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

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

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(% p

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by c

ount

) L-Cystine 2HCl

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Milled L-Cystine 2HCl is completely soluble within minutes

cum

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ive

part

icle

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Fre

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(% p

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by c

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)

cum

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icle

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)

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)

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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)

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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 )

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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

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Industrial Golden

Standard in QC / QA / Reg

Years of Expertise in

Media Development

Expertise in Solid Powder Production