[转移贴]The Impact of Cell Culture Medium on Cell Line and Process

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原帖由论坛会员pipi302发表于 2009-9-19 12:43

The Impact of Cell Culture Medium on Cell Line and Process Development Timelines
Animal-component free (ACF) medium sped up cell-line developmentbyeliminating the adaptation period from serum-containing medium usedinearly development to ACF medium used for high-titer production.  
ABSTRACT
Cellculture medium plays a significant role in determining thecelldensity, viability, and productivity of a robust manufacturingprocess.Current regulatory requirements prefer bioprocesses that do notuse anyanimal-derived components throughout cell line and cellculturedevelopment. However, using media containing serum for earlystages ofcell line development is still a common practice for derivingstableproduction cell lines for manufacturing. The ideal medium shouldbeable to support all activities in the cell line and processdevelopmentstages for maintaining consistent cell populations andproduct quality.Here, we describe such performance by anin-house-developed, universalanimal component free (ACF) medium thatcan support transfection,amplification, subcloning, banking, andproduction of the differentChinese hamster ovary (CHO) cell linesgenerally used for recombinantprotein production. By using this ACFmedium, designated as MTCM, therewas an overall reduction in cell linedevelopment time for two distinctexpression systems by eliminating theadaptation period betweenserum-containing medium to ACF medium.Furthermore, by using the samebasal medium throughout cell line andprocess development, transitionalchanges in growth characteristics,productivity, and heterogeneity inthe cell populations weresimultaneously kept to a minimum. Finally, arobust and high yieldingupstream production platform technology forhuman monoclonal antibodies(HuMAbs) was developed by using MTCM bothas basal and feed media in fedbatch cultures. This accelerated CHOcell culture optimization that ledto 6 g/L HuMAb production andfacilitated an efficient processintegration of upstream anddownstream.
Productivityenhancement by CHO cell culture to several grams/L has beenrevolutionizing the biomanufacture of recombinant therapeuticantibodies.1The development of antibodies and their derivatives withclose to 200clinical trials largely depends on the knowledge gatheredabout CHOexpression systems, which has enabled the biotech industrytoaccelerate the development of its pipeline candidates. It isalsoforecasted that CHO production systems will play a crucial roleinfuture biomanufacturing processes because of their capabilitytoproduce high expression titers,1–3  in some cases equivalent to thoseof transgenic animals (approximately 10 g/L), along with desirablepost-translational modifications.  The development of cell culturemedium has been one of the major efforts contributing to thesignificant improvements in product titer in the past by supportinghigh cell densities and extending culture longevity in fed-batch mode.Often, production medium for high titer processes is different fromthat used during the earlier stages of cell line development, includingtransfection and subcloning, where single-cell derived colonies need togrow. Medium with serum is often used at these early cell linedevelopment stages for both adherent as well as suspension cellcultures. These recombinant cell lines are then slowly adapted to ACFmedia or chemically defined (CD) media during the later stages ofdevelopment, for inoculum expansion and recombinant protein production.These transitional phases can take a considerable amount of time forcell lines to adapt to such medium shifts and for process developmentscientists to select stable, high producers from a diversifiedpopulation. Here, we describe upstream process development using aproprietary medium throughout the cell line and process developmentphases.
  Materials and Methods

  Transfection and Cell Line Development
Transfections of different CHO host cell lines were carried out using asingle expression plasmid containing the heavy and light chain genes ofa human antibody. A BioRad Gene pulser was used to electroporate theDNA into either a dhfr-deficient (dhfr–, Expression System I) or dhfr-containing (dhfr +, ExpressionSystemII) CHO cell line. Transfected cells were plated in 96-wellplates andantibody levels in media with selection pressure weremeasured using ahuman IgG kappa/gamma sandwich ELISA. The highestproducing cell lineswere expanded and single-cell cloned by limitingdilution.
  Subcloning and Cell Bank Preparation
Analiquot of actively growing cells was diluted in complete growthmediato yield 0.25, 0.5, or 1.0 cell/well, and 10–20 x 96-wellflat-bottomtissue culture plates were plated. Distinct clones that wereconfirmedto have originated from single cell/well were evaluated toidentify thehighest IgG expressing clones. A clone possessing thehighestproductivity was expanded to prepare a research cell bank.Master cellbanks and manufacturer's working cell banks were furtherestablishedfrom research cell banks.
Cell Culture Process and Analysis
Recombinant CHO cells were grownin shake flasks or 5- to 85-L scalestirred tank bioreactors (fromApplikon, Bellco, or Bioengineering) inbatch and fed-batch modes. Thetemperature and CO2 level were controlledfor cell culture incubators. The pH, dissolved oxygen (DO), agitation,and gas flows were also controlled if cells were cultured inbioreactors. In fed-batch mode, feed media were added according to thefeeding schedules that were tailored to the specific recombinant cellline. Cell density and cell viability were measured using Cedex(Innovatis), Vicell (Beckman Coulter) cell counters or by a manual handcount using the Trypan blue exclusion method. Antibody concentrationswere determined by Protein A HPLC (Waters).
Medarex proprietary media for research, development, and large-scalemanufacturing were formulated in both powder and liquid forms from amedia vendor.  
Copy number analysis
Genomic DNA was purified from transfected and untransfected CHO celllines. Gene-specific primers and a fluorescent probe for a TAQmanmethod were developed by Applied Biosystems using the following twoassays. A murine dhfr assay was used to determine the number of dhfr copies of the expression plasmid integrated into the CHO genome. A rodent gapdh assay was used to determine the number of genomes present in the same sample tested for dhfrfor normalization. The reactions were thermal cycled and data wascollected using the ABI Prism 7300 Sequence Detection System (AppliedBiosystems).  
Results and Discussion



Figure 1. Genealogy of commonly used CHO host cell lines
A universal medium that can supporttransfection, selection, and amplification, along with cell cultureoptimization for high productivity, has time and cost advantages. Sucha medium not only reduces overall timelines for cell culturedevelopment but also provides a uniform process development platform byminimizing the changes caused by intermittent adaptation processes. Aconcerted effort to develop a Medarex proprietary medium, MTCM A, aspart of an integrated cell line and cell culture process development,was carried out for a HuMAb CHO production platform. This medium wasfurther optimized by adjusting the levels of nutrient requirements forour cell lines, and an improved second-generation medium, MTCM B, wasderived. These two versions of MTCM media, with minor changes incomposition, can support transfection, amplification, subcloning, andproduction, and were successfully used for the development of differentbut related CHO cell lines for HuMAb production. The genealogy of theCHO host cell lines that are generally used for recombinant expressionis depicted in Figure 1.  Reduction in Cell Line Development Timelines



Figure 2.  Effect of using a single medium throughout cell line development for Expression Systems I and II
Expression System I with CHO dhfr– host cell lines. Standard transfection and amplification of a CHO dhfr–cellline are performed by plating cells in serum containingcommerciallyavailable media after electroporation and subsequentlyincreasing themethotrexate (MTX) concentration to 500 nM in the same96-well plates.These cell lines may be further amplified to 2 μM MTXin batch cultures.The process has included two rounds of subcloning,occurring before andafter adaptation to the serum-free commerciallyavailable medium,followed by adaptation to MTCM medium. This entireprocess takesapproximately 12 months (Figure 2A). Conditions were then developed toaccelerate the process in the commercial medium without serum rightfrom the initiation of cell line development, a process that includedoptimizing transfection efficiency. This shortened the cell linedevelopment timeline by up to four months and eliminated the need for alengthy serum-weaning process and one round of subcloning (Figure 2A).However, using only MTCM media during the entire process of cell linedevelopment would eliminate media transitions and potentially reducethe timeline to seven months for a 2 μM MTX amplified cell line (Figure2A).
Expression System II with CHO dhfr+ host cell lines. Asimilar process could be carried out with Expression System II, whichdoes not require amplification. Initial protocols used serum in acommercially available medium at the transfection stage, which was thenremoved during adaptation. In a manner similar to Expression System Iabove, the conditions were optimized using the same commerciallyavailable medium without serum throughout transfection and subcloning.As Figure 2B indicates, by using only MTCM media, we could acceleratethe cell line development timeline to five months. In addition, furtherreduction in the timeline by another month could be achieved using bulktransfection and selection methods.  
In both cases, pre-adapted host cell banks in MTCM media were generatedin a serum-free freezing mixture. Transfection with thesewell-characterized host cell banks eliminated media transitions andreduced the timelines for cell line development, as illustrated inFigure 2.  
Amplification Using MTCM Media


Figure 3.  Productivity and gene copy number analysis duringamplification and subcloning for a HuMAb cell line derived fromExpression System I
Ina controlled experiment for ExpressionSystem I (Figure 3), CHOcolonies in 96-well plates were graduallyamplified to 1 μM MTX in acommercial medium (parental 1 μM) andsubcloned (clone 1 μM). Theparental line was heterogeneous, asindicated by the increase in genecopy number from 18 to 40 uponsubcloning. On the other hand, theefficiency of MTCM media foramplification was monitored by amplifyingup to 500 nm MTX and subcloned(Figure 3). This process yielded a cellline (clone 0.5 μM) with 23 genecopies and better volumetricproductivity compared to the parental cellline (parental 0.5 μM).Several MTCM-derived clones were pooled andamplified to 2 μM MTX. Thepooled culture was increased from an averageof 29 gene copies per cellat 500 nm MTX (pool 0.5 μM) to 34 copies at 2μM MTX (pool 2 μM).Subcloning of the bulk amplified pool resulted incell lines (clone 2μM) producing over 500 mg/L in batch culture with 47and 54 gene copiesper cell. Copy number analysis clearly demonstratedthe increase ingene copies and productivities in correlation withincreasingresistance to higher MTX concentration in MTCM medium. Inaddition,bulk amplification was also successful with MTCM media, asmonitored byqPCR copy number analysis.  Subcloning Efficiency Using MTCM Media


Table 1.  Cloning efficiency of MTCM on recombinant cell lines derived from Expression Systems I and II
CHO-derivedHuMAb cell lines were plated at0.25, 0.5, and 1.0 cell/well and wereused to measure the subcloningefficiency of MTCM first- andsecond-generation media. Even when platedat 0.25 cell/well,recombinant cell lines generated from both expressionsystems were ableto grow to 10 to 90% cloning efficiency (Table 1).High-titer cellculture processes can be successfully developed furtherfrom thesesubclones after appropriate upstream process development. Ina case ofExpression System I using MTCM A medium, MTCM mediumoutperformed twocommercially available ACF media when the same parentalcell line wassubcloned by limiting dilution. Approximately 227 colonieswereobtained from MTCM medium by plating at 1.0 cell/well in 20 x 96wellplates (Table 1), whereas no colonies appeared in the othertwocommercial ACF media (not shown). As shown in Table 1, thesetwoversions of MTCM media supported high efficiency cloning forCHOrecombinant cell lines derived from both expression systems.  Preparation of Cell Banks with MTCM Media


Table 2. Use of MTCM for cell bank preparation of different CHO recombinant cell lines from Expression Systems I and II
Cryopreservationof cells for manufacturingwas performed with the two versions of MTCMmedia for differentrecombinant cell lines. These banks wereperiodically monitored to checkrecovery of the cells for furthersubculturing and the inoculumexpansion process for upstreammanufacturing. Freezing the cells atvarying densities, from 1 to 3 x 107cells/mL, was successful in afreezing mixture containing either MTCM Aor MTCM B and dimethylsulfoxide. These cells were thawed with >85%viability for all CHOcell lines tested (Table 2). One of the cellbanks was evaluated forrecovery and propagation after five years andwas found to have the sameperformance as at the time of banking. Todate, more than 20 recombinantCHO cell banks have been made in MTCMmedia and used in differentmanufacturing processes. Inoculum expansionprocesses for manufacturingare consistent and robust with the banksprepared and stored in theseACF basal media. Using ACF media fromearly stages of cell linedevelopment also reduces safety concerns fromexogenous contaminants.  HuMAb Production with MTCM Media


Figure 4.  Comparison of first and second generation MTCM basal media for a HuMAb production using Expression System I
HuMAbbatch processes with MTCM A and B weredeveloped that can yield up to 1g/L without feed addition. In general,MTCM B supports better cellgrowth and longevity of the culture, and asa result, leads to betterproductivity in batch and fed-batch processes.For example, Figure 4shows that, cell culture processes in MTCM B notonly reached higherpeak cell density, but also expressed higherantibody levels for bothbatch and fed-batch compared to those in MTCMA.

Figure 5.  Fed-batch processes in bioreactors using Expression SystemII for two different HuMabs. A) MTCM B used as basal medium, combinedwith MTCM feeds for cell line 1 from Expression System II, with an11-fold increase in productivity from batch to fed-batch. B) MTCM Bused as basal medium, combined with MTCM feed for cell line 2 fromExpression System II, with a 10-fold increase in productivity frombatch to fed-batch.
Feedformulations and feed strategies havebeen developed by tailoring themto one or two basic generic processeswith MTCM A or B basal media toreach high expression levels. In mostcases, when high productivity isdemanded in short timelines, theseprocesses only need minimaloptimization time. These fed-batch processesrange from 2 to 6 g/LHuMAb production with MTCM basal and feed mediacombinations for celllines derived from these two expression systems,which can increaseproductivity up to 11 fold compared to batch cultures(Figures 5 and6).

Figure 6.  Fed-batch processes in bioreactors for Expression System Ifor two different HuMAbs. MTCM B used as basal medium, combined withMTCM feeds for two cell lines from Expression System I, with a 7-foldincrease in viable cell density from batch to fed-batch.
High yield processes also can be developedusing commercially available chemically defined basal media along withMTCM feed media (Figure 7). Up to a 13-fold increase in productivitywas observed when using commercial basal medium with MTCM feed and feedstrategies from our generic processes. However, it is reported thatthere are usually only 2–5 fold productivity gains from media andprocess optimization efforts.10

Figure7. Combination of commercial chemically defined basal media withMTCMfeed media for two different HuMAbs. A) Chemically defined CHObasalmedium combined with MTCM feed for a cell line from ExpressionSystem Iwith 2,000 nm methotrexate (MTX), showing a 10-fold increaseinproductivity from batch to fed-batch. B) Chemically defined CHObasalmedium combined with commercial feed, compared to MTCM feed for acellline derived from Expression System II, showing a 3-fold and13-foldincrease, respectively, in productivity from batch to fed-batch.
UsingMTCM as a feed medium in combinationwith commercial basal media fromdifferent vendors or using MTCM as abasal medium with differentcommercial feed media demonstrated the broadapplicability of MTCMcompositions for high yield upstream processes.  Process Development Strategies and Advantages

The biotechnology industry depends on robust manufacturing cell linesto produce biologics. The timelines for deriving a stable manufacturingcell line suitable for consistent process scale-up requires thoroughscreening and subcloning of the initial transfectant population. In thecase of suspension dhfr–CHO cell cultures, amplification procedures using MTX prolongs thisprocess. However, the recombinant cell lines derived from ExpressionSystem II have a more straightforward development time, which can takeapproximately seven months to adapt from serum-containing transfectionto serum removal and subcloning. Through careful development of MTCMmedium that can support different activities, we were able tosignificantly reduce the timeline from transfection to cell lineselection for both expression systems (Figure 2). Recombinant celllines derived from both expression systems can use the same mediumwithout serum right from the transfection stage. Using one mediumthroughout all upstream process development phases also helps retainthe higher productivity carried from early clones by eliminating mediashifts.


Table 3. Comparison of MTCM media costs to commercially available media for different manufacturing scales
Arobust CHO-based high yielding platformtechnology was established forHuMAb manufacturing. With this strategy,media inventory became muchmore streamlined for different processdevelopment stages as well asfor manufacturing. Basal mediumcompositions can be manufactured inboth liquid and powder formulationsthat support growth and productionequally well. The powder form of bothbasal and feed media has beensuccessfully manufactured up to the 450-kgscale and shown to be stablefor more than two to three years, which hasfacilitated convenientinventory maintenance for multiple processes ofdifferent HuMAbmolecules. Powder formulations provide a convenientmeans of transportand storage of large batches for commercial-scalemanufacturing andalso decrease the costs associated with testing andreleasing multiplemedia lots. Overall, manufacturing efficiency andtimelines arefavorably influenced for a wide variety of antibodycandidates and alsoresulted in more operation-friendly processes byusing common mediarequirements for different CHO cell lines. Inaddition, knowledge ofmedium formulations provides better insightsduring upstream processoptimization. Furthermore, using in-house mediacan significantlyreduce the manufacturing cost of goods, and mediumcost can be loweredas much as 60% compared to commercially availablemedia (Table 3).  Conclusion

Overall, we significantly reducedresource requirements andsubstantially shortened cell line developmenttimelines by optimizing aproprietary cell culture medium, MTCM, forall upstream process phasesincluding transfection, amplification,subcloning, banking, andproduction. Medium changes are some of themost influential factorsaffecting the heterogeneity of cellpopulations with respect topost-translational or secondarymodifications. Integrating mediumscreening into the process as earlyas the clone selection step focusesprocess development efforts, notonly on deriving high productivityclones, but also on maintainingcomparable biochemical profiles of themolecule for desired therapeuticpotency.11
Using a single cell culture mediumfrom early-stage cell linedevelopment all the way through high-titerproduction significantlyaffected the process development strategies toobtain stable, robustproduction cell lines and high yielding cellculture processes. Itallows for more predictable feed streams fordownstream processes andenables an efficient integration of processtechnologies.12 It alsoleads to effective regulatorystrategies by supporting economicalprocesses starting at earlyclinical phase production.
ALAHARI ARUNAKUMARI, PhD, is the senior director of process development and the corresponding author, XIAO-PING DAI, PhD, is the assistant director of bioprocess development, JOEL GOLDSTEIN, PhD, is the associate director of transfectoma development, CLAUDIA KLOTH, PhD, is the senior manager of upstream process validation, bioprocess development, HAILE GHEBREMARIAM is the assistant director, cell line development, GLENN MACISAAC is a scientist II, cell line development, and MELISSA WAGNERis a scientist I, cell line development, all at Medarex Inc.,Bloomsbury, NJ, aarunakumari@medarex.com [aarunakumari@medarex.com]
908.479.2647.
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