Here are some key points to consider for raw material selection to control endotoxin contamination:- Select raw materials from reputable suppliers with a strong quality system in place- Qualify suppliers based on their bioburden/endotoxin control processes and limits - Establish specifications for raw materials based on intended use and risk assessment- Include bioburden and endotoxin limits in material specifications- Require supporting documentation like certificates of analysis from suppliers- Perform identity and quality testing of raw materials upon receipt- Consider additional testing like bioburden screening for higher risk materials- Store and handle materials appropriately to prevent contamination - Establish retest/requalification periods based on material stability and risk
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In this webinar, you will learn: Sources of endotoxin contamination Contamination control strategy Endotoxin removal strategies Detailed description: Endotoxin, a lipopolysaccharide (LPS), is a type of pyrogen and is a component of the exterior cell wall of Gram-negative bacteria. To ensure safety on patient’s endotoxin content in the drug should always be controlled. In a biological processing it may emanate from facility, utility, raw materials, process, and personnel. In this webinar we discuss the regulatory norms, strategies for prevention & removal of endotoxin to ensure that the final drug product is safe.
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Similar to Here are some key points to consider for raw material selection to control endotoxin contamination:- Select raw materials from reputable suppliers with a strong quality system in place- Qualify suppliers based on their bioburden/endotoxin control processes and limits - Establish specifications for raw materials based on intended use and risk assessment- Include bioburden and endotoxin limits in material specifications- Require supporting documentation like certificates of analysis from suppliers- Perform identity and quality testing of raw materials upon receipt- Consider additional testing like bioburden screening for higher risk materials- Store and handle materials appropriately to prevent contamination - Establish retest/requalification periods based on material stability and risk
Similar to Here are some key points to consider for raw material selection to control endotoxin contamination:- Select raw materials from reputable suppliers with a strong quality system in place- Qualify suppliers based on their bioburden/endotoxin control processes and limits - Establish specifications for raw materials based on intended use and risk assessment- Include bioburden and endotoxin limits in material specifications- Require supporting documentation like certificates of analysis from suppliers- Perform identity and quality testing of raw materials upon receipt- Consider additional testing like bioburden screening for higher risk materials- Store and handle materials appropriately to prevent contamination - Establish retest/requalification periods based on material stability and risk (20)
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Here are some key points to consider for raw material selection to control endotoxin contamination:- Select raw materials from reputable suppliers with a strong quality system in place- Qualify suppliers based on their bioburden/endotoxin control processes and limits - Establish specifications for raw materials based on intended use and risk assessment- Include bioburden and endotoxin limits in material specifications- Require supporting documentation like certificates of analysis from suppliers- Perform identity and quality testing of raw materials upon receipt- Consider additional testing like bioburden screening for higher risk materials- Store and handle materials appropriately to prevent contamination - Establish retest/requalification periods based on material stability and risk
- 1. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. ENDOTOXIN CONTROL STRATEGY IN BIOMANUFACTURING Somasundaram G (Som) Senior Consultant, Asia Pacific Global BioPharm CoE Subhasis Banerjee Ph.D. Principal Application Expert, APAC Bioprocessing Customer Applications
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
- 3. Disclaimer: Views expressed in this talk constitute our professional opinion
- 4. Agenda 1 2 3 Endotoxin & sources of contamination Contamination Control Strategy (CCS) Raw material selection 4 Removal of endotoxin 5 Case Studies
- 5. Endotoxin & sources of contamination
- 6. What are Pyrogens? Substances that induce fever when Ingested into the body by various routes Origin/Source Microbial - Bacteria Non-microbial - Inorganic colloids, steroids The most frequently encountered pyrogen in pharmaceutical processing is : "Endotoxin" - material associated with the outer cell wall of gram negative bacteria. 6 Endotoxin control strategy in biopharma manufacturing
- 7. Endotoxins are continually shed from bacteria during its life cycle; they are also released when the bacterial cell dies 7 Sources of Endotoxin Contamination Endotoxin control strategy in biopharma manufacturing Image : wakopyrstar.com Probability of contamination exists wherever bacteria are present - air, water, food etc.
- 8. Physico - Chemical Characteristics of Endotoxins Innermost region composed of lipid A Hydrophobic Responsible for pyrogenicity Outer region composed of polysaccharides Hydrophilic Structure Composition Complexes of Lipopolysaccharides (LPS) containing lipids, carbohydrates and proteins Purified form (LPS) does not contain protein 8 Endotoxin control strategy in biopharma manufacturing
- 9. Physico-Chemical Characteristics of Endotoxins Size Their amphiphilic nature leads to aggregation in solution Aggregates vary in size based on solution chemistry - 10,000-1000,000 Daltons has been observed Smallest irreducible size is found to be > 10,000 Daltons* Charge At pH > 2, endotoxins are negatively charged and behave as anions The negative charge results due to the presence of acidic phosphate and carboxyl groups in the molecule * K. J. Sweadner et al., Applied and Environmental Microbiology, 34, 1977, p382. 9 Endotoxin control strategy in biopharma manufacturing
- 10. Why Depyrogenate? Prevention of endotoxin contamination, though ideal, cannot always be ensured Endotoxins are not removed during the sterilization step (sterile filtration or autoclaving) Sensitivity of mammals to Endotoxins is extremely high trace contamination levels can induce severe reactions ranging fever to death – (0.1 ng of LPS / kg body weight required to create an adverse reaction (1 EU/ml) 10 Endotoxin control strategy in biopharma manufacturing
- 11. Assays for Endotoxin (USP issue <23>, Chapter <151>, Chapter Endotoxin Test <85>) Pyrogen Test Measures the temp. rise of rabbits following the intravenous injection of a test solution Designed for products that can be tolerated by the test rabbit Dose: must not exceed 10 ml/kg injected; injection time not to exceed 10 minutes Bacterial Endotoxin Test Uses a reagent Limulus Amebocyte Lysate (LAL) which reacts with endotoxins to form a clot Endotoxin levels are measured by recording the spectrophotometric light absorbance at suitable wavelengths Tests can be End-Point (gel clot) or kinetic (turbidimetric & colorimetric) The rFC method is now a compendial method in the European and Chinese Pharmacopoeias. The Japanese Pharmacopeia allowed the rFC method as an alternative in its XVIII edition and is moving toward a compendial status in a couple of years. Also, the South Korean Agency conducted an independent study with manufacturers and found equivalency between rFC and LAL. Overall, rFC has been accepted in more than 60 countries. 11 Endotoxin control strategy in biopharma manufacturing
- 12. Poll Question
- 14. • Why & What is Contamination Control Strategy • Understand the impact of bioburden excursions • Recognize the sources of bioburden • Develop strategies to mitigate risk Endotoxin control strategy in biopharma manufacturing 14 Contamination control strategy (CCS)
- 15. CCS – Definition & Regulatory Drivers 15 “A planned set of controls for microorganisms, pyrogens and particulates, derived from current product and process understanding that assures process performance and product quality”. The controls can include parameters and attributes related to active substance, excipient and drug product materials and components, facility and equipment operating conditions, in-process controls, finished product specifications, and the associated methods and frequency of monitoring and control. EU GMP Annex 1 Draft 2020: ‘A contamination control strategy should be implemented across the facility in order to assess the effectiveness of all the control and monitoring measures employed’ CCS is mentioned 44 times in the update PIC/S GMP Part I, 2021: ‘Cross-contamination should be prevented for all products by appropriate design and operation of manufacturing facilities. The measures to prevent cross contamination should be commensurate with the risks. Quality Risk Management principles should be used to assess and control the risks’ ‘5.10 At every stage of processing, materials and products should be protected from microbial and other contamination’ Endotoxin control strategy in biopharma manufacturing
- 16. 16 Contamination Control Strategy: Holistic program that encompasses concepts within the context of the entire manufacturing facility and process Facility Design: Designed according to GMP Regulations / Guidelines, Best practice and process requirements Personnel: Gowning regime with personnel monitoring program, suitable limits and action plan, training and culture Cleaning and Disinfection/Sterilisation: Consistent removal of soiling and microbial load deactivation Environmental Monitoring: Monitor viable and non- viable, suitable limits and action plan Process Simulation: End to end testing including intervention assessment and worse case stressing Process Design: Process controls including GMP and GEP to ensure product is microbial/pyrogen, chemical, non-viable and cross-contamination free. What is a Contamination Control Strategy Process Design Process Simulation Environmental Monitoring Cleaning & Disinfection/Sterlisation Personnel Facility Design Contamination Control Strategy Endotoxin control strategy in biopharma manufacturing
- 17. Poll Question
- 18. 18 Microbial Byproducts Endotoxin Exotoxin Lipopeptides Flagellin DNA Extracellular proteases Bioburden Bacteria Fungi Mycoplasma Virus TSE Agents Why : Bioburden and their byproducts Scope Endotoxin control strategy in biopharma manufacturing
- 19. Facility & Environment Equipment Processes Materials Utilities Personnel Each source contributes to the process bioburden profile Sources of bioburden 19 Staphylococcus Bacillus Non-fermenting Gram Negative rods Aspergillus Source: Public domain CDC/ Robert Simmons Endotoxin control strategy in biopharma manufacturing
- 20. Many routes for microbial ingress Bioburden excursions are often the result of Improper cleaning, storage, or sanitization Suboptimal system design Aseptic connections Sampling Lapses in aseptic technique Intensive risk assessments can prevent many of these contaminations or excursions Key Points Sources of bioburden/endotoxins Endotoxin control strategy in biopharma manufacturing 20
- 21. Downstream Risk profile & control strategies differ throughout the process Secondary Clarification Chromatography Protein A Final Filling Final Sterile Filtration Concentration & Formulation Bulk Storage and Transport Viral Inactivation Chromatography CEX Virus Filtration Clearance Ultrafiltration / Diafiltration Bioreactor Primary Clarification MCB WCB Seed Train Raw Materials Filtration Bioburden Reduction Bioburden Reduction Chromatography AEX Bioburden Reduction Bioburden Reduction Final Fill Risk Areas Upstream Downstream Endotoxin control strategy in biopharma manufacturing 21
- 22. Assess Mitigate Monitor Risk Mitigation Strategies Endotoxin control strategy in biopharma manufacturing 22
- 23. 23 Assess Risks Key Points Characterize the microbial profile of the process Utilize a combination of assessment tools A cross-functional team is crucial to the process Your bioburden/Endotoxin risk mitigation strategy should address Patient safety Drug supply Business risk
- 24. Risk Mitigation Material Considerations Bioburden profile What microorganisms are present? How many? Variation over time? Toxin producing? Spore formers? Material Origin Material consistency Supplier transparency Quality management system Quality philosophy Material Characteristics Growth Promoting Bacteriostatic Bactericidal
- 25. 25 Downstream Monitoring What do I test for? Where? Why? Chromatography Protein A Bulk Storage and Transport Viral Inactivation Chromatography CEX Virus Filtration Clearance Ultrafiltration / Diafiltration Bioburden Reduction Bioburden Reduction Chromatography AEX Bioburden Reduction Bioburden Reduction Bioburden Virus Mycoplasma Endotoxin V M B E B E B E B E V E V E B E B E M Monitor Endotoxin control strategy in biopharma manufacturing
- 26. Poll Question
- 28. Control of Endotoxin Contamination: raw materials • Inspection and Testing (LAL) of Contamination Sources: Utilities Water: WFI quality, Less than 0.25 EU/ml. • Incoming Raw Materials • In process Chemicals/Buffers • Certificates • Sampling etc. • No beta-glucan interference for false-positive results 28 Endotoxin control strategy in biopharma manufacturing
- 29. WFI specifications as per USP Chapter <1231> https://www.pharmaguideline.com/2011/03/specification-for-water-for-injection.html Endotoxin control strategy in biopharma manufacturing Sampling & testing of WFI 1. Description Clear, colorless & odorless liquid 2. Total Organic Carbon Note more than 500ppb 3. Conductivity (at 25’C) Less than 1.3mS/cm 4. Bacterial Endotoxins Less than 0.25EU/ml 5. Microbial limit test a. Total aerobic microbial count b. Pathogens i. Escherichia coli ii. Salmonella sp. iii. Pseudomonas aeruginosa iv. Staphylococcus aureus <10cfu/100ml Should be absent Should be absent Should be absent Should be absent 6. Acidity or alkalinity No red or blue color produced 7. pH Between 5.0 & 7.0 8. Ammonium <0.2ppm 9. Calcium and Magnesium A pure blue color is produced 10. Heavy metals <0.1ppm 11. Chlorides The solution shows no change in appearance 12. Nitrates <0.2ppm 13. Sulphates The solution shows no change in appearance 14. Oxidizable substances The solution remains faintly pink 15. Residue on evaporation <0.001% https://www.sigmaaldrich.com/IN/en/product/mm/pyr0matkit
- 30. Raw materials selection : COQ Endotoxin control strategy in biopharma manufacturing 30 Cell culture media (Add BPM product COA) Sterilizing grade filter
- 31. 31 1X0 devices flushed with water/buffer, as indicated (600 LMH, 50 L/m2 or 25 L/m2 with buffer) Format Beta-glucan LAL assay (pg/mL)1 water buffer X0HC < 25.3 < 80 X0SP < LOQ < LOQ β-(1-3) Glucan Significance Operational β-Glucan can cause false positive in traditional Limulus Amebocyte Lysate (LAL) test for detecting endotoxins. Regulatory β-Glucans are considered as “process-related impurities” categorized under downstream derived impurities as per International Conference on Harmonization (ICH) Topic Q6B. Patient Safety β-Glucan levels in patient’s blood streams are monitored for detection of fungal and bacterial infections. • No beta-glucans to interfere with limulus amoebocyte lysate (LAL) testing for bacterial endotoxins Raw materials selection : beta-glucan Endotoxin control strategy in biopharma manufacturing
- 32. Handling Transport of materials in the facility Testing Sampling Transfer into different packaging Storage conditions Weighing Sieving Crushing Sifting Process Flow Raw Materials Each step may introduce contamination into the process Water transfer (cleaning, compounding) Compounding Mixing Hold times Dispensing Sampling Room Cleaning Equipment Cleaning Personnel Hygiene How do I assess the risk of these parameters?
- 33. Prevent • Remove animal derived components • Caution! Serum-free does not mean mycoplasma free • Consider chemical free • Recombinant alternatives to serum • r-Insulin, r-Transferrin & r-albumin • Select raw material quality grade • Pharmaceutical grade versus analytical grade • Audit vendor Mitigate • Pre-treat components • Choose treatments effective for viral and bacterial reduction Detect • Screen raw material with rapid tests – Caution! Sample sizes versus kg to tons of material Prevent Raw Material Contamination Raw Material Selection
- 35. Methods of Endotoxin Removal Non-Filtration Depyrogenation achieved by inactivation of the molecule Examples of Processes: Acid/Base or Polymyxn B Treatment, Hydrogen Peroxide (Oxidation), Heat (Moist and Dry) Pyrogenicity of the molecule is 'lost' by structural changes; 'the altered molecule' may still be present Filtration: Depyrogenation achieved by removal of the molecule Examples of Processes: Size Exclusion - Separation based on size (RO &UF) Adsorption - Separation based on charge or hydrophobic interaction (MF or depth filtration AEX chromatography Binding and elute chromatography Depends on the isoelectric point of the protein pH range stability of the protein 35 Endotoxin control strategy in biopharma manufacturing
- 36. Endotoxin Removal by Filtration: Size Exclusion Principle - effective 'pore' size of the membrane filter is smaller than the effective size of the endotoxin Membrane "Pore" Rating - membranes are available in a range of pore sizes that have a correspondence to the MWs of marker proteins they can retain - classified into ultrafiltration, nanofiltration & reverse Osmosis Membrane Mol. Weight Cut Off (kilodaltons) 100 kD 1000 kD 0.1 kD 1 kD 10 kD Membrane Process Reverse Osmosis Nanofiltrati on Ultrafiltration "Loose" "Tight" 36 Endotoxin control strategy in biopharma manufacturing
- 37. Endotoxin Removal by Filtration: Size Exclusion - Endotoxin Aggregation State Solution Endotoxin Aggregation Form Size Water Vesicle Submicron Saline 0.9% Vesicle Submicron MgCl2 5 mM Vesicle Submicron EDTA 5 mM Micelle 300-1000 kD 1% Sodium deoxycholate Micelle or Sub Unit down to 10-20 k Daltons 2% Sodium deoxycholate & 5mM EDTA (Worst Case) Sub unit 10-20 k Daltons K. J. Sweadner et al., Applied and Environmental Microbiology, 34, 1977, p382. Endotoxin Size Depends on Solution Chemistry! How big is the endotoxin molecule? 37 Endotoxin control strategy in biopharma manufacturing
- 38. Endotoxin Removal by Filtration: Size Exclusion - membrane selection Membrane Flux LRV 100 kD 1000 kD 1 kD 10 kD Endotoxin Aggregate Range Soln. 1 Soln. 2 Aggregation State of Endotoxin - high LRV High Process Flux - Affects System Size Ease of passage of Other Solutes - buffer salts, sugars, amino acids etc. Membrane/solvent compatibility 38 Endotoxin control strategy in biopharma manufacturing
- 39. Endotoxin Removal by Filtration Size Exclusion - what do you choose? Depends on knowledge of endotoxin aggregation size & extent of LRV desired - pilot testing at worst case conditions. Classically, ultrafiltration using a 10 kD membrane is a widely employed process. A more open ultrafiltration may give the benefits of higher flux - leads to lower system size/cost 39 Endotoxin control strategy in biopharma manufacturing
- 40. A 100 kD UF membrane may also be used to remove endotoxins if they are aggregated to a size >> 100 kD Nanofiltration (0.1-1 kD) may be used for endotoxin removal from solutions with very low MW solutes (NaCl, amino acids) & higher filtrate purity. Reverse Osmosis (< 0.1kD) is good only for removal of endotoxins from water 40 Endotoxin control strategy in biopharma manufacturing Endotoxin Removal by Filtration Size Exclusion – additional comments
- 41. Ultrafiltration (TFF): Effectiveness in Depyrogenation Solution 0.22 m 0.025 m 1,000 kD NMWCO 100 kD NMWCO 10 kD NMWCO Water 0 > 4 > 4 > 4 > 4 0.9% NaCl 0 > 4 > 4 > 4 > 4 5 mM MgCl 0 > 4 > 4 > 4 5 mM EDTA >= 4 > 4 > 4 1% Na Cholate 1 2 > 5 2% Na Cholate & 5 mM EDTA 0 0 > 5 1% Na Deoxycholate 0 0 > 5 A Range UF Membranes (10 - 1000 kD) can retain Endotoxins depending on Solution Condition (LRV on Membranes) 41 Endotoxin control strategy in biopharma manufacturing Internal data
- 42. Endotoxin Removal by Filtration: Adsorptive Forces Electrostatic - Due to charge/charge interaction; Unlike charges attract and like charges repel Dispersion or Van der Waals - Forces that exist between all atoms & molecules; quantum mechanical in origin; generally weak Hydrogen Bonding - Ability of a H atom bound to electronegative atom (O) to attract other electronegative atoms; unique electrostatic attraction Hydrophobic interactions - Entropic in origin; results from the need for water molecules to maximize their conformational disorder. 42 Endotoxin control strategy in biopharma manufacturing
- 43. Endotoxin Adsorption: Results Membrane Liquid Filtered EU/cm2 LRV Charged Durapore®, 0.2 micron Water > 5 x 105 > 5.0 Charged Durapore®, 0.2 micron Mannitol > 400 >1.5 Charged Durapore®, 0.2 micron Water with 150 mM NaCl very low very low Hydrophobic Durapore®, 0.2 micron Water with 150 mM NaCl >1630 > 3.35 Hydrophobic Durapore®, 0.45 micron Water with 150 mM NaCl 1630 3.1 43 Endotoxin control strategy in biopharma manufacturing
- 44. Endotoxin Removal by Filtration Limitations of Adsorption Processes Adsorption processes (charged & hydrophobic) can proceed only until the membrane capacity is not exhausted when adsorption sites are filled, breakthrough occurs In general, adsorption processes depend on solution chemistry pH, Ionic Strength, competitive adsorption by other solutes 44 Endotoxin control strategy in biopharma manufacturing
- 45. 45 Endotoxin Removal by AEX chromatography Anion exchange High ionic strength Low ionic strength Non-binding sample constituents Chromatogram Endotoxin control strategy in biopharma manufacturing
- 46. o Endotoxin pI 2-4 o For basic proteins pI > 7 , ET binds to AEX matrix at neutral pH eluting at a higher conductivity than proteins o For acidic proteins : ET may co-adsorbed along resulting in product loss o Use of detergents in the wash step facilitates ET removal by chrome Endotoxin control strategy in biopharma manufacturing 46 Endotoxin Removal by AEX chromatography
- 47. Clearance vs. Yield Anion exchange chromatography Bind & Elute separation Both AEX resins show similar performance: • 2.5 – 3.0 LRV endotoxin clearance • 85 – 90% BSA yield Endotoxin control strategy in biopharma manufacturing Feed: Endotoxin 5.0E+05 EU/mL + BSA 5 mg/mL Fractogel® DEAE & Eshmuno® Q at 3.4mL CV (10cm BH) the elution pool was analyzed for endotoxin LRV and BSA yield. E.Coli endotoxin (pI ~2) and BSA(pI ~4.8) as model protein spiked into buffer. Loading feed was formulated at pH5 and no salt for both binding.
- 48. Clearance vs. Yield Anion exchange chromatography Flow-Through separation Fractogel® DEAE shows better performance: • Endotoxin clearance 1.4 LRV at DEAE and 0.9LRV at Q • ≥ 87% flowthrough of BSA Endotoxin control strategy in biopharma manufacturing Feed: Endotoxin 5.0E+05 EU/mL + BSA 5 mg/mL Fractogel® DEAE & Eshmuno® Q at 3.4mL CV (10cm BH) the elution pool was analyzed for endotoxin LRV and BSA yield. E.Coli endotoxin (pI ~2) and BSA(pI ~4.8) as model protein spiked into buffer. Loading feed was formulated at pH5 with 8-10 mS/cm sodium chloride.
- 49. Poll Question
- 50. Case studies
- 51. Case Study Bioburden/Endotoxin excursions in the Protein A Pool Chrom Protein A Bulk Storage and Transport Viral Inactivation Chrom CEX Virus Filtration Clearance Ultrafiltration Diafiltration Bioburden Reduction Bioburden Reduction Chrom AEX Bioburden Reduction Bioburden Reduction Situation • Spore-forming bioburden alert-level excursions in the Protein A pool over several campaigns Root Cause • Failure to recognize a trend in the pattern of excursions • Sanitization solution was not sporicidal • Sub-optimal sanitization process Corrective and Preventative Actions • Scale down studies with a new sanitizer and optimization of sanitization conditions • Process scale verification Endotoxin control strategy in biopharma manufacturing
- 52. Case Study Bioburden action-level excursions of in the UF/DF step Chrom Protein A Bulk Storage and Transport Viral Inactivation Chrom CEX Virus Filtration Clearance Ultrafiltration Diafiltration Bioburden Reduction Bioburden Reduction Chrom AEX Bioburden Reduction Bioburden Reduction Situation • Bioburden and endotoxin exceeded action levels in multiple batches • Intensive investigation of the process and support areas Root Cause • Bioburden formation in the TFF cassettes due to inadequate cleaning and storage processes Corrective and Preventative Actions • Improve cleaning and storage processes • Sterilization or sanitization of buffer tanks • Assessment of the water for injection (WFI) system and transfer lines • Introduction of bioburden reducing filters • Validation of hold times and storage conditions • Revision of bioburden limits based on process capability Buffer, Sanitizer, and Storage Solutions Operations WFI Operation Suvarna K. et. al. “Case Studies of Microbial Contamination in Biologic Product”, American Pharmaceutical Review 14(1) January/February 2011. Endotoxin control strategy in biopharma manufacturing
- 53. Case Study Sporadic bioburden action-level excursions Chrom Protein A Bulk Storage and Transport Viral Inactivation Chrom CEX Virus Filtration Clearance Ultrafiltration Diafiltration Bioburden Reduction Bioburden Reduction Chrom AEX Bioburden Reduction Bioburden Reduction Situation • Sporadic mixed bioburden excursions at multiple points in the downstream process Root Cause • Aseptic connections of equipment and sampling devices Corrective and Preventative Actions • Short term: • Retrained operators in aseptic techniques • Long term: • Reduced the number of aseptic connections • Implemented sterile to sterile connectors and steam to sterile connectors. • Introduced a facility-wide sterile sampling system Endotoxin control strategy in biopharma manufacturing
- 54. 54 Conclusion • Endotoxins have been & going to be one of the challenges in Biopharma manufacturing. • If not controlled, they could pose serious consequences to the manufacturer & finally to the patient. • As described, endotoxin control should be ensured for the safety of the drug • Selection of Raw materials with low endotoxin levels should be used/preferred • Bioburden control & monitoring plays a major role in reducing endotoxins in the process/product • Downstream Unit operations should be optimized for robust control of Bioburden & Endotoxins
- 55. © 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. The vibrant M, Millipore® Durapore®, Fractogel® and Eshmuno® are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. THANK YOU Somasundaram G. Dr. Subhasis Banerjee
- 56. Q & A
Editor's Notes
- This slide is mandatory for use in webinars. Place immediately after the title slide
- The compendial test described in this USP uses the lysate from the blood cells of horseshoe crabs (known as the LAL method, standing for limulus amebocyte lysate). An alternative known as the Recombinant Factor C (rFC) method that uses only synthetic reagents, as opposed to animal-sourced ones, was developed, and patented in 1997, and has been successfully implemented since 2018.
- Q1. Which of the following sentences are correct: All endotoxins are pyrogens All pyrogens are endotoxins Endotoxins are not pyrogens
- Thank you Si-Ying & to all of you who have participated in the poll questions. Hello Everyone & its my pleasure to take you through section 2 & 3. Let’s begin with section 2 on contamination control strategy. As we heard from Dr.Subhasis the sources of endotoxins are from Gram negative bacteria, controlling/eliminating them from the process is an ideal strategy, which is nothing but CCS.
- In this section, I will address Why & What is Contamination Control Strategy Understand the impact of bioburden excursions Recognize the sources of bioburden Develop strategies to mitigate risk
- A deliberate and well-documented contamination control strategy is one of the new focuses in the Annex I document. The main additions and revisions are in 2.1. “The manufacture of sterile products is subject to special requirements in order to minimize risks of microbial, particulate and pyrogen contamination.” and in 8.2 “Primary packaging containers and components should be cleaned using validated processes to ensure that particulate, pyrogen and bioburden contamination is appropriately controlled.” For pyrogens such as endotoxins, additional special controls for cleaning equipment and tooling, water and filtration system should be in place. The pharmaceutical washing process should be a validated process with a defined cycle per item, which results in product that meets specified limits for bacterial endotoxin, bioburden, particulate matter and, if applicable, silicone oil level. The process validation study data should demonstrate that the pharmaceutical washing process is capable of reducing the endotoxin content by at least 99.9% (3.0 log10).
- CCS is an holistic program that encompasses concepts within the context of the entire manufacturing facility & process. It involves Facility design, Personnel working in cleanroom, Cleaning & Disinfection, EM, Process Simulation & Process Design. The CCS is a culmination of an exercise to identify activities designed to prevent microorganisms, pyrogens, and particulates contamination in the product, the facility, and supporting processes used to manufacture the product. Manufacturers can formulate their contamination control strategy based on information in the quality target product profile or in the critical quality attributes, in the facility and in the processes used to manufacture and transport the product. The strategy implementation involves executing the strategic plan and managing the implementation by priority overtime should it be deployed. The evaluation of the efficiency and effectiveness of the contamination control strategy implemented is confirmed by analyzing and trending the various quality performance parameters related to contamination control. The strategy evaluation allows the manufacturer to identify a new strategic plan to support improvement goals or new measures/controls to achieve the desired result, minimizing the contamination risk.
- Q2: Is your company looking into the aspect of Closed Processing? Yes, I need a consultation. No, not currently
- Thank you Si-Ying & to all the participants. So what are we talking about here? Although, today’s presentation is focused on Endotoxins, it is important to consider all microorganisms specifically on fungal and bacterial bioburden, though Mycoplasma, Virus and Transmissible Spongiform Encephalopathies, or TSE, are special cases to consider particularly in upstream mammalian processes. In addition to considering the impact of viable cells capable of proliferation you need to consider cellular byproducts of microorganisms because they too can also affect product characteristics as well as pose risks to patient safety, as mentioned by Dr.Subhasis.
- So where does bioburden come from? Everywhere! We may not like to think about it but people are one of the largest sources of contamination in pharmaceutical processes. Data suggests that greater than Ten Percent of particles generated by humans contain bacteria. That might not sound like much but in a resting state an average person sheds 100,000 particles/minute, which translates to around 10,000 colony forming units of bacteria per minute. As important as personnel are the other inputs to the process and each source contributes to bioburden profile that is unique to your process. Even if you have two very similar processes in different parts of your facility, you will likely see a slightly different profile and different trends. The identification of your bioburden to Genus and species is important as it can suggest the source. For example, Molds are suggestive of environmental contamination in the air as well as cardboard and wooden pallets used to ship material. Bacillus is often suggestive of environmental contamination. Staphylococcus, or Propianibacterium point to human contamination And non-fermenting gram negative rods such as Stenotrophomonas, Burkholderia, or Ralstonia point investigators toward water systems or raw materials with high water content.
- We talked about the origin of bioburden, but where and how does it affect your process? There are a variety of risk areas throughout the process and the amount of risk and the degree of control will vary throughout the process. Further, the drivers will change throughout the process. Upstream is treated as an aseptic process designed to protect the bioreactor, business drivers often inform the control strategy. If a batch goes down due to contamination in the upstream stage there is no direct impact on patient safety (if you don’t consider product supply). Downstream is typically treated as a low bioburden process designed to protect the drug substance however many steps are open operations or contain materials such as chromatography resins and tangential flow filtration devices that are re-used multiple times. Typically these components cannot be sterilized and require sanitization and storage solutions In Drug Product Formulation and Fill for biologics is an aseptic process where the filter-sterilized material receives no further sterilization upon filling into its final container. At this highly critical phase of the process patient safety is of critical concern. This is evidenced by the high degree of regulatory focus on this part of the process.
- Risk Mitigation is a 3 phase approach Assessment Mitigation Monitoring
- Lets talk about some of Key points in Risk Assessment. There are many components in assessing the risk in your process and it can take a considerable investment in time to do it properly. Risk assessments, properly performed, can save you time and agony of dealing with excursions. There is no one-size-fits all solution to assessing your process. Utilize a combination of tools , techniques, and data. Ensure you document and justify thoroughly. And a cross-functional team is essential to critically challenge your assessment. If you do these risk assessment exercises, will they be perfect? Will your process be forever devoid of excursions? No. but it doesn’t mean your Contamination control strategy is a failure. Revisiting your strategy is part of a continuous improvement program. You will need to keep assessing and improving over time.
- I mentioned earlier that people are considered one of the largest contributors of microorganisms in pharmaceutical processes. There are numerous trainings and publications that address people within pharmaceutical processes Raw materials or feed materials to the process are another large contributor. There are three key areas to think about when examining materials. First is the origin of the material and how the supplier handles it. Many raw materials are produced in industrial settings that are very different than a controlled pharmaceutical process. What is your suppliers quality philosophy? How robust is their quality management system?. Do you know from lot to lot what you are getting? The material may have trace impurities but if it is well characterized and consistent from batch to batch that material may be appropriate. Second, what are the characteristics of the material? Is it growth promoting to microorganisms? Bacteriostatic? Bactericidal? If the material is growth promoting, your approach to addressing risk could be very different than for a material that is bactericidal. Thirdly, what is the bioburden profile of the material? We talked about bioburden profile previously. Based upon your risk assessment you may decide a particular material might not be suitable and that you may need to choose a different supplier, you may also decide that the material requires special handling or processing before it is fit for use in your process.
- Just as example of Monitoring, lets consider Downstream monitoring for bioburden/Endotoxin occurs at several points in the downstream process. These should be established based upon your risk assessment. You will need to establish your designated sampling locations, bioburden levels and document why when and how they will be sampled and tested. You will also need to develop a plan for when you have adverse trends and excursions. This plan should outline how you will investigate, who is involved, what is the risk to the patient or product, how you will address corrective and preventative actions. Consider samples at critical process steps such as before the filtration step or after hold times and around the chromatography preparation operations such as after removal and WFI rinse of the storage solution and sanitization solution from the resin. Also consider sampling the equilibrium and elution buffers. Certainly there will be expectations for sampling of the bulk drug substance.
- Q3. What is your current sampling procedure for Endotoxins? Open sampling. Closed sampling into a bag. Closed sampling into a conical tube No samples, in-line monitoring only I do not know
- Beta glucan blocking buffers do not always completely block all beta glucan (specificity). To say the use of blocking buffers in “…will reduce any effects of glucans…” is not true. (Roslansky and Novitsky, Sensitivity of Limulus amebocyte lysate (LAL) to LALreactive glucans, Journal of Clinical Microbiology, Nov. 1991, p. 2477-2483.) This seems self-evident; however it is not clear the way that this is written that one would need the beta glucan buffer for the test and not for the rFC assay.
- “Our companys” in-house data Charge based removal available
- Q4: Endotoxins have a isolectric point in the range of following pH: 2-4 5-7 8-9
- In this particular case, there were several instances of bioburden excursions over the alert levels with Bacillus, a spore forming bacterium. This occurred over several campaigns. The root causes were three-fold: A failure to recognize an adverse trend in the pattern of excursions which as time went on became more severe. And a non-sporicidal sanitization solution coupled with a less than optimal sanitization procedure were also contributing factors. Chromatography resins are the ideal environment for microbial growth and biofilm formation The protein A step is often an area of focus because it is being fed with material that is relatively nutrient-rich (from a microbial standpoint) and many steps may still be open such as column packing. Further protein A tends to be harder to sanitize as sanitizers that are highly effective in reducing bioburden used in other chrom steps can negatively affect impact resin effectiveness or resin life. Protein A resin can also be degraded by microbial proteases. The Corrective actions in this case took a long time because sanitizer candidate kill kinetics studies on resin spiked with bacteria were conducted with the target sanitizer—it takes time to cultivate the bacterial spikes conduct the studies and wait for the bacterial count results. Scaled-down studies needed to be performed with a new sanitizer to optimize the sanitization conditions were required. Finally process-scale verification was required.
- This second case study comes from a paper by Survarna. In this case results showed that bioburden and endotoxin exceeded the action levels in multiple batches. The bacteria were identified as Sphingomonas species, Stenotrophomonas maltophilia, Ralstonia pickettii, and Staphylococcus species. The nature of this bioburden was suggestive of biofilm formation so the investigation needed to be expanded to examine not only the process area but also the support areas for the Water for Injection and the Buffer, Sanitizer, and Storage Solutions Operations. The root cause was biofilm formation in the TFF cassettes due to inadequate cleaning and storage processes. The corrective and preventative actions put in place had to address the entire facility. Cleaning and storage processes required improvement. The Sterilization or sanitization of buffer tanks was assessed as was the WFI system and transfer lines. And the bioburden limits were also addressed based upon process capability. Hold times and storage conditions in downstream processing are extremely important and you can be expected to produce data to justify your hold times and storage conditions, in this particular case they needed to be validated. Lastly bioburden reducing filters were installed in the process. These filters were added as a future preventative action after a thorough investigation and several other steps were taken. If you have a bioburden problem, simply installing filters to correct the problem without an investigation and identification of the underlying issue will be problematic and will be scrutinized. Once you have corrected the underlying issues, filtration is an excellent preventative measure.
- In this third case study there was sporadic bioburden excursions—do you see a trend with the previous case studies? It has to do with trending. A single isolated excursion needs to be examined but bioburden monitoring is really about trends over time. In this case the issue was excursions at multiple points in the process. The predominant Genus was Staphylococcus suggesting a human source. The root cause in this investigation pointed to lapses in aseptic technique with aseptic connections that were made at many points in the process and to the house-assembled sampling devices. The corrective and preventative actions were divided into short term and long term First operators were retrained in aseptic techniques. Second, the team chose to reduce the number of aseptic connections to those most necessary and to implement sterile to sterile connections to connect single use components or steam-to sterile connectors to connect single use components to stainless steel connections. Thirdly the team eliminated the house-assembled sampling systems to single-use fully validated sterile sampling systems throughout the facility.