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Technical Bulletin
Cell Culture Media Manufactured in the imMEDIAte ADVANTAGE™ Pin Mill Demonstrates Chemical Equivalency to Media Manufactured in the Small Scale Pin Mill and Continuous Pin Mill
Figure 1. The Kemutec Universal Mill, model 6H.
IntroductionIn July 2008, SAFC imMEDIAte ADVANTAGE™ (IA) group installed a state-of-the-art, small-scale pin mill capability for the manufacture of quick-turnaround, non-cGMP dry powder cell culture media (DPM). This latest pin mill addition, termed the IA Pin Mill, provides seamless scale-up of non-cGMP prototype formulations to the cGMP Small Scale Pin Mill (SSPM) and large-scale commercial Continuous Mill (CM).
In 2012, SAFC expanded the capacity of the IA Pin Mill to better meet customer demand. The IA Pin Mill is now capable of producing 0.50 kg to 20 kg of non-cGMP DPM.
The IA Pin Mill consists of the same stainless steel pin mill design as its large-scale cGMP counterparts along with the same:
• Processflow:pre-blend,pinmill,post-blend
• Nitrogen gas: utilized to cool the mill and transport components after particle size reduction
• Milling temperature: criteria is monitored: <40 C
Pin mills are a type of high-speed impact mills consisting of a feed inlet, a set of two disks with concentric rings of pins and a product outlet. The pre-blended media components are fed into the mill and enter the center of the disk where they are centrif-ugally propelled outward and reduced in size by the rotating intermeshing pins. The raw materials are mixed in a blender both before entering and after leaving the pin mill chamber to ensureaconsistentfeedintothemillandahomogenousfinalproduct after being milled. Figure 1 shows a photograph of the IA Pin Mill equipment, which is a Universal Mill, model 6H, manufactured by Kemutec (Bristol, PA).
AspartofthequalificationoftheIAPinMillitwasnecessaryto demonstrate that the DPM it manufactured was chemically homogeneous and comparable to the cGMP SSPM- and CM-manufactured DPM. This was established via chemical analysis comparing two different lots (of disparate sizes) of IA Pin Mill-produced DPM against the SSPM and CM control DPM.
SAFC3050 Spruce StreetSaint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900Fax: 1-888-289-8449Global Email: [email protected]
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SAFC3050 Spruce StreetSaint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900Fax: 1-888-289-8449Global Email: [email protected]
Homogeneity To demonstrate that the IA Pin Mill manufacturing process fully and uniformly blends a medium’s components to achieve a homogeneous product (i.e., any given component is present at the same concentration at any given location throughout the batch), multiple samples (6 – 14 aliquots) throughout the lot were pulled, hydrated and sent for quantitative analysis of amino acids, vitamins and zinc, one of the trace metals in the medium at a very low level of ~20 ppm). Percent relative standard deviation (standard deviation divided by the mean and multiplied by 100%, or %RSD) was chosen as the pass/fail metric to assess homogeneity, and was used to quantify the variation between samples in a given population (the magnitude of the %RSD value is inversely proportional to the homogeneity of the product). There will be an inherent amount of variability present due to sample preparation and the analytical method itself, which will be larger for components which are present in smaller quantities. Therefore, based on preliminary work and information from the testing laboratories regarding assay variability, an acceptance criterion of <10% RSD for the amino acids and vitamins and <15% RSD for the trace metal zinc was chosen to indicate a homogeneous product.
Figures 2 and 3 show the homogeneity results obtained as part of the initial validation for a SAFC proprietary chemically definedmedium(EX-CELL® CD CHO Serum-Free Medium for CHO Cells) at two different batch sizes – a 600 g batch size representing the low end of the IA Pin Mill capacity and an 11 kg batch size, representing the high end of the capacity. To validate the expanded capacity of IA Pin Mill, the amino acid, vitamin and zinc analytical testing was repeated. This validation was performed on two different lot sizes, 11 kg and 21 kg, and utilized the same pass/fail criteria as the initial validation. All of the components met the %RSD criteria for being homogeneously distributed throughout the IA Pin Mill media independent of batch size. The data demonstrate that the manufactured media formulation will contain the same amounts of individual components, within the normal assay variability range, regardless of being produced via the IA Pin Mill (0.50 kg to 10 kg), cGMP Small Scale Pin Mill (10 kg to 100 kg) or large-scale cGMP Continuous Mill (300 kg to 4000 kg).
Figure 2 Figure 3
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SAFC3050 Spruce StreetSaint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900Fax: 1-888-289-8449Global Email: [email protected]
Figures 4 and 5 show the homogeneity results on a SAFC proprietary,chemically-definedmedium(EX-CELL® CD CHO) at two different batch sizes- an 11 kg batch, representing the mid-range of the IA Pin Mill capacity and a 21 kg batch, representing the high end of the capacity. All of the components evaluated in Figures 4 and 5 met the %RSD criteria for being homogeneously distributed throughout
the IA Pin Mill media, independent of batch size. The data demonstrated that the manufactured media formulation will contain the same amounts of individual components, within the normal assay variability range, regardless of being produced in a small IA Pin Mill batch (11 kg) or a large IA Pin Mill batch (21 kg). Combined with the data from Figures 2 and 3, this work demonstrates that SAFC’s IA lab can produce homogeneous packaged lots from 0.50 kg to 20 kg.
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Asp Th
r
Ser
Asn Glu
Pro Gly
Ala
Val
Cys
tine
Met Ile Leu
Tyr
Phe Tr
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Lys
His
Arg
Amou
nt (%
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Amino Acids
Amino Acid Homogeneity: Red = IA 11 kg, Black = IA 21 kg
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Pyridoxine Riboflavin Thiamin Nicotinamide Hydroxy-L-Proline
Ethanolamine Zinc
Amou
nt (%
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Media Components
Vitamin and Trace Metal Homogeneity: Black=IA 11kg, Red=IA 21 kg
Figure 4
Figure 5
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SAFC3050 Spruce StreetSaint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900Fax: 1-888-289-8449Global Email: [email protected]
Biological Comparison A comparison of the IA Pin Mill media versus the large-scale CM media was performed using cell culture studies with the CHO (Chinese Hamster Ovary) cell line B13-24, a derivative of theDX-B11celllinethatsecretesthehumanimmunoglobulinIgG. The studies evaluated cell growth over multiple passages, culturelongevity,viability,andantibodyproductioninEX-CELL®
CD CHO media. Figures 4, 5 and 6 illustrate that IA pin-milled producedmediaexhibitednosignificantdifferencesincellgrowth density, longevity, viability and IgG production when comparedtothecGMPpin-milledcontrol.Table1quantifiesand summarizes the average percent errors seen in Figures 6, 7, and 8.
Figure 7:Figure 6:
Figure 6 Figure 7
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SAFC3050 Spruce StreetSaint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900Fax: 1-888-289-8449Global Email: [email protected]
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Warranty, Limitation of RemediesSAFC warrants to the purchaser for a period of one year from date of delivery that this product conforms to its specifications. Other terms and conditions of this warranty are contained in SAFC written warranty, a copy of whichisavailableuponrequest.ALLOTHERWARRANTIES,EXPRESSEDORIMPLIED,INCLUDINGTHEIMPLIEDWARRANTYOFMERCHANTABILITYANDFITNESSFORAPARTICULARPURPOSE,AREEXCLUDED. Innocase will SAFC be liable for any special, incidental, or consequential damages arising out of this product or the use of this product by the customer or any third party based upon breach of warranty, breach of contract, negligence, strict tort, or any other legal theory. SAFC expressly disclaims any warranty against claims by any thirdpartybywayofinfringementorthelike.THISPRODUCTISINTENDEDFORPURPOSESDESCRIBEDONLYANDISNOTINTENDEDFORANYHUMANORTHERAPEUTICUSE.
Additional Terms and Conditions are contained in the product Catalog, a copy of which is available upon request.
©2012SAFC. All rights reserved. SAFCandSIGMA-ALDRICHare trademarks of Sigma-AldrichCo. LLC,registeredintheUSandothercountries.EX-CELLandimMEDIAteADVANTAGEaretrademarksofSigma-AldrichCo.LLC,registered in theUSandothercountries.Purchasermustdeterminethesuitabilityof theproduct(s)for their particular use. Additional terms and conditions may apply. Please see product information on the SAFC website at www.safcglobal.com and/or on the reverse side of the invoice or packing slip.
Issued June 2012 T101 1008 0109 0410
Average Percentage Error
Lot No.Growth and Viability
(Figure 6)Longevity and Viability
(Figure 7)IgG Production
(Figure 8)
8H0387-2 – 0.8 – 3.8 7.9
8H0387-4 2.6 – 4.9 4.7
8H0387-7 3.0 – 4.8 5.8
8H0389-1 4.4 8.8 – 14.2
8H0389-7 0.6 5.8 18.7
8H0289-14 0.2 7.0 – 6.2 Table 1: A summary of the average percent errors for the 8H0387 and 8H0389 lots (versus the cGMP control) for the three experiments performed.
Summary Upon evaluation of the analytical and biological studies, it has been demonstrated that the imMEDIate ADVANTAGE™ Pin Mill is an effective and scaleable dry powder manufacturing capability that can quickly produce quality, homogenous cell culture media equivalent to its cGMP large-scale Continuous Mill counterpart. The imMEDIAte ADVANTAGE Pin Mill capability can rapidly manufacture dry powder media formulations that effectively allows process development groups to meet their application/milestone-specifictimelines,whilealsoremovingmanufacturing variables to ensure consistency and seamless scale-up to large-scale manufacturing.
Table 1
Figure 8:
Figure 8
Figure 8:
