Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality

Perfusion Seed Cultures Improve Biopharmaceutical Fed-Batch Production

Capacity and Product Quality

William C. Yang, Jiuyi Lu, and Chris KwiatkowskiCell Culture Development, Biogen Idec Inc., Research Triangle Park, NC 27709

Hang YuanManufacturing Sciences, Biogen Idec Inc., Cambridge, MA 02142

Rashmi Kshirsagar and Thomas RyllCell Culture Development, Biogen Idec Inc., Cambridge, MA 02142

Yao-Ming HuangCell Culture Development, Biogen Idec Inc., Research Triangle Park, NC 27709

DOI 10.1002/btpr.1884Published online February 15, 2014 in Wiley Online Library (wileyonlinelibrary.com)

Volumetric productivity and product quality are two key performance indicators for any bio-pharmaceutical cell culture process. In this work, we showed proof-of-concept for improvingboth through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed trainbioreactor stage immediately prior to the production bioreactor stage, to minimize the consump-tion of perfusion media for one CHO cell line and then successfully applied the optimized perfu-sion process to a different CHO cell line. Exponential growth was observed throughout the N-1duration, reaching >40 3 106 vc/mL at the end of the perfusion N-1 stage. The cultures weresubsequently split into high-seed (10 3 106 vc/mL) fed-batch production cultures. This strategysignificantly shortened the culture duration. The high-seed fed-batch production processes forcell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processesreached the same titer in 17 days. The shortened production culture duration potentially gener-ates a 30% increase in manufacturing capacity while yielding comparable product quality. Whenperfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of theactive protein were obtained, compared to the low-seed process. This, combined with corre-spondingly lower levels of the inactive species, can enhance the overall process yield for theactive species. Using three different CHO cell lines, we showed that perfusion seed cultures canoptimize capacity utilization and improve process efficiency by increasing volumetric pro-ductivity while maintaining or improving product quality. VC 2014 American Institute ofChemical Engineers Biotechnol. Prog., 30:616–625, 2014Keywords: CHO cell culture, ATF, perfusion, fed-batch, capacity, antibody, fusion protein,product quality

Introduction

The production bioreactor is one of the bottlenecks in thecell culture stage of biologic manufacturing. Traditional fed-batch production processes consist of an unproductivegrowth phase where cell mass accumulates followed by amore productive stationary phase where the majority of thedrug product is generated. That unproductive growth phaselengthens run duration and lowers volumetric productivity,which leads to inefficient production bioreactor utilizationand reduces the output rate. Conventional cell culture devel-opment strategies for generating more productive cultures

include cell line engineering,1,2 optimization of media andfeed,3–5 and optimization of process parameters such as tem-perature, pH, and osmolality.6–8 However, the above strat-egies can increase resource burden and increase developmenttime, especially when new cell lines and new chemical com-ponents are introduced to an existing clinical or commercialprocess. Thus, there is a need for a more disruptive platformstrategy to improve volumetric productivity without changingthe cell line or the media and feed formulations.

We propose to achieve higher volumetric productivity anddebottleneck production bioreactor usage by shifting thegrowth phase from the production stage into the N-1 seedtrain stage and enable higher seed densities in the N fed-batch production stage.9,10 As it is difficult to sustain highcell densities using traditional batch seed train cultures, wepropose the use of perfusion in the N-1 seed train bioreactor.

Additional Supporting Information may be found in the online versionof this article.

Correspondence concerning this article should be addressed toW. C. Yang at [email protected]

616 VC 2014 American Institute of Chemical Engineers

Perfusion allows us to remove waste and add nutrients toreach the high cell densities required for seeding the produc-tion reactor at a higher seeding density. At the end of theperfusion N-1 stage, we split the cells into high-seed fed-batch production bioreactors. High-seed fed-batch productionbioreactors theoretically deliver the same titer in a shorteramount of time, leading to increased seed train occupancyper run and reduced production bioreactor occupancy perrun. The shorter production duration and higher volumetricproductivity would yield more production batches per cam-paign and lead to increased manufacturing capacity with fewchanges to the fed-batch production bioreactor equipment.

However, high volumetric productivity is not the onlyimportant aspect of a biopharmaceutical process. The effect ofincreasing productivity on product quality must also be con-sidered, and its importance depends on the stage of the bio-pharmaceutical product’s life cycle. For many years, biologicmanufacturers have introduced process-improving changes torecombinant proteins at various life cycle stages ranging fromclinical development to postcommercialization in order torealize process economics, new technology, and yield orcapacity increases.1–3,11,12 These changes must result in criti-cal quality attribute profiles that are comparable to previouslygenerated clinical or commercial material and ultimately can-not change the protein-of-interest’s efficacy and safety, suchas antibody aggregation, charge heterogeneity, and glycosyla-tion.11 Alternatively, these types of improvements can also beimplemented during early stage process development, beforethe product even enters clinical manufacturing. In theseinstances, product quality comparability is less of an issuebecause there is no comparator molecule. Thus, the improve-ments obtained by implementing new technologies in theearly stages of product development can change product qual-ity attributes for the better and lead to superior and more effi-cient processes and products, such as enhanced proteinfolding to obtain higher yield of the active species. Productquality comparability is highly dependent on the protein-of-interest. As a result, it is important for a new manufacturingtechnology to possess the capability and flexibility to eitherpreserve product quality or change it.

There are many cell retention devices available for enablingperfusion cell culture,13 but the use of perfusion at manufactur-ing scale has been largely unexplored by literature. Inclined cellsettlers have been used at manufacturing scale,9 but there arescale-up drawbacks associated with inclined settlers, such as along residence time and low separation efficiency.13 Thesedrawbacks can limit the effectiveness of inclined settlers at highcell densities and limit the maximum achievable cell density.Alternatively, a filtration-based perfusion process can addressmany of the concerns with inclined settlers. Alternating tangen-tial flow (ATF) perfusion has the good scaling properties of fil-tration and has been used in continuous culture to support veryhigh cell densities.14 Filtration scale-up has been well character-ized over the years15 and has been reduced to parameters suchas bioreactor-ATF exchange rate to filtration rate ratio, filterflux, and flow per fiber.16 One of the primary scale-up chal-lenges of filtration is filter fouling. However, the self-cleaningbackflush action of the ATF system and the short duration ofthe perfusion N-1 stage should minimize those concerns.

Thus, the goal of this work was to generate a diverse set ofbench scale proof-of-concept data showing that ATF perfusionN-1 can generate high cell densities, improve facility through-put, and yield acceptable product quality in order to projecttangible benefits and justify an investment for scale-up and

implementation at manufacturing scale. To this end, we usedthree different CHO cell lines producing three differentrecombinant proteins to show that high density ATF perfusionN-1 technology coupled with high-seed fed-batch productioncan increase titer and maximize manufacturing capacity com-pared to our traditional cell culture process of a low densitybatch N-1 followed by a low-seed fed-batch production.

First, we showed that our ATF-based process enabled expo-nential cell growth to >60 3 106 vc/mL, compared to previ-ously demonstrated cell densities of 16–24 3 106 vc/mL.9,10

After showing the growth potential of the ATF system, wetook a manufacturing-centered development approach toimprove workflow and logistics by optimizing the perfusionN-1 process to consume less than three bioreactor volumes ofperfusion media. This was accomplished through media devel-opment and biocapacitance probe feedback control to auto-matically adjust perfusion flow rates in real-time based on cellgrowth. Minimizing the volume of perfusion media is impor-tant in defining a manufacturing-friendly process as lower vol-umes require smaller media tanks, occupy less floor space,and generate less waste. Our optimized perfusion N-1 processstill enabled CHO cells to grow to >40 3 106 vc/mL.

We then used the optimized perfusion N-1 process to inocu-late the high-seed fed-batch production cultures at 10 3 106 vc/mL seed density, allowing us to obtain the same or higher har-vest titer in a shorter amount of time. This provided proof-of-concept for improving manufacturing output and capacity bygreater than 30%. The high potential gains in facility throughputprojected by our results could have significant implications forthe infrastructure planning of a biologic manufacturer.

Our optimized perfusion N-1 process not only resulted ingains in facility throughput but also resulted in desirable prod-uct quality attributes for all three of our proteins-of-interest.For cell lines A and B, the high-seed process yielded the sameproduct quality as the low-seed process, addressing compara-bility concerns of switching processes. For cell line C, whichproduces a fusion protein prone to misfolding, the high-seedfed-batch process resulted in superior protein folding com-pared to the low-seed process, which was something that hadnot been previously demonstrated using perfusion technology.Together, this work portrays a realistic and holistic view onthe application of perfusion technology in cell culture manu-facturing by showing its applicability and advantages to threeof our internal cell lines.

Materials and Methods

Cell lines and media

Three different CHO cell lines producing three differentrecombinant protein products were used in this study. Celllines A and B produced monoclonal antibodies and cell lineC produced an Fc fusion protein. Proprietary chemicallydefined basal and feed media were used in this study.17

Seed cultures

All three cell lines were thawed and grown as previouslydescribed.17,18 Cells were passaged in 1 or 3 L shake flasks(Corning, NY) every 3–4 days using 13-concentrated basalmedia with incubator settings of 36�C and 5% CO2.

Cells from the shake flasks were then used to inoculatebench-scale 5-L glass Applikon bioreactors (Foster City,CA) equipped with Finesse DeltaV controllers (San Jose,

Biotechnol. Prog., 2014, Vol. 30, No. 3 617

CA) in either the traditional batch N-1 mode or the perfusionN-1 mode for the culture stage preceding the production bio-reactor. For both seed train bioreactor modes, temperaturewas controlled at 36�C. pH was controlled between 6.9 and7.3 using 1 M sodium carbonate (batch mode) or 0.5 Msodium hydroxide (perfusion mode) and CO2 sparging.Sodium hydroxide was used as the base for perfusion modepH control to minimize the CO2 accumulation due to theHenderson–Hasselbalch relationship. Agitation variedbetween 200 and 400 rpm as a function of increasing cellmass.

For traditional batch N-1, 13-concentrated basal mediawas used. There was no feed, only base addition for pH con-trol. Dissolved oxygen was controlled at 30% using cascadeair and oxygen sparging. The N-1 bioreactor had a 2.5-Lworking volume and the culture duration was 3 days. At theend of the N-1 stage, cells were split into traditional low-seed fed-batch production bioreactors at a target seedingdensity of 0.4 3 106 or 1.0 3 106 vc/mL.

For perfusion N-1, 13- and 23-concentrated basal mediawere used, depending on the cell line and application. Weused ATF (ATF4, Refine Technology, Pine Brook, NJ) per-fusion technology with 0.2 or 0.5 mm filters to retain thecells but not the protein product. The perfusion media ratesranged between a cell-specific perfusion rate (CSPR) of 0.05and 0.12 nL cell21 day21, depending on the cell line and themedia. The ATF exchange rate varied between 2 and 3 L/min. The volumetric perfusion flow rate, equal to the ATFfilter permeate rate, was automatically controlled by a bioca-pacitance probe19 (Aber, Aberystwyth, UK). Weight-basedfeedback control via a Sartorius Signum scale (Sartorius,Gottingen, Germany) was used to deliver fresh perfusionmedia to the reactor to maintain a total working volume of 4L throughout the duration of the perfusion N-1 stage. Dis-solved oxygen was controlled at 50% using air and oxygensparging to compensate for the residence time inside theATF unit. The perfusion N-1 culture duration was 5–7 days.At the end of the perfusion N-1 stage, cells were split intohigh-seed fed-batch production bioreactors.

Fed-batch production cultures

The fed-batch production bioreactors were also performedin 5-L Applikon bioreactors with a 2.5 L initial working vol-ume. All fed-batch production bioreactors for all cell linesused the same 13-concentrated basal and feed media. Feedmedia additions commenced on Day 2 or Day 3 for the low-seed process and on Day 0 of the high-seed process. Feedwas administered every 24 h; the feed volume was propor-tional to the integral of viable cells (IVC) of the correspond-ing process.

For cell lines A and B, temperature was controlled at35�C, pH was controlled between 6.9 and 7.3 using 1 Msodium carbonate and CO2 sparging, and agitation variedbetween 300 and 400 rpm. The low-seed fed-batch produc-tion duration was 17 days, while the high-seed fed-batchproduction duration was 12 days. The high-seed productionbioreactors for cell lines A and B were seeded at 10 3 106

vc/mL (253 the seed density of the corresponding low-seedproduction bioreactors).

For cell line C, the low-seed process consisted of a 6-dayperiod, where the temperature was controlled at 35�C fol-lowed by an 8-day period, where the temperature was con-

trolled at 30�C. The high-seed cell line C process wasseeded at 10 3 106 vc/mL (103 the seed density of the cor-responding low-seed process). The high-seed process wascarried out for 12 days and the temperature was controlled at30�C starting from Day 0. The pH for both high- and low-seed was controlled between 6.9 and 7.3 using 1 M sodiumcarbonate and CO2 sparging, and agitation varied between200 and 300 rpm.

Offline analytical methods

Bioreactors were sampled daily. Metabolic parametersincluding glucose, glutamine, glutamate, lactate, ammonium,sodium, potassium, and calcium were measured using theNOVA Flex (NOVA Biomedical, Waltham, MA). Viablecell density (VCD) and viability were measured using Try-pan blue exclusion via an automatic Cedex Cell Counter(Roche Diagnostics, Penzberg, Germany). pH and pCO2

were measured using a NOVA pHOX Analyzer (NOVA Bio-medical, Waltham, MA). Supernatant samples were sterilefiltered and stored at 2–8�C for further titer and productquality analyses. IVC was determined from the area underthe VCD curve and was estimated using a sum of trapezoidsapproximation across the desired time interval.

Analysis of protein concentration and product quality

Protein concentrations (titers) for cell lines A, B, and Cwere measured using a high-performance liquid chromatog-raphy (HPLC) system (Waters, MA) with a UV detector anda Protein G affinity column (Applied Biosystems, CA). Theaverage volumetric productivity, Qp, (mg L21 day21) or theaverage amount of product produced per day can bedescribed by the following equation:

Qp5P

D

where P (mg/L) is the product titer at a certain run time(D) in days.17

Small scale purification was performed on the harvest cellculture fluid using Protein A affinity chromatography and thecorresponding product quality assays were performed on theProtein A eluate. For the antibodies produced by cell lines Aand B, the percentage of the main monomer peak in the Pro-tein A eluate was measured using the LabChip GXII (PerkinElmer, Waltham, MA) under nonreducing impurity profilingconditions. Imaging capillary isoelectric focusing (ICIEF) wasused to measure the antibody charge heterogeneity. Hydrophilicinteraction ultraperformance liquid chromatography (HILIC-UPLC) was used to measure the antibody N-glycan profile.

For the Fc fusion protein produced by cell line C, hydro-phobic interaction chromatography (HIC) using an HPLCsystem was performed on the Protein A eluate to quantifythe active and inactive levels of the recombinant protein.

Results

Perfusion N-1 optimization

Cell line A was used as the model system for perfusion N-1 optimization. In the first iteration of perfusion N-1, we per-formed a 7-day N-1 perfusion culture using 13-concentratedproprietary chemically defined basal media. The CSPR was0.1 nL cell21 day21. Cells grew exponentially to >60 3 106

vc/mL and maintained high viability (Figure 1A). However,

618 Biotechnol. Prog., 2014, Vol. 30, No. 3

the initial perfusion N-1 process was suboptimal due to highaccumulation of pCO2 and high consumption of the perfu-sion media (Figure 1C,D).

We first sought to reduce the volume of perfusion mediaperfused by reducing the CSPR. A lower CSPR, reduced themedia consumption, but led to stunted growth and did notreduce pCO2 accumulation. Furthermore, the 0.05 nL cell21

day21 CSPR had to be increased to 0.07 during the culturedue to poor growth.

After observing poor results at 0.05 nL cell21 day21 CSPRusing 13-concentrated media, we used a 23-concentrated ver-sion of our basal media. The more concentrated media, com-bined with a higher N-1 seed density, which allowed usshorten the culture duration from 7 to 5 days, enabled the useof a lower 0.05 nL cell21 day21 CSPR for exponential growthto >40 3 106 vc/mL. To seed the N-1 higher, we progres-sively increased the starting seed densities of each of the pre-ceding inoculum passages. Furthermore, the shorter cultureduration kept pCO2 lower than 100 mmHg and the 23-concentrated media resulted in the consumption of less thanthree bioreactor volumes of perfusion media (Figure 1). On thefifth and final day of the perfusion N-1 stage, we split the cellsinto high-seed fed-batch production bioreactors.

Cell line A high-seed fed-batch production

High-seed (10 3 106 vc/mL) fed-batch production cultureswere fed daily using our proprietary chemically defined feedmedia. If we overlay Day 0 of the high-seed process withDay 5 of the low-seed process, the performance of the high-seed cultures tracks that of the low-seed culture in VCD,viability, glucose consumption, lactate generation, and pro-tein production (Figure 2). The high-seed process was able

to reach the same harvest titer as the low-seed process, gen-erating 5 g/L of antibody in just 12 days, compared with 17days for the low-seed process. Furthermore, protein aggrega-tion, charge heterogeneity, and glycosylation were notaffected by high-seed culture conditions (Table 1).

Cell line B high-seed fed-batch production

Cell line A’s optimized perfusion N-1 process using 23-concentrated media, higher N-1 seed density, and shorter cul-ture duration was then applied to cell line B with similar suc-cess. Growth in the perfusion N-1 stage was exponential andresembled that of the optimized process for cell line A, alsogenerating growth to approximately 40 3 106 vc/mL in 5days. At the end of the 5-day N-1 stage, cells were split intohigh-seed (10 3 106 vc/mL) fed-batch production bioreactorcultures. For cell line B, the higher seeding density resulted inmuch higher growth (Figure 3A). Since the cells were fedbased on growth, the overall glucose consumption and lactategeneration trended well with that of the low-seed cultures,which were also fed based on growth (Figure 3B,C). Like cellline A, the high-seed process for cell line B generated thesame amount of protein in five fewer days (Figure 3D). Fur-thermore, these efficiency gains did not come at the expenseof product quality, as aggregation, charge heterogeneity, andglycosylation of the high-seed harvest material were similar tothat of the low-seed harvest material (Table 1).

Impact on increasing manufacturing capacity

By lengthening the N-1 seed train stage and seeding theproduction bioreactor higher, we can shorten the productionstage and create more balance between seed train and pro-duction bioreactor occupancies. This provides similar or

Figure 1. Optimization of perfusion N-1 cultures for cell line A.

Shortening the process duration, lowering the CSPR, and concentrating the perfusion media lead to better growth (A, B), lower pCO2 (C), and lowerperfusion volume consumption (D). All data were N 5 1. Open markers in (A) denote cell viability. � 5 13 Media, 0.1 CSPR; � 5 13 Media,0.05–0.07 CSPR; ~5 23 Media, 0.05 CSPR.


greater product output by weight in less time. This woulddebottleneck the production bioreactor and allow for moreruns to be performed in the time allotted for a campaign,and thus, higher levels of protein production given the sameconstraints.

The extent of the debottlenecking obtained by implement-ing a perfusion N-1 process depends on the duration of theseed and production stages as well as the facility layout. Forinstance, if one seed train (S) vessel feeds one production(P) vessel (P/S ratio 5 1), the production vessel wouldremain the bottleneck in a 5-day perfusion N-1/12-day fed-batch production scenario (Figure 4A). If one seed train ves-sel feeds two production vessels (P/S ratio 5 2), the bottle-neck is then shifted to the N-1 stage (Figure 4B). As the P/Sratio increases to 3, the bottleneck increasingly shifts towardthe seed train and the benefit of switching to perfusion N-1seed culture diminishes (Figure 4C). By plotting the outputas a function of the number of production vessels per seedtrain, we see that the benefit obtained by having multipleproduction vessels per seed train plateaus regardless ofwhether the seed cultures are batch or perfusion (Figure 4D).

Even the traditional, short batch seed train becomes the bot-tleneck at P/S ratios above 3. To maximize the number ofbatches, the optimal P/S ratio for a 3-day N-1/17-day N pro-cess (low-seed) is 3 while the optimal ratio for a 5-day N-1/12-day N (high-seed) is 2.

Based on the facility layout models in Figure 4, we cancalculate the increase in output as a result of switching tothe perfusion N-1/high-seed fed-batch for cell lines A and B.A P/S ratio 5 2 indicates that the facility is designed for oneset of seed train bioreactors to support two production bio-reactors. One perfusion N-1 run would exclusively seed pro-duction bioreactor A and a second subsequent perfusion N-1run using that same seed train bioreactor days later wouldseed production bioreactor B. As Biogen Idec manufacturingfacilities are either P/S 5 1 or P/S 5 2, we can implementperfusion N-1 to lengthen the N-1 stage and shorten the pro-duction stage to obtain more batches in the same time. Basedon those assumptions and the high-seed fed-batch data forcell lines A and B, we can potentially increase manufactur-ing output by greater than 30% depending on the facility ofchoice (Table 2). A greater relative change in capacity is

Figure 2. High-seed cell line A fed-batch production cultures.

High-seed growth (A), metabolism (B, C), and protein production (D) track that of the corresponding low-seed process. All data were at least N 5 2.Open markers in (A) denote cell viability. � 5 Low-seed process; � 5 High-seed process.

Table 1. Normalized Product Quality Attributes for Cell Lines A and B

Cell Line N-1 Format N Seed Density GXII Major Peak

Charge Heterogeneity N-glycan Profile

%Acidic %Main %Basic %G0F %G1F %G2F

A Batch Low 100.0 6 0.2 100 6 2 100 6 1 100 6 22 100 100 100A Perfusion High 97.3 6 0.5 90 6 2 110 6 1 118 6 47 100 6 1 110 6 7 122 6 10B Batch Low 100 100 6 7 100 6 20 100 6 6 100 100 100B Perfusion High 99.7 6 0.1 119 6 2 130 6 23 81 6 2 100 6 2 102 6 2 101 6 4

100% represents the average for the low-seed production processes for cell lines A and B and the values for the high-seed production processes wereportrayed relative to 100%. All data were N 5 2, except the low seed N-glycan data. All attributes were obtained using the Protein A eluate. Puritymeasured by LabChip GXII protein profiling, charge heterogeneity measured by ICIEF, and N-linked glycosylation measured by HILIC-UPLC do notdiffer greatly between low-seed and high-seed harvest material for cell lines A and B.


obtained using our P/S 5 1 facilities, while a greater absolutemass of protein produced is obtained using our P/S 5 2facilities.

Cell line C perfusion N-1

Perfusion N-1 was also performed with cell line C. Theoptimized perfusion N-1 process could not be effectivelyused with cell line C due to poor growth and poor viability

(Figure 5). Thus, we reverted to the original formulation of13-concentrated basal media as the perfusion media with ahigher CSPR of 0.10–0.12 nL cell21 day21. Using theseparameters, the cell line C perfusion N-1 stage reached >353 106 vc/mL in 6 days with high viability (Figure 5). OnDay 6, the perfusion N-1 culture was split into high-seedfed-batch production bioreactors at 10 3 106 vc/mL.

Cell line C demonstrated that perfusion N-1 process devel-opment does not differ greatly from traditional process

Figure 3. High-seed cell line B fed-batch production cultures.

High-seed growth (A), metabolism (B, C), and protein production (D) track that of the corresponding low-seed process. All data were at least N 5 2.Open markers in (A) denote cell viability. � 5 Low-seed process; � 5 High-seed process.

Figure 4. Effect of facility layout, seed train duration, and production bioreactor duration on manufacturing capacity.

The annual capacity/output (number of batches per year) was calculated from an empirical equation (Supporting Information Appendix A). High-lighted cells correspond to the output for the low-seed (3-day N-1/17-day N) and high-seed (5-day N-1/12-day N) production regimes. Campaignlength was assumed to be 365 days and bioreactor turnaround time was assumed to be 2 days. The annual capacity in terms of production batches isshown for P/S ratios of 1 (A), 2 (B), and 3 (C). Panel (D) shows the annual capacity of the low-seed and high-seed process for cell lines A and B asa function of the P/S ratio. Increasing the number of production vessels per seed train results in diminishing returns as the bottleneck shifts to theseed train.


development. Optimized platform processes can be devel-oped based on representative cell lines, but there will alwaysbe programs and cell lines for which the platform process isnot well suited. If we find that a certain cell line is not ame-nable to our optimized perfusion N-1 process, we revert tothe use of a 0.1 nL cell21 day21 CSPR with 13 media. Ifcell line specific optimization is required, we would thenanalyze the spent media to rebalance the perfusion media foramino acids, vitamins, and glucose and optimize the CSPRto create a more balance between nutrient delivery and wasteremoval.

Cell line C high-seed fed-batch production and impact onproduct quality

The Fc fusion protein-of-interest produced by cell line Cis prone to misfolding, which leads to accumulation of theprotein in its inactive form. As lower temperatures are morethermodynamically favorable for protein folding,20 a com-mon protein expression technique for alleviating misfoldingis to reduce the culture temperature.21,22 Thus, the low-seedcell line C process uses a change in temperature from 35�Cto 30�C during the culture. As the VCD of the low-seed pro-cess on the day of the temperature change is approximatelythe same VCD as the seed density of the high-seed process,the high-seed process was directly inoculated at a 30�C cul-ture temperature on Day 0. This resulted in different growth,metabolism, and protein production profiles (Figure 6).

By directly culturing the cell line C high-seed process at30�C, we produced a greater fraction of the active speciescompared to that of the low-seed process (Table 3). The use ofthe 0.5 mm hollow fiber filter enabled the inactive protein pro-duced during the higher temperature 36�C perfusion N-1 stageto perfuse out of the N-1 vessel. Thus, there was very little car-ryover of the inactive species when the perfusion N-1 was splitinto high-seed fed-batch production. The high-seed culturecould then commence fed-batch production at a more thermo-

dynamically favorable temperature of 30�C starting on Day 0without sacrificing cell mass. As expected, the lower produc-tion temperature reduced the amount of inactive species,which in turn boosted the level of the active species.

Discussion

Using three different CHO cell lines producing three dif-ferent recombinant proteins, we demonstrated proof-of-concept for increasing manufacturing output and proteinproduct quality through the use of perfusion N-1 technology.Our results suggest that we can potentially increase outputby 30% through the implementation of perfusion N-1 fol-lowed by high-seed fed-batch cultures, depending on theprotein-of-interest. These calculated gains exceed that ofPohlscheidt et al.,9 who reported a 12–19% increase in man-ufacturing output through the implementation of inclined-settler perfusion N-1 cultures. Similarly, Padawer et al.10

showed that the use of perfusion N-1 and subsequent high-seed fed-batch production cultures yields the same titer in 8days as the low-seed culture does in 14 days. This is compa-rable to the 17-day to 12-day production process truncationthat we obtained for cell lines A and B in this work. How-ever, we reached higher cell densities and projected greatergains in manufacturing output with our perfusion N-1 pro-cess. Furthermore, we applied the technology to change theproduct quality for cell line C such that it could potentiallylead to increases in overall process yield.

Although titer and output are key performance indicators,product quality attributes also carry much weight in the bio-pharmaceutical industry. As we did not introduce a new cellline or any new media components, the increase in volumet-ric productivity for cell lines A and B did not compromiseproduct quality: antibody aggregation, charge heterogeneity,and glycosylation between low-seed and high-seed cultureswere very similar overall. Although the charge heterogeneityhad a wider degree of variation, with up to 20% increases in

Table 2. Theoretical Gains in Fed-Batch Production Output Obtained by Implementing Perfusion N-1 Cultures for Cell Lines A and B at Biogen

Idec Manufacturing Facilities

N-1 FormatN SeedDensity

HarvestTiter (g/L)

VPR g L21

day21 P/S RatioProduction BR

Harvest Volume (L)Batchesper Year

kg mAb perYear (kg)

PercentIncrease (%)

Batch Low 5 0.29 1 2,000 18 180Perfusion High 5 0.42 1 2,000 24 240 133Batch Low 5 0.29 2 15,000 35 2,625Perfusion High 5 0.42 2 15,000 44 3,300 126

Assumptions made for the calculations: campaign length is 1 year, maximum number of batches per 1-year campaign slot, 2-day turnaround time perbioreactor.

Figure 5. Cell line C perfusion N-1 cultures.

Open markers in (A) denote cell viability. � 5 23 Media, 0.05–0.10 CSPR; � 5 13 Media, 0.10–12 CSPR.


acidic and basic species, this should not greatly affect com-parability. Acidic species could be formed through deamida-tion, glycation, or C-terminal lysine cleavage. Conversely,basic species could be formed through oxidation or amida-tion. However, differences in charge heterogeneity do not nec-essarily result in differences in efficacy or safety. There was arecent study that isolated acidic, basic, and main species of anantibody and showed that the charge variants did not affectthe in vitro potency, FcRn binding affinity, or pharmacoki-netics in rats.23 Thus, the 20% variation between the low-seedand high-seed process would likely yield similar results to thecomparative evaluation performed by Khawli et al. The abilityto generate a product quality profile that is comparable to anexisting low-seed process greatly increases the attractivenessof implementing perfusion N-1. Not only can perfusion N-1and high seed production be applied to improve the output ofnew processes but also the tandem could be applied toimprove the output of legacy processes.

Conversely, perfusion N-1 also has the ability to changeproduct quality for the better, which was observed for cellline C. Due to product quality concerns, the ideal culture tem-perature for cell line C is 30�C. However, we could not oper-ate the low-seed process entirely at 30�C because the lowtemperature does not allow enough cell mass to accumulatefor adequate productivity. Perfusion N-1 enabled us to accu-

mulate cell mass prior to fed-batch production culture andallowed us to seed higher and directly culture cell line C at30�C. As a result, we obtained higher levels of the active spe-cies. The correspondingly lower levels of the inactive speciesare also significant because the purification yield of cell lineC’s product is highly dependent on the removal of inactivespecies. During the purification process for this particular pro-tein, portions of the active species must be sacrificed in orderto effectively remove the inactive species; the more inactivespecies that require removal, the lower the recovery yield ofthe active species. Thus, the modest increase in the active spe-cies of cell line C belies its impact on downstream operationsand its significance on improving the overall process.

Although we have shown many benefits offered by perfu-sion N-1 technology, it does come with risks and complex-ities.9 Should this concept be implemented at manufacturingscale, the facility would require a refit to accommodate thesizable ATF unit itself as well as its many associated perfu-sion media mixing and storage tanks. Furthermore, there areoperational risks and complexities associated with the newperfusion process, such as preparing the ATF filter, steriliza-tion, connection to the N-1 vessel, and cycling of the variousperfusion media storage tanks.

Although a short, nonsteady-state perfusion N-1 processadds operational complexity, that complexity is relatively

Figure 6. High-seed cell line C fed-batch production cultures.

High-seed production results in a completely different growth (A), metabolism (B, C), and protein production (D) profile compared to the corre-sponding low-seed process due to the differences in starting temperature. All data were N 5 1. Open markers in (A) denote cell viability. � 5 Low-seed process; � 5 High-seed process.

Table 3. Product Quality Attributes and Active Titer for Cell Line C

N Seed Density %Active Species %Inactive Species Total Titer (g/L) Active Titer (g/L) %Active Titer Increase

Low 54.1 45.9 2.1 1.1High 64.3 35.7 2.0 1.3 118%

HIC-HPLC was used to determine the relative percentage of active and inactive protein. All data were N 5 1. The two species sum to 100%. Activetiter is defined as the proportion of the total titer that comprises the active species. Directly seeding cell line C at 30�C greatly enhances the productionof active species, which correspondingly increased the amount of active titer. The lower temperature also reduced the level of inactive species.


low compared to that of a continuous, steady-state produc-tion perfusion process.14 Compared to steady-state produc-tion perfusion processes, the short run duration and lowerVCDs of our optimized perfusion N-1 process would notfoul the filter, create mass transfer problems, lead to highlevels of foaming, produce levels of CO2 that would necessi-tate stripping, nor would it require cell-bleeds to maintainsteady-state. Moreover, the equipment and operations for thesubsequent high-seed fed-batch production process wouldremain largely unchanged.

We demonstrated proof-of-concept for perfusion N-1 usingbench scale bioreactors. Prior to determining whether thetechnology is suitable for implementation at manufacturingscale, a sensible next step would be to scale-up the perfusionN-1 process in a 1,000-L bioreactor using the appropriatelysized ATF10. This will provide insight into the scalabilityand operational complexities of ATF perfusion N-1 cultures.

Scale-up will also provide us another opportunity to addressCO2 accumulation as higher levels of dissolved CO2 has beenobserved in large-scale cultures compared to bench-scale cul-tures.24,25 Although the shorter duration of our optimized per-fusion N-1 process generated lower absolute pCO2 values, thehigher N-1 seed resulted in higher cell mass in the sameamount of time which led to a higher pCO2 evolution rate.Thus, CO2 accumulation could potentially resurface duringscale-up. Methods for reducing pCO2 include a second spargerfor open pipe sparging, changing the concentration of or elim-inating the use of bicarbonate26 and modulating the bubblesize through new sparger designs.27,28

In addition to scale-up studies, future studies should alsofocus on improving the productivity of the high-seed fed-batch production stage. We obtained the same titer as thelow-seed process for cell lines A and B, but the integratedcell mass of the high-seed process was higher for both celllines. Thus, this decrease in specific productivity should beinvestigated in future experiments as there is the potential tounlock even greater volumetric productivities.

Subsequent amino acid analyses of the cell line A and Bhigh-seed fed-batch cultures did not reveal nutrient depletionor gross accumulation. Furthermore, waste accumulation wassimilar between the low-seed and high-seed production proc-esses. Lower specific productivities have been observed athigher cell densities in the literature and were partly attrib-uted to the greater levels of waste produced by high celldensities29 or autocrine factors secreted by high cell den-sities.30 Perhaps the higher cell mass of the high-seed pro-cess yielded higher levels of an unidentified waste productoutside the typically measured ammonium and lactate thatconsequently lowered specific productivity. Analyzing thecell cycle using flow cytometry could also reveal additionalfactors that reduce specific productivity, such as oxidative ormetabolic stress.31

As most of the process optimization in this work was per-formed on the perfusion N-1 stage, devoting time and resourcestoward optimizing the high-seed fed-batch production (processparameter changes or media rebalancing) may bear additionalfruit. Optimization of the high-seed fed-batch production stageto prolong culture duration, while maintaining specific produc-tivity would provide further improvements in titer.

Conclusions

In summary, we showed that we can increase manufactur-ing capacity and improve product quality through the use of

perfusion seed cultures. The perfusion N-1 strategy forincreasing manufacturing capacity is more economical andflexible than building or acquiring additional large scalemanufacturing facilities. We increased seed train occupancyper run and reduced production bioreactor occupancy perrun, although the production bioreactor remains the bottle-neck. Further optimization may bring the ratio closer tounity. Finally, we also showed that the combination of perfu-sion N-1 culture and high-seed fed-batch production yieldsproduct quality that is equal or superior to that producedfrom traditional batch N-1 culture and low-seed fed-batchproduction. Based on our results, perfusion N-1 is an effec-tive tool available to address output bottlenecks and productquality concerns that may arise in the production of biophar-maceutical compounds.

Acknowledgments

The authors thank Biogen Idec Analytical Developmentand Cell Culture Development’s High Throughput AnalyticalTeam for performing the Protein G and product quality anal-yses. They also thank Weimin Lin, An Zhang, Weiwei Hu,Anne McCullough, Earl Pineda, and John Bonham-Carter(Refine Technology) for helpful technical discussions andLam R. Markely for review of the manuscript.

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Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality