effect of raw materials and production processes on dry powder media quality
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Effect of raw materials and production processes on dry powder media qualityTRANSCRIPT
Effect of raw materials and production processes on dry powder media quality
DMEM-F12 production: a case studyDMEM-F12 production: a case study
Dr. ZIMMER AlineCell Culture World CongressMunich, 29/02/2012
Agenda
DMEM-F12 media characterization1
• Physico-chemical characterization
• Cellular performance testing (CHO-S cells)
• Fingerprinting
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• Fingerprinting
• Performance prediction
Quality improvement - case studies2
Perspectives in future media preparation3
Impact of raw materials on dry powder media quality
Physico-chemical
propertiesProcessing
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Raw material quality
Purity Packaging concept
Impact of production processes on dry powder media quality
Milling process
Blending process
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Dry powder media quality
Quality of raw
materials
Packaging concept
Benchmarking of DMEM-F12• Recipe containing 50 raw materials (inorganic salts, amino acids, vitamins, glucose, lipids...)
INORGANIC SALTS AMINO ACIDS VITAMINS OTHERS
Chloride saltsCalcium / Magnesium Potassium / Sodium
Unpolar / hydrophobicAla / Met / Leu / Ile /
Pro / Phe / Trp
Biotin Calcium pantothenate
DextrosePutrescine
Sodium pyruvate
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Potassium / Sodium Pro / Phe / Trp Sodium pyruvate
Phosphate saltsSodium dibasic
/ monobasic
NeutralTyr / Thr / Gln / Gly / Ser / Cys / Cystine /
Asn
Choline chloride Cyanocobalamin
Folic acid Linoleic / lipoic acid
Sulfate saltsCupric / Ferrous
Magnesium / Zinc
BasicLys / Arg / His
i-inositolNiacinamidePyridoxal HCl
ThymidineHypoxanthine
Nitrate saltsFerric nitrate
AcidGlu / Asp
Pyridoxine HCI Riboflavin
Thiamine HCl
Phenol redSodium bicarbonate
Benchmarking of DMEM-F12• Recipe containing 50 raw materials (inorganic salts, amino acids, vitamins, glucose, lipids...)
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• Comparison of dry powder media from several suppliers regarding:• Appearance• pH• Osmolality• Humidity• Particle Size• Cellular Performance
Raw materials: the Fundamentals of Media
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Visual differences of DMEM-F12 dry powder media fro m different suppliers
• Supplier A and B: white appearance
• Merck Millipore media: pinkish color (resulting from Vitamin B12)���� gentle production process
Quality of powdered Media …… starts with a reliable production process
Supplier A lot
Merck Milliporelots
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Commercial DMEM-F12 powder showing non homogeneous blending
Homogeneity
Gentle production process preserves critical raw ma terials from degradation
• Pinkish color: marker for mild raw material treatment
• Brownish / yellow color: degradation of a raw material during the production process detected by UPLC
Supplier A Merck Millipore
UPLC Analysis
pH value observed after solubilisation of DMEM-F12from different suppliers
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Reconstituted media from supplier S exhibits a lowe r pH value
Osmolality observed after solubilisation of DMEM-F12from different suppliers
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Reconstituted media from supplier S exhibits a lowe r osmolality
Turbidity analysis of DMEM-F12 from different suppli ers
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All tested DMEM-F12 are soluble without precipitati on
Humidity in DMEM-F12 dry powder media
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DMEM-F12 particle size analysis by air jet sieving
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Two types of particle size reduction effects are obs erved based on the processing technology
DMEM-F12: CHO-S performance testing
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50% difference in viable cell density between the l owest and the highest performer medium
Serial amplification assay of commercial DMEM-F12 d ry powder media
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Constant performance of Merck Millipore media
Agenda
DMEM-F12 media characterization1
• Physico-chemical characterization
• Cellular performance testing (CHO-S cells)
• Fingerprinting
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• Fingerprinting
• Performance prediction
Quality improvement - case studies2
Perspectives in future media preparation3
Near Infrared spectroscopyFast, simple and non destructive method to measure physicochemical properties of powders via light absorption
Applications:- Quality assessment- Identification of raw materials- Process control (homogeneity)
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- 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
Principle component analysis (PCA) of dry powder me dia: Clustering commercial media by NIR
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PCA of DMEM (blue), DMEM-F12 (red) and RPMI 1640 (g reen)
Analysis by NIR-Spectroscopy (NIR-Spectrometer Bruk er, Vektor MPA)
Supplier C-1
Supplier C-2Supplier B
Supplier S
Principle component analysis (PCA)Correlation of cellular performance and NIR analysi s
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Merck Millipore
Supplier C-3Supplier B
Supplier A
PCA of DMEM-F12. Analysis by NIR-Spectroscopy (Bruk er, Vektor MPA)
Agenda
DMEM-F12 media characterization1
• Physico-chemical characterization
• Cellular performance testing (CHO-S cells)
• Fingerprinting
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• Fingerprinting
• Performance prediction
Quality improvement - case studies2
Perspectives in future media preparation3
Solubility:
L-Cystine dihydrochloride: 50 mg/mL in 2M HCL(CAS: 30925-07-6)
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L-Tyrosine disodium salt: 50 mg/mL H2O(CAS: 69847-45-6)
L-Cystine dihydrochloride
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Raw material
Milled raw material
Solubility of L-Cystine dihydrochloride
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milled in PBS not milled
@ 0.12 g/L
Complete Solubilisation of Media …
L-Cystine 2HCl is partially insoluble under defined media preparation conditions
… is part of raw material and processing evaluation
cum
ulat
ive
part
icle
di
strib
utio
n
Fre
quen
cy
(% p
artic
les
by c
ount
) L-Cystine 2HCl
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Milled L-Cystine 2HCl is completely soluble within minutes
cum
ulat
ive
part
icle
di
strib
utio
n
Fre
quen
cy
(% p
artic
les
by c
ount
)
cum
ulat
ive
part
icle
di
strib
utio
n
(% p
artic
les
by c
ount
)
Particle size (µm)
Particle size (µm)
Milled L-Cystine 2HCl
L-Tyrosine Disodium Salt
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Raw material
Milled raw material
Particle size distribution L-Cystine HCl
L-Cystine HCl L-Cystine HCl (milled)
26
Acc
umul
ated
par
ticle
s (%
)
particle diameter (µm)
particle size reduction by factor 5.5
Particle size distribution L-Tyrosine 2Na salt – Sup plier 1
L-Tyrosine 2Na salt L-Tyrosine 2Na salt (milled)
27
accu
mul
ated
% p
artic
les
particle diameter (µm)
particle size reduction by factor 2.7
Particle size distribution of raw materials
5.5 x
2.7 x
Size reduction
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The effect of milling is dependent from :- The type of raw material- The starting size of particles
2.7 x
Effect of the raw material source on the particle s ize distribution
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The effect of milling is dependent from :- The starting size of particles- The raw material source (supplier)
L-Tyrosine 2Na salt limit of solubility (supplier 2 )
30
1 g/L (milled) 4 g/L (milled) 4 g/L (not milled)
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.
~ 20 fold the concentration in DMEM-F12
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
Rawmaterialquality
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• The production process is directly linked with batch to batch consistency and cellular performance
• Milling and blending process• Packaging
• The combination of raw materials and the production process is mandatory for excellent dry powder media performance
Production process
Cellular performance
Acknowledgments
Research and development• Dr. Joerg von Hagen• Maria Wehsling• Ronja Müller• Marc Vieten
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Production & Operations• Tanino Scaramuzza• Frank De Groot• Jochen Bausch
Quality control & Analytics• Joachim Eichner• Dr. Robert Schiwon• Christoph Hoffman
Leading Pharma Raw Material Production
High Quality Raw Materials
Lifted synergies for production of mammalian cell culture media
33
Industrial Golden
Standard in QC / QA / Reg
Years of Expertise in
Media Development
Expertise in Solid Powder Production