Parameters (CPPs).
   • Develop control strategy.

2. Stage 2 – Process Qualification

   • Conduct three consecutive validation runs.
   • Demonstrate process consistency.

3. Stage 3 – Continued Process Verification (CPV)

   • Routine monitoring of CPPs and CQAs.

Reference:
FDA Guidance on Process Validation, 2011 (Page 8).

---

38. What is risk-based validation in biologics manufacturing?

Risk-based validation focuses on high-risk areas using Quality Risk Management (QRM) principles.

Risk assessments follow ICH Q9: Quality Risk Management.

Reference:
ICH Q9: Quality Risk Management, 2005 (Page 10).

---

39. How is cleanroom classification done for biologics facilities?

Cleanrooms follow ISO 14644-1 classification based on airborne particle count.

Routine monitoring includes viable (bacteria, fungi) and non-viable (particles) testing.

Reference:
EU GMP Annex 1: Manufacture of Sterile Medicinal Products, 2022 (Page 35).

---

40. What are the key environmental monitoring parameters in a biologics facility?

Alert and action limits must be defined based on risk assessment.

Reference:
USP <1116>: Microbial Control in Cleanrooms (Page 18).

Section 3: Aseptic Processing, Contamination Control & Process Validation

41. What are the key considerations for aseptic processing in biologics manufacturing?

Aseptic processing is critical for biologics to prevent contamination. Key considerations include:

• Cleanroom Classification: Manufacturing must occur in controlled areas (ISO 5, 7, 8).
• Personnel Gowning: Strict gowning protocols to minimize contamination risks.
• Sterile Filtration: Biologics are typically filtered using 0.2 µm sterilizing-grade filters.
• Environmental Monitoring (EM): Real-time monitoring of viable and non-viable particles.
• Aseptic Process Simulation (Media Fill): Regulatory requirement to demonstrate sterility assurance.

Reference: FDA Guidance on Sterile Drug Manufacturing (2023), Section 5.2

---

42. How is contamination control managed in biologics manufacturing?

Contamination control involves:

• Strict Environmental Controls: Temperature, humidity, and pressure differentials are monitored.
• Closed Systems: Use of single-use bioreactors to reduce contamination risks.
• Personnel Training: Regular training on aseptic techniques.
• Disinfection Program: Use of validated disinfectants rotated to prevent microbial resistance.

Reference: EU Annex 1 on Sterile Manufacturing, Section 4.1

---

43. What is an aseptic process simulation (APS), and why is it required?

APS (or media fill study) tests the aseptic process by replacing the product with a sterile microbiological growth medium. It is required to:

• Demonstrate that the aseptic workflow does not introduce contamination.
• Validate operator aseptic techniques.
• Assess sterility assurance under worst-case conditions.

Regulatory Requirement: Must be performed at least semi-annually per FDA 21 CFR 211.167(a).

Reference: PDA Technical Report No. 22 on Media Fill Validation, Page 10

---

44. What is the difference between terminal sterilization and aseptic processing?

Reference: USP <1211> Sterilization and Sterility Assurance, Page 14

---

45. How is environmental monitoring (EM) performed in biologics facilities?

EM ensures compliance with aseptic conditions:

• Viable Monitoring: Settling plates, contact plates, and air samplers for microbial counts.
• Non-viable Monitoring: Laser particle counters for airborne particles.
• Trending & Investigation: Data is reviewed for out-of-trend (OOT) excursions.
• Action Limits:
  • ISO 5: ≤ 1 CFU/m³ (viable particles)
  • ISO 7: ≤ 10 CFU/m³

Reference: EU Annex 1, Table 1: Cleanroom Classification Limits

---

46. What is a smoke study, and why is it required?

A smoke study (or airflow visualization study) is required to:

• Verify that unidirectional airflow in aseptic areas effectively removes contaminants.
• Ensure that airflow does not create turbulence that can lead to contamination.

Regulatory bodies require documented video evidence of smoke studies, especially in filling lines and isolators.

Reference: FDA Aseptic Processing Guide, Section 3.2

---

47. What is the difference between clean-in-place (CIP) and sterilize-in-place (SIP)?

Reference: PDA Technical Report No. 29 on Cleaning Validation, Page 25

---

48. How is viral clearance validated in biologics manufacturing?

Viral clearance is validated using:

• Viral Inactivation:
  • Low pH treatment (inactivates enveloped viruses).
  • Detergent-based viral inactivation.
• Viral Removal:
  • Filtration: 20 nm virus-retentive filters.
  • Chromatography: Removes viruses via ion-exchange or size-exclusion chromatography.

Regulatory expectation: ≥ 4-log reduction for high-risk viruses.

Reference: EMA Guideline on Viral Safety Evaluation, Page 18

---

49. How are bioburden and endotoxins controlled in biologics?

• Bioburden: Controlled using pre-filtration, strict water system control (WFI), and environmental monitoring.
• Endotoxins: Removed by:
  • Depyrogenation (Heat > 250°C)
  • Ultrafiltration (< 10 kDa filters)
  • Chromatographic purification

Reference: USP <85> Bacterial Endotoxins Test, Page 6

---

50. What is filter integrity testing, and why is it important?

Sterilizing filters (0.2 µm) must be integrity tested before and after use to confirm sterility assurance. Methods include:

• Bubble Point Test: Determines the pressure required to force air through a wetted filter.
• Forward Flow Test: Measures gas flow through a wetted filter at constant pressure.
• Diffusion Test: Detects integrity breaches by measuring gas diffusion rates.

Reference: PDA Technical Report No. 26 on Filtration Validation, Page 32

Section 4: Facility Design, Process Control & Case Studies

61. What are the key facility design considerations for a Biological Product Manufacturing Facility?

A Biological Product Manufacturing Facility must be designed to ensure contamination control, compliance with regulatory requirements, and efficient process flow. Key considerations include:

• Segregation of Processing Areas: To prevent cross-contamination, areas for cell culture, purification, formulation, and fill-finish must be separated.
• HVAC & Cleanroom Design: Classified areas (ISO 5, 7, 8) with proper airflow, pressure differentials, and HEPA filtration.
• Personnel and Material Flow: Unidirectional movement of personnel, materials, and waste to minimize contamination risks.
• CIP/SIP Systems: Automated Cleaning-in-Place (CIP) and Sterilization-in-Place (SIP) for equipment.
• Environmental Monitoring System (EMS): To continuously monitor critical parameters such as temperature, humidity, and differential pressure.
• Single-use Technology (SUT): Reducing cleaning validation challenges by implementing disposable bioreactors and tubing.

Reference: FDA Guidance on Sterile Drug Manufacturing (2023), Section 6.3

---

62. How is process control implemented in biologics manufacturing?

Process control in biologics manufacturing ensures product consistency, efficacy, and safety. Key elements include:

• Process Analytical Technology (PAT): Real-time monitoring of critical parameters like pH, oxygen levels, and nutrient concentrations.
• Automation & SCADA Systems: Automated data collection and real-time process adjustments.
• Risk-Based Approach: Failure Mode and Effects Analysis (FMEA) to identify and mitigate risks in production.
• Real-time Release Testing (RTRT): Advanced analytics to release batches without waiting for full QC testing.
• Deviation Management System: Investigating and documenting process deviations to ensure compliance.

Reference: ICH Q10 Pharmaceutical Quality System, Section 4.2

---

63. What is a case study example of a contamination event in biologics manufacturing?

Case: Mycoplasma Contamination in CHO Cell Culture

• Issue: A leading biopharmaceutical company detected mycoplasma contamination in CHO (Chinese Hamster Ovary) cell cultures, leading to batch failures.
• Root Cause Analysis:
  • Contaminated raw materials (serum supplements).
  • Inadequate testing of incoming raw materials.
  • Insufficient filtration control.
• Corrective Actions:
  • Implementation of high-throughput mycoplasma testing.
  • Switch to serum-free media.
  • Enhanced supplier qualification procedures.
• Outcome: Reduced contamination incidents by 95% over 12 months.

Reference: PDA Technical Report No. 50 on Mycoplasma Contamination, Page 18

---

64. How is cross-contamination prevented in multi-product biologics facilities?

Cross-contamination prevention strategies include:

• Dedicated Production Suites: Separate HVAC, equipment, and personnel flow for different products.
• Sequential Production Batches: Cleaning validation between different product batches.
• Airflow Control: Unidirectional airflow and pressure differentials to prevent mixing of airborne contaminants.
• Validated Cleaning Protocols: Use of worst-case challenge conditions for cleaning validation.
• Electronic Batch Records (EBR): Prevents mix-ups by digitally tracking material use and processing steps.

Reference: EMA Guideline on Cross-Contamination in Shared Facilities, Section 3.4

---

65. What is the role of data integrity in biologics manufacturing?

Data integrity ensures that all manufacturing and testing data are complete, consistent, and accurate. Key principles:

• ALCOA+ Principles: Data should be Attributable, Legible, Contemporaneous, Original, Accurate, Complete, Consistent, Enduring, and Available.
• Audit Trails: Every modification in electronic records must be traceable.
• Access Controls: Restricted access to critical manufacturing and QC data.
• Data Review & Trending: Statistical tools are used to detect out-of-trend (OOT) results.
• Regulatory Compliance: Compliance with 21 CFR Part 11 (FDA) and EU Annex 11 for computerized systems.

Reference: FDA Guidance on Data Integrity and Compliance, Section 5

---

66. How does risk management apply to biologics manufacturing?

Risk management helps identify, evaluate, and mitigate risks in the manufacturing process. Approaches include:

• Quality Risk Management (QRM) per ICH Q9:
  • Risk Assessment: Identifying risks to product quality.
  • Risk Control: Implementing mitigation strategies.
  • Risk Review: Continuous monitoring and improvement.
• Failure Mode and Effects Analysis (FMEA): Evaluating failure points in equipment, process, or raw materials.
• HACCP (Hazard Analysis and Critical Control Points): Applied for contamination risk in biologics processing.

Reference: ICH Q9 Quality Risk Management, Page 12

---

67. What are the key challenges in downstream purification of biologics?

Downstream processing challenges include:

• Host Cell Protein (HCP) Removal: Requires effective chromatography and ultrafiltration.
• Aggregates & Particulates: Must be removed using advanced filtration and SEC (Size-Exclusion Chromatography).
• Viral Clearance: Must achieve at least 4-log reduction via viral filtration and inactivation steps.
• High Process Variability: Even slight changes in pH or salt concentration can impact yield.

Reference: PDA Technical Report No. 41 on Purification Strategies, Page 21

---

68. What is the impact of continuous bioprocessing on biologics manufacturing?

Continuous bioprocessing offers benefits such as:

• Increased Productivity: Continuous perfusion culture provides higher cell densities than batch processes.
• Reduced Footprint: Smaller bioreactors with high efficiency replace large batch systems.
• Improved Process Control: Real-time monitoring allows for immediate adjustments.
• Lower Costs: Reduced raw material consumption and labor costs.

Reference: ISPE Continuous Manufacturing Guide, Section 7

---

69. What is the regulatory expectation for viral clearance validation?

Viral clearance studies must demonstrate that manufacturing processes remove or inactivate potential viral contaminants.

• Key Methods:
  • Filtration (Virus Removal): Nanofiltration removes viruses >20 nm.
  • Low pH Treatment: Inactivates enveloped viruses.
  • Chromatography Steps: Capable of reducing viral load.
• Regulatory Expectation:
  • FDA & EMA require ≥ 4-log reduction for high-risk viruses.
  • Validation studies must use model viruses to challenge process steps.

Reference: FDA’s Points to Consider in the Manufacturing of Biotech Products, Section 3.5

---

70. What is the difference between biosimilars and biologics in terms of manufacturing?

Reference: FDA Biosimilar Guidance, Section 4