Extractables from single-use bioreactors were found to negatively impact cell culture performance. Testing identified various extractable molecules, including degradation products of the antioxidant tris(2,4-di-tert-butyl-phenyl)phosphite. Bis(2,4-di-t-butyl-phenyl)phosphate (bDtBPP) in particular was shown to inhibit cell growth. The findings suggest controlling raw materials and manufacturing processes to minimize extractable contaminants that harm cells.
In-House Microbial Isolates in Compendial Testing: Regulatory RequirementsRobert Westney
This presentation provides an overview of the current regulatory expectations for the use of in-house microbial isolates in compendial testing. It reviews regulatory, compendial and industry references on the topic. Importantly, it also provides a strategy for selection of these isolates.
In-House Microbial Isolates in Compendial Testing: Regulatory RequirementsRobert Westney
This presentation provides an overview of the current regulatory expectations for the use of in-house microbial isolates in compendial testing. It reviews regulatory, compendial and industry references on the topic. Importantly, it also provides a strategy for selection of these isolates.
Recombinant Factor C (rFC) is a recombinantly manufactured protein used for the detection of bacterial endotoxins. They can be cloned and expressed in yeast, Escherichia coli and mammalian cells. It is an alternative to the Limulus amebocytelysate (LAL) which is widely used for the detection of lipopolysaccharides (LPS). However, rFC is a better alternative as it is animal free, simpler, more specific and sensitive as it strongly binds to the LPS and detect their presence.
Cell Culture Media Filtration – Filter Selection and SizingMilliporeSigma
The purpose of this application note is to provide estimated filtration areas for different sterilizing-grade filters with a panel of media used for Chinese Hamster Ovary (CHO) cell culture processes.
ISO 10993 Biological Evaluation of Medical Devices UpdateNAMSA
The ISO 10993 Biological Evaluation of Medical Devices Update covers the revisions/updates that were discussed at the TC194 meeting in Mishima, Japan in April of 2014.
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.
Biocompatibility - ability of material to elicit an appropriate biological response on a given application in the body.
The ability of a material to perform with an appropriate host response in a specific application", Williams' definition.
"The quality of not having toxic or injurious effects on biological systems".
Smear preparation:
A drop of water is placed in the centre of a slide
One loopfuls of organisms is transferred to the centre of slide
Spread the organisms over the slide
The smear is allowed to dry
Slide is passed through flame several times to heat-kill and fix organisms
A bacterial stain is stained with crystal violet (fuchsin, methylene blue) 1 min
Observe under microscope
Basic Dyes
Methylene Blue
Crystal Violet
Carbol Fuchsin
Safranin
Malachite Green
Acidic Dyes
Picric Acid
Nigrosin
India Ink
Eosin
differrential statining
Two or more reagents Distinguish
Bacterial groups
Specific Structures
Example
Gram stain
Acid Fast Stain
Aim of the Presentation
1. Study the biodegradation process of pharmaceutical raw materials.
2. Purification of the biodegradation enzymes.
3. Identification of the biodegradation products.

Cell Culture Media Filtration – Filter Selection and SizingMilliporeSigma
The purpose of this application note is to provide estimated filtration areas for different sterilizing-grade filters with a panel of media used for Chinese Hamster Ovary (CHO) cell culture processes.
The Application of Statistical Design of Experiments for Mathematical Modelin...realjimcarey
A full factorial statistical design was used to mathematically model the process for growing the E. coli cell line (BL21(DE3)/ pET17b::gfpuv. The experimental factors of mixing (RPM), temperature, glucose concentration, and tryptic soy broth concentration were included in the shaker flask study. Optical density measurements were used as the means of quantifying cell growth. During the exponential growth phase, the process showed a statistically significant dependence upon mixing, temperature, and tryptic soy broth concentration. The interaction between mixing and temperature was also found to have a statistically significant effect upon the exponential growth rate. Interestingly, the glucose concentration did not exhibit a statistically significant effect upon this growth phase. Optical density measurements taken at seven individual time points throughout the experiment were also used to model the system during different growth phases. It was interesting to note that mixing initially exhibited a negative effect upon growth rate, but as the growth rate accelerated, it had a positive effect. In the early growth phase, tryptic soy broth concentration had the largest positive effect, while temperature dominated most phases of cell growth. As expected, higher temperatures favored higher growth rates. From these data, mathematical models were constructed that may be used to predict the growth rate within the experimental bounds explored in this study.
Recombinant Factor C (rFC) is a recombinantly manufactured protein used for the detection of bacterial endotoxins. They can be cloned and expressed in yeast, Escherichia coli and mammalian cells. It is an alternative to the Limulus amebocytelysate (LAL) which is widely used for the detection of lipopolysaccharides (LPS). However, rFC is a better alternative as it is animal free, simpler, more specific and sensitive as it strongly binds to the LPS and detect their presence.
Cell Culture Media Filtration – Filter Selection and SizingMilliporeSigma
The purpose of this application note is to provide estimated filtration areas for different sterilizing-grade filters with a panel of media used for Chinese Hamster Ovary (CHO) cell culture processes.
ISO 10993 Biological Evaluation of Medical Devices UpdateNAMSA
The ISO 10993 Biological Evaluation of Medical Devices Update covers the revisions/updates that were discussed at the TC194 meeting in Mishima, Japan in April of 2014.
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.
Biocompatibility - ability of material to elicit an appropriate biological response on a given application in the body.
The ability of a material to perform with an appropriate host response in a specific application", Williams' definition.
"The quality of not having toxic or injurious effects on biological systems".
Smear preparation:
A drop of water is placed in the centre of a slide
One loopfuls of organisms is transferred to the centre of slide
Spread the organisms over the slide
The smear is allowed to dry
Slide is passed through flame several times to heat-kill and fix organisms
A bacterial stain is stained with crystal violet (fuchsin, methylene blue) 1 min
Observe under microscope
Basic Dyes
Methylene Blue
Crystal Violet
Carbol Fuchsin
Safranin
Malachite Green
Acidic Dyes
Picric Acid
Nigrosin
India Ink
Eosin
differrential statining
Two or more reagents Distinguish
Bacterial groups
Specific Structures
Example
Gram stain
Acid Fast Stain
Aim of the Presentation
1. Study the biodegradation process of pharmaceutical raw materials.
2. Purification of the biodegradation enzymes.
3. Identification of the biodegradation products.

Cell Culture Media Filtration – Filter Selection and SizingMilliporeSigma
The purpose of this application note is to provide estimated filtration areas for different sterilizing-grade filters with a panel of media used for Chinese Hamster Ovary (CHO) cell culture processes.
The Application of Statistical Design of Experiments for Mathematical Modelin...realjimcarey
A full factorial statistical design was used to mathematically model the process for growing the E. coli cell line (BL21(DE3)/ pET17b::gfpuv. The experimental factors of mixing (RPM), temperature, glucose concentration, and tryptic soy broth concentration were included in the shaker flask study. Optical density measurements were used as the means of quantifying cell growth. During the exponential growth phase, the process showed a statistically significant dependence upon mixing, temperature, and tryptic soy broth concentration. The interaction between mixing and temperature was also found to have a statistically significant effect upon the exponential growth rate. Interestingly, the glucose concentration did not exhibit a statistically significant effect upon this growth phase. Optical density measurements taken at seven individual time points throughout the experiment were also used to model the system during different growth phases. It was interesting to note that mixing initially exhibited a negative effect upon growth rate, but as the growth rate accelerated, it had a positive effect. In the early growth phase, tryptic soy broth concentration had the largest positive effect, while temperature dominated most phases of cell growth. As expected, higher temperatures favored higher growth rates. From these data, mathematical models were constructed that may be used to predict the growth rate within the experimental bounds explored in this study.
When it comes to the cloud, Gartner may have said it best:
“By 2020, a corporate ‘no-cloud’ policy will be as rare as a corporate ‘no-internet’ policy is today.”
If your organization is still skeptical of the cloud, now is the time to take a closer look. Faster implementation timelines and reduced maintenance costs are just two reasons why the cloud is becoming the standard across all industries.
In our webinar, we dispelled common concerns and explored the benefits of operating in the cloud. We also provided real-world examples of companies that have taken the leap and discovered just how much better business works in the cloud.
Magdalena Zawisławska, Mara Falkowska, Frame-based analysis of synesthetic metaphors in Polish, 6th UK Cognitive Linguistics Conference, School of Linguistics and English Language at Bangor University, Bangor, 19-22.07.2016.
Manage Your Existing Mobile Apps with AEM Mobilearumsey
Discover how AEM Mobile can be used to manage ALL of your mobile applications. These slides accompany a lab that was created for Adobe Summit 2016.
Associated GitHub proejct:
https://github.com/Adobe-Marketing-Cloud-Apps/aem-mobile-hybrid-reference
Primary focus of the conference is analysing the market of pharma industries, growth of the market in global and spending money by pharma industries in market, different kind of media are using to promote the products to reach customers directly. The conferences provide a platform to detail the research work of expertise and analysts from various scientific backgrounds and the same can be perceived by young researchers and students. The conference mainly aims to provide knowledge on pharma marketing In course of research work and therefore pharma market 2016 would be a perfect venue to share and develop knowledge and innovative thoughts on pharma marketing.
Back2 basics - A Day In The Life Of An Oracle Analytics QueryChristian Berg
Oracle BI is in use for years, has gone through many iterations, and is widespread across projects around the globe. Millions of analyses have been created, deployed, and run billions of times. The actual lifecycle of an Oracle BI query from its inception through to the effective delivery of results may therefore be regarded as a given commodity not requiring a closer look. Few people have regarded an OBI query from this point of view: "How does it get constructed? How does it get translated between the different architectural components and the RPD layers? How, through what, and where does its lifecycle get influenced? Where can we as users/developers/administrators actively intervene with its makeup and structure? Why do we genuinely have to have an understanding of the subtleties of the lifecycle to create successful BI solutions?"
David A. Weil, Ph.D, senior applications scientist with Agilent Technologies, presented "Identification of Potential Bioactive Leachables and Extractables from Plastic Lab Ware by using GC and LC Separation Methods linked with MS Detection."
Process Intensification for future bioprocessingMilliporeSigma
Watch the interactive recording here: https://bit.ly/2OdLYwX
Process optimization and upstream intensification led to smaller, more efficient biomanufacturing facilities becoming more commonplace, with smaller facilities comprised primarily of single use or hybrid technology capable of producing significant amounts of drug product. Such changes, however, bring new challenges, like managing the supply of huge amounts of cell culture media or buffers within smaller footprints. In this webinar two topics will be addressed that help to intensify upstream and downstream processes and address the challenges of future facilities.
Bulk powders of cell culture media (CCM) or single chemicals often show physical disadvantages. CCM powders with fine particles show high dust formation and poor flowability. In addition, dissolution is time consuming due to floating of light particles on the water surface. For the pursued intensification of upstream processing, media preparation times are becoming a serious bottleneck. This mainly accounts for the much higher media consumption or higher concentrated media formulations for future continuous upstream processes. Granulated material can overcome limitations with CCM powder, while additionally being a viable option to reduce caking of bulk chemicals like buffers.
Buffer production for downstream processing remains a significant portion of the facility footprint, labor needs and equipment cost. As downstream operations are essentially product-mass-based, increased productivity in upstream will lead to a proportional increase in demand for downstream buffers. Merck KGaA Darmstadt, Germanty R&D has their expertise in concentration of buffers to improve and streamline buffer management.
Effect of UV Treatment on the Degradation of Biodegradable Polylactic AcidCatherine Zhang
In this study, an alternative composting method of biodegradable polylactic acid was proposed, capable of reducing the molecular weight by over 80% in 90 minutes.
Process equipment characterization – how standardized extractables data suppo...MilliporeSigma
View the recording here: https://bit.ly/35KIwBb
Biopharmaceutical Industry recently increased adoption of Single-Use systems and components in manufacturing process operations. Drug manufacturers are responsible for the characterization of SU components and systems used for the production to ensure patient safety. SUS Suppliers are encouraged by BPOG and BPSA to provide comprehensive extractables data package to support drug manufacturer’s E&L assessments.
This webinar will give an overview of the E&L evaluation workflow and practical study approaches from both supplier and end-user perspective, in accordance with the latest industry’s standards and upcoming USP <665> requirements. Case studies will be presented on how the data from suppliers are used to mitigate risk associated to SU materials, highlighting the key role of collaboration between the supplier and the drug manufacturer.
Process equipment characterization – how standardized extractables data suppo...Merck Life Sciences
View the recording here: https://bit.ly/35KIwBb
Biopharmaceutical Industry recently increased adoption of Single-Use systems and components in manufacturing process operations. Drug manufacturers are responsible for the characterization of SU components and systems used for the production to ensure patient safety. SUS Suppliers are encouraged by BPOG and BPSA to provide comprehensive extractables data package to support drug manufacturer’s E&L assessments.
This webinar will give an overview of the E&L evaluation workflow and practical study approaches from both supplier and end-user perspective, in accordance with the latest industry’s standards and upcoming USP <665> requirements. Case studies will be presented on how the data from suppliers are used to mitigate risk associated to SU materials, highlighting the key role of collaboration between the supplier and the drug manufacturer.
A Manufacturer’s Perspective on Innovations in BiomanufacturingKBI Biopharma
A presentation by Abhinav A. Shukla, Ph.D., KBI's Vice President of Process Development & Manufacturing delivered at the IBC’s Biopharmaceutical Development & Production Week, Huntington Beach, CA (2013)
Concentrate! Green and orange don't always clashJames Sherwood
A presentation suitable for A-level students and science teachers about the application of green chemistry, illustrated using the example of citrus waste as an opportunity to make renewable fuels, chemicals, and materials.
Considerations to Extractables and Leachables Testing SGS
How to organize Extractables Assessments? FDA continues to issue Warning Letters to companies that fail to properly complete Design Verification, Design Validation, and Process Validation, and recently to include failures of manufacturers in Risk Management. The evaluation of extractables and leachables has become an increasingly important aspect in the Quality by Design (QbD) initiative of the FDA in the area of drug product design, including materials used in the drug product production process and container and closure systems used for product packaging. This presentation provides general approaches and practical aspects in E&L testing.
Anti Microbiological Assay Test or Antibiotic Assay Test of Pharmaceutical Pr...ijtsrd
In this paper, we are going to discuss the anti microbiological assay of the antibiotics. Aim of this paper is to predict the potency of the antibiotic preparation in reference with the working standard of the antibiotic and using the mathematical model in order to obtain the potency of the preparation in regards to its claim. Faiz Hashmi "Anti-Microbiological Assay Test or Antibiotic Assay Test of Pharmaceutical Preparation Containing Antibiotics using Cylinder Plate Method'" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-5 , August 2019, URL: https://www.ijtsrd.com/papers/ijtsrd27940.pdfPaper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/27940/anti-microbiological-assay-test-or-antibiotic-assay-test-of-pharmaceutical-preparation-containing-antibiotics-using-%E2%80%98cylinder-plate-method%E2%80%99/faiz-hashmi
Bio based products 1/2: Feedstocks and formulation, certification workshop [p...James Sherwood
This workshop uses the example of a bio-based shampoo to demonstrate the use of biomass feedstocks, the bio-refinery concept, certification, and bio-based product standards. This is the presenter version and is up to date as of November 2014.
For annotated handouts featuring detailed descriptions of the slides please visit http://www.slideshare.net/JamesSherwood2/bio-based-products-22-feedstocks-and-formulation-certification-workshop-annotated-handouts
The purpose of this presentation is to give the audience in the workshop an opportunity to learn about the various aspects of biomass use in the chemical industry. The chosen scenario is the production of a shampoo formulation. The participants are given a variety of numbered options concerning biomass selection and the types of certification that can be used. By the end of the workshop the participants will have filled in a 4-digit code with 48 possible solutions. The implications of each decision during the workshop can then be discussed. The last 48 slides are not part of the presentation but describe each possible result of the workshop options, which can be reached using the links on slide 51. A detailed description of how the results are obtained is provided to the audience in their handouts.
Bio based products 1/2: Feedstocks and formulation, certification workshop [p...
Nashed-Samuel_Yasser
1. Extractables from Single Use
Bioreactors and Impact on Cell
Culture Performance
Yasser Nashed-Samuel, Ph.D.
Process Development
Amgen, Inc. – Thousand Oaks, CA,
USA
2016 PepTalk
Single-Use Technologies and Continuous
Processing
San Diego, CA – January 18-22
2. Overview
• Extractable/Leachable potential impact
• Inconsistent cell culture yield in Single Use
Bioreactors/Bags (SUB)
• ID Extractables
• Extractable impact on cell culture
2
3. Extractables and Leachables Definitions
• Extractables - chemicals that migrate from the
product-contact material (container) into a
solvent at elevated temperatures.
(Accelerated Condition)
• Leachables - chemicals that migrate from the
container into a formulated drug during
normal storage/usage conditions.
(Normal Condition)
Solvent
Drug
4 °C
Heat
4. Biologics (Injectables) are in the Highest
Risk Category
Guidance for Industry-Container Closure Systems for Packaging Human Drugs and Biologics (1999)
5. Manufacturing Process of Biologics
5
Cell Culture
Drug Product in:
Syringe, Vial,
Device, IV
Leachable Toxicology Risk
(Proximity to Drug Product)
All product contact materials can leach: Polymers,
rubber, steel, glass, connectors, etc..
7. Single Use Bioreactors (SUB) for Cell
Culture
• Polymer bag
• Sterile / disposable
• Reduce cleaning/sterilization
validation steps
• Reduce cross contamination
• Manufacturing flexibility
• Reduce long term cost
• Maintenance, parts, steel, labor
• Short term development costs
7
Picture of representative single use bioreactor (from Sartorius website)
8. Cell Growth Inconsistency in SUBs
Batch/Run by date
ViableCellDensity(VCD)
• Decreased yield = less profit
• Potential root cause(s)
• Media
• Leached material from Bag?
• Innovative idea to non-Extractable people
Lower yield
9. Hypothesis:
SUB Leachable(s) Inhibits Cell Growth
• Get information from vendor
• Perform Extractable study and ID Extractables
• Spike in individual water soluble Extractables into Cell
Culture process using bags from “good” lots.
• Measure cell growth
9
Extractables*
Spike in Cell
Culture
Measure Cell
Growth
*Vendor data/information from extractables testing
10. Finishing
OEM
Vendors
Polymer
• Resins
Additives
• Stabilizers
• Processing aids
Pellets
Extrusion
• Film
• Sheet, Coating
Finishing/Assembly
• Printing, Fittings
• Gluing
• Etc..
Bag
suppliers
Etc..
Material Information Chain
Polymer
Producers
Compounding Conversion
Biotech
Device/
Packaging
companies
Additives
End User
Polymer information Performance
Evaluation
Engineering
E/L information from suppliers is challenging/limiting
12. Sources of Extractables/Leachables
from SUBs
• Film, Ports, Tubing, etc.
• Additives
• Polymer degradents, monomers
• Processing agents
• Label migration
• Residual from tools
• Sterilization (Gamma irradiated)
• Heat sealed
• Variability in manufacturing
12
Picture of representative single use bioreactor
13. Extraction Conditions of Intact Bag
13
Sample
Extraction
Surface
Solvents
Solvent
Vol.
Time @ Temp
Analysis
Solvent control N/A A, B N/A N/A
HPLC
LCMS
GCMS
ICPMS
2 days @ 50 °CIntact Bag
Bag
interior
A, B
≤ 5% bag
capacity
• Bag from commercial process unavailable.
• Evaluated bags from different suppliers
• (premise: materials from suppliers likely similar)
• Extract intact bag interior (closed system)
• Add solvent
• Close bag
• Heat
15. Bag Extractables
• Identified 20-40+ extractable molecules per bag
• Database (30%)
• Structure Elucidation (70%)
• Antioxidants
• Plasticizers
• Organic acids
• Miscellaneous additives
• Nylon related components
• Polymeric related materials
• Inorganic elements
• Extractable profile similar and different for each bag
vendor
15
21. 21
γ irradiation
of Bags
Heat/
Hydrolysis1,2
bDtBPP(4) Formation Due to Sterilization
(gamma irradiation)
P
O O
O
OH
OHP
OH
O
O
OP
OH
O
O
OH
P
O O
O
O
Antioxidant A
1. J. Sep. Sci. 2010, 33, p3463
2. Packag. Technol Sci. 1999, 12, p119
(Literature)
21
3
21
Oxidized A
bDtBPP
(4)
22. HPLC: Water Soluble Extractables
• Water soluble extractables likely to have ‘higher’ impact
to cell culture performance, if any.
22
min5 7.5 10 12.5 15 17.5 20 22.5 25
mAU
200
300
400
500
600
700
800
900
1000
Bag_solvnet B
Water
Solvent_B
Bag extracted with Water
1
* *
23
4
**
* = identified
23. Extractable Detrimental Impact on Cell
Culture
23
HO
OH
O
HO
O
O
OO
Amgen Confidential. PDA J Pharm Sci Tech 2013, 67(2) p123
• Spike extractables at ~ 1ppm
into cell culture medium
OP
OH
O
O
Extractable compounds
OP
OH
O
O
OH
OHP
OH
O
O
bDtBPP
(4)
4
3
1
bDtBPP (4) is detrimental to cell growth
24. bDtBPP Impact on Cell Culture Lines
24Amgen Confidential. PDA J Pharm Sci Tech 2013, 67(2) p123
• bDtBPP spiked into 9 different CHO cell culture lines
• bDtBPP impact on VCD/Viability varies
Most impacted Least impacted
25. Options
• Do not use antioxidant A for bags
• Change sterilization method
• Control the manufacturing process
• Restrict SUB to specific cell lines
• Future work
• Understand the Biochemistry/Mechanism
26. Summary/Conclusion
• Hypothesis: Extractable(s) impacts cell culture
performance
• Extractables from intact bags were identified
• Poor cell culture performance correlated to an antioxidant
tris(2,4-di-tert-butyl-phenyl)phosphite (A) degradant:
Bis(2,4-di-t-butyl-phenyl)phosphate (bDtBPP)
• Currently, antioxidant A presents in many polymer films.
Industry is now aware of bDtBPP.
P
O O
O
Antioxidant A
OP
OH
O
O
Antioxidant degradant: bDtBPP
Detrimental to cell growth
27. Acknowledgements
Process Development
• Hans Lee
• Kiyoshi Fujimori
• Mike Ronk
• Jian Liu
• Yanxin Luo
• Ping Yeh
• Pavel Bondarenko
• David Semin
• Janet Cheetham
27
Cell Culture
• Heather Nunn
• Matt Hammond
• Mirna Mujacic
• Carole Heath
• Guy Caspary
• Dina Fomina-Yadlin
• Kathy Maggiora
• Rebecca Hernandez
• Dave Treiber