Effect of raw materials and production processes on dry powder media quality

1. Effect of raw materials and production
processes on dry powder media quality
DMEM-F12 production: a case study
Dr. ZIMMER Aline
Cell Culture World Congress
Munich, 29/02/2012

2. Agenda
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DMEM-F12 media characterization
• Physico-chemical characterization
• Cellular performance testing (CHO-S cells)
• Fingerprinting
• Performance prediction
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3
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Quality improvement - case studies
Perspectives in future media preparation
Cell Culture World congress, 29/02/2012

3. Impact of raw materials on dry
powder media quality
Physicochemical
properties
Processing
Packaging
concept
Purity
Raw
material
quality
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Cell Culture World congress, 29/02/2012

4. Impact of production processes
on dry powder media quality
Blending
process
Milling
process
Quality of
raw
materials
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Cell Culture World congress, 29/02/2012
Dry
powder
media
quality
Packaging
concept

5. Benchmarking of DMEM-F12
• Recipe containing 50 raw materials (inorganic salts, amino acids, vitamins,
glucose, lipids...)
INORGANIC SALTS
VITAMINS
OTHERS
Chloride salts
Calcium / Magnesium
Potassium / Sodium
Unpolar / hydrophobic
Ala / Met / Leu / Ile /
Pro / Phe / Trp
Biotin
Calcium pantothenate
Dextrose
Putrescine
Sodium pyruvate
Phosphate salts
Sodium dibasic
/ monobasic
Neutral
Tyr / Thr / Gln / Gly /
Ser / Cys / Cystine /
Asn
Choline chloride
Cyanocobalamin
Folic acid
Linoleic / lipoic acid
Sulfate salts
Cupric / Ferrous
Magnesium / Zinc
Basic
Lys / Arg / His
i-inositol
Niacinamide
Pyridoxal HCl
Thymidine
Hypoxanthine
Nitrate salts
Ferric nitrate
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AMINO ACIDS
Acid
Glu / Asp
Pyridoxine HCI
Riboflavin
Thiamine HCl
Phenol red
Sodium bicarbonate
Cell Culture World congress, 29/02/2012

6. Benchmarking of DMEM-F12
• Recipe containing 50 raw materials (inorganic salts, amino acids, vitamins,
glucose, lipids...)
• Comparison of dry powder media from several suppliers regarding:
• Appearance
• pH
• Osmolality
• Humidity
• Particle Size
• Cellular Performance
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Cell Culture World congress, 29/02/2012

7. Raw materials: the Fundamentals of Media
Visual differences of DMEM-F12 dry powder media from different suppliers
• Supplier A and B: white appearance
• Merck Millipore media: pinkish color (resulting from Vitamin B12)
gentle production process
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Cell Culture World congress, 29/02/2012

8. Quality of powdered Media …
… starts with a reliable production process
Supplier A lot
Merck Millipore
lots
UPLC Analysis
Homogeneity
Commercial DMEM-F12
powder showing non
homogeneous blending
Supplier A
Merck Millipore
• Pinkish color: marker for mild raw material treatment
• Brownish / yellow color: degradation of a raw material during the
production process detected by UPLC
Gentle production process preserves critical raw materials from degradation
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Cell Culture World congress, 29/02/2012

9. pH value observed after solubilisation of DMEM-F12
from different suppliers
Reconstituted media from supplier S exhibits a lower pH value
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Cell Culture World congress, 29/02/2012

10. Osmolality observed after solubilisation of DMEM-F12
from different suppliers
Reconstituted media from supplier S exhibits a lower osmolality
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Cell Culture World congress, 29/02/2012

11. Turbidity analysis of DMEM-F12 from different suppliers
All tested DMEM-F12 are soluble without precipitation
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Cell Culture World congress, 29/02/2012

12. Humidity in DMEM-F12 dry powder media
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Cell Culture World congress, 29/02/2012

13. DMEM-F12 particle size analysis by air jet sieving
Two types of particle size reduction effects are observed based on
the processing technology
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Cell Culture World congress, 29/02/2012

14. DMEM-F12: CHO-S performance testing
50% difference in viable cell density between the lowest and the
highest performer medium
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Cell Culture World congress, 29/02/2012

15. Serial amplification assay of commercial DMEM-F12 dry
powder media
Constant performance of Merck Millipore media
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Cell Culture World congress, 29/02/2012

16. Agenda
1
DMEM-F12 media characterization
• Physico-chemical characterization
• Cellular performance testing (CHO-S cells)
• Fingerprinting
• Performance prediction
2
3
16
Quality improvement - case studies
Perspectives in future media preparation
Cell Culture World congress, 29/02/2012

17. Near Infrared spectroscopy
Fast, simple and non destructive method to measure physicochemical
properties of powders via light absorption
Applications:
- Quality assessment
- Identification of raw materials
- Process control (homogeneity)
- Chemical quantitative analysis of complex
mixtures
• One NIR spectrum is a fingerprint from a raw
material or a mixture
• Combination with multivariate data analysis
analysis of large and complex datasets
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Cell Culture World congress, 29/02/2012

18. Principle component analysis (PCA) of dry powder media:
Clustering commercial media by NIR
PCA of DMEM (blue), DMEM-F12 (red) and RPMI 1640 (green)
Analysis by NIR-Spectroscopy (NIR-Spectrometer Bruker, Vektor MPA)
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Cell Culture World congress, 29/02/2012

19. Principle component analysis (PCA)
Correlation of cellular performance and NIR analysis
Supplier C-1
Supplier S
Supplier C-2
Supplier B
Supplier C-3
Merck Millipore
Supplier A
PCA of DMEM-F12. Analysis by NIR-Spectroscopy (Bruker, Vektor MPA)
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Cell Culture World congress, 29/02/2012

20. Agenda
1
DMEM-F12 media characterization
• Physico-chemical characterization
• Cellular performance testing (CHO-S cells)
• Fingerprinting
• Performance prediction
2
3
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Quality improvement - case studies
Perspectives in future media preparation
Cell Culture World congress, 29/02/2012

21. Solubility:
L-Cystine dihydrochloride: 50 mg/mL in 2M HCL
(CAS: 30925-07-6)
L-Tyrosine disodium salt: 50 mg/mL H2O
(CAS: 69847-45-6)
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Cell Culture World congress, 29/02/2012

22. L-Cystine dihydrochloride
Milled raw material
Raw material
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Cell Culture World congress, 29/02/2012

23. Solubility of L-Cystine dihydrochloride
milled
in PBS
@ 0.12 g/L
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Cell Culture World congress, 29/02/2012
not milled

24. Complete Solubilisation of Media …
L-Cystine 2HCl
cumulative particle
distribution
Frequency
(% particles by count)
… is part of raw material and processing evaluation
L-Cystine 2HCl is partially
insoluble under defined media
preparation conditions
Milled L-Cystine 2HCl
Particle size (µm)
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Cell Culture World congress, 29/02/2012
cumulative particle
distribution
Frequency
(% particles by count)
Particle size (µm)
Milled L-Cystine 2HCl is
completely soluble within
minutes

25. L-Tyrosine Disodium Salt
Milled raw material
Raw material
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Cell Culture World congress, 29/02/2012

26. Particle size distribution L-Cystine HCl
Accumulated particles (%)
L-Cystine HCl
L-Cystine HCl (milled)
particle size reduction by factor 5.5
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particle diameter (µm)

27. Particle size distribution L-Tyrosine 2Na salt – Supplier 1
accumulated % particles
L-Tyrosine 2Na salt
L-Tyrosine 2Na salt (milled)
particle size reduction by factor 2.7
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particle diameter (µm)

28. Particle size distribution of raw materials
Size reduction
5.5 x
2.7 x
The effect of milling is dependent from :
- The type of raw material
- The starting size of particles
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29. Effect of the raw material source on the particle size
distribution
The effect of milling is dependent from :
- The starting size of particles
- The raw material source (supplier)
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30. L-Tyrosine 2Na salt limit of solubility (supplier 2)
1 g/L (milled)
4 g/L (milled)
4 g/L (not milled)
~ 20 fold the concentration
in DMEM-F12
Increased solubility of milled L-Tyrosine 2Na salt.
Possible addition of at least 1 g/L L-Tyrosine 2Na salt in a chemically
defined cell culture media with an amino acid load of > 9 g/L.
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31. Perspectives in future media preparation
• The raw material quality is key for optimal
media performance on cellular level
• Raw material selection
• Raw material pre-processing
• Supply chain
• The production process is directly linked
with batch to batch consistency and cellular
performance
Raw
material
quality
Production
process
• Milling and blending process
• Packaging
• The combination of raw materials and the
production process is mandatory for excellent
dry powder media performance
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Cell Culture World congress, 29/02/2012
Cellular
performance

32. Acknowledgments
Research and development
• Dr. Joerg von Hagen
• Maria Wehsling
• Ronja Müller
• Marc Vieten
Production & Operations
• Tanino Scaramuzza
• Frank De Groot
• Jochen Bausch
Quality control & Analytics
• Joachim Eichner
• Dr. Robert Schiwon
• Christoph Hoffman
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Cell Culture World congress, 29/02/2012

33. Leading Pharma Raw Material Production
Lifted synergies for production of mammalian cell culture media
High Quality
Raw Materials
Industrial
Golden
Standard in
QC / QA / Reg
Years of
Expertise in
Media
Development
Expertise in Solid
Powder Production
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