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    RP 1416 en 00

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    Reprinted from Volume 141, 12 February 2015 ELSEVIER STEM SISTER AUT onlay Complete Clarification Solution for Processing High Density Cell Culture Harvests Sladjana Tomic, Lise Besnard, Benjamin Farst, Rainer Reithmeier, Rolf Wichmann, Pierre Schelling, Christian Hakemeyer On-line Access via: www elsevier.com/locate/seppur ‘Ne spony i asumed by Hovis eos aay jury nor nage vo pasos er ope) as amar of pedo Tn, ngieoe or eters o om any se operation of ary methods, pes, insets, or Wess ond inthe materi een. eos of apd advance inthe edcal sence port inepende!verekon of hagnore and rg degen be made Sepurcon od Ptcation Technolgy 14 (2015) 268-275, Contents lets avaiable at ScioncaDiect Separation and Purification Technology journal homepage: www.clsevi r-com/locate/soppur Complete clarification solution for processing high density cell culture harvests Deo Sladjana Tomic**, Lise Besnard’, Benjamin First®, Rainer Reithmeier‘, Rolf Wichmann, Pierre Schelling®, Christian Hakemeyer” Somat Sms oa 0, eM, 2 3) awa, ee “nea en oman ot pment ae Does ohne 7 shear “anna rua on at Boar Rah eps mR? 377 Pre Corny horioy of oma ngnering eperme 9 Becherl nd Cel Engineering 1 Den ner, rt Fp ra 5, 4227 Doran, Germay ‘ei ene 15 Gerber 2014 ‘expression systems have not oly edo cll cultures with very hgh densty and increased prot tes, but also to fed steams with 2 high proportion of solids apd colli that reduce the efclency ofthe Separation step ses with tale-ap exit and instalation limit the atractvenes of centration. ‘whereas larieation sey by depth ation canbe costly duet igh ir areas reqaed. Therefore, an efcent manufartare process must be developed to overcome these resticions In tis study We ‘eres Soa sion Dyesent a complete separation solution that combines cel harvest pretentment vith a poleationic Peco focusing agent (yaalydmetylaamonium chord or pDADNTAC) followed by dep ation sing depth flters that were specially developed for filtration of ecuateé or precptated Feed Streams Muliple antibody feed streams treated wth pOADMAC and subsequent tere sing pe- treatment adapted depth filters resulted in mproved removal of alsa colli, increased clarfeation "roughput, ecient reduction of DNA and high process ye. The residual level of pDADMAC inthe fluates was asesed by surtace pasion resonance specrascpy. Overall. this next generation ela ‘on salton presented herein led to aroust igh yield economical separation process with enhanced Impurity removal hat canbe readily incorporated iv current cafiation patos. 1 tntroduction| ‘Downstream purification of antibodies requires the clarification ‘of harvested cell culture broth to remove plugging components land enable economic reuse of chromatography columns for many ‘yees. Traditional clarification of mammalian cell culture harvests Consists of one ara combination of several separation technologies, ‘such as centrifugation, tangential flow tration (TFF) and normal flow ftaton (NFR). Inorder to increase the productivity, intense efforts in mamma- lin expression systems have been invested. While in the past decades eel culture processes of monoclonal antibodies resulted in titers of about 1 gil, productivities exceeding 10 g/L are now reported {1}. Tis is generally combined with cell density levels ‘of >20 million cells and cell viability <50% [23]. In such * caeponing er Te: 4 985673603 no ass: jana tomiconereroupc Tome). pear 1015 seppurz0r4 2.02 153s sem 14 ther BA abs eee harvests the high proportion of sold biomass (cell, cell debts and colloids) and high content of soluble byproducts (host cel pro- {eins and DNA) reduces the performance of traditional separation technologies. Disk stack centrifuges requite greater discharge frequency andjor acceleration rates and produce larger pellets, potentially leading to yield issues. In addition, che rise in colloid Toads with high biomass and low cell viability arvests result in ay plugging of the subsequent depth fier [4] Likewise, open ‘channel TF cassettes exhibit lower x and capacty due wo higher Solid content, hence requiring higher filtration area. NFF filters equire more internal volume to hold the increased biomass and ‘may experience erler breakthrough of fine particulates. This leads toa lower capacity ofthe biaburden filter or the need to imple ment a second dept fer device “To overcome these limits of traditional separation technologies various pretreatment approaches based on precipitation or occu Tation techniques have been éeveloped and evaluated over the years [5]. While precipitation relies on lowering the solubility of a Toma partion a fet Techno 1 201) 28-275, solutes inorder to create solid particles, flocculation relies on the azzrezation of particles Flocculation occurs if the van-der-Weals [traction between particles ourveghs the electrostatic repulsion according to DLVO theory (Deraguin, Landau, Verwey and (Overbeek) Floculating agents canbe use to tigger the aggree3- tion of dispersed pariculates into larger-size clusters. Typically, positively charged polymers ae used as occulating agents (eg polyamines, polyethylenimine, polyallyiimethylammyonium Chloride (pUADMAC, Fig. 1) or chitosan), The flocculation mechs nism using polycationic polyelectrolytes has been extensively studied (67) They are widely used in wastewater treatment and paper manufacturing. but recent studies have looked at applying Aoculation in biopharmaceutical applications (88). However. 2 setting or centrifugation step is often used in order to separate Maccuated material, because traltional depth fkers cannot handle high solid feed streams. ‘The objective ofthis study was to develop an efficent separa tion solution that comprises cell harvest flocculation, followed by depth tration, Multiple antibody cel culture ids were treated ‘with POADMAC and tered ciectly through pretreatment adapted depth filters, without any additional adjustment step. ‘The separation effectiveness for pDADMAC-mediated flocs was benchmarked with the performance ofa tractional depth tation approach. In addition, pDADMAC dose-effect on harvest particle Size distribution was assessed to provide a beter understanding fof the pOADMAC Nleculation mechanism 2. Materials and methods 21, Cell culture ond protein expression Antibodies used in this study were prduced in Chinese hamster ovary (CHO) cell lines, which reached a ell density of 1.3-1.6 10” total cells, turbidity of "1000 NTU (21:10 ils= tion with water gave for mAbt 1830 NTU and for mAb2 1880 NTU) and viability at harvest of 37% and 70% for mAbt and ‘Abo, respectively. mAbI titer was 0 g/L, whereas mAb2 was expressed atthe level of 15g. The monoclonal antibody mAb3 feed stream (12> 10" total celsfml with 80% vibilty and turbidity >1000 NTU) was solely treated with pDADMAC in order toanalyze the particle size distribution upon flocculation. 22. Rocultion of el culture ‘The optimum dose of PPADMAC (obtained as purified 10% solu- tion from Merck KGaA, Darmstadt, Germany) was Rist defined in 2 screening experiment where 40™mL of the representative cell culture harvest were mixed with increasing amounts ofthe poly ‘ationic polymer ranging from 0 to 0.2% wt. Ate incubation for Simin st room temperature, the locculated cal culture was centrifuged at 20003 for 5-10 min Hereaus Instruments Hana, Germany), followed by filtration using 0.2 jim Milex® ters. Centrate turbidity of the various samples was determined using a portable rurbidimeter model 2100P 1SO (Hach, Berlin, Germany). Frtered supematants were submitted for product concentration. HCP and DNA analysis and compared to non-treated, centrifuged feed 23. tration experiments ‘The optimized flocculation condition, which was judged mainly bby means of centrae turbidity, was applied on 3 larger volume of feed. A portion of the cell culture harvest of about 4L was treated with pDADMAC and carfed using pretreatment adapted depth Ivers (Carisolve" 20MS, OMS and! GOHX graded depth filters {all uPOD device format of 23 cm, Merck KGaA, Darmstadt, Germany). 20MS is designed to clarify smaller aggregated Dariles (of 20 um), 40MS for medium aggregated particles (of 440m) and GOHX for larger aggregated particles (of 60 um). In parallel, the untreated cell culture harvest was filtered using traditional depth filters featuring diatomaceous earth layers of Aitferent pore size (ilistak DOHC and KOH depth fers, Merck KGaA. Darmstadt, Germany). The traditional depen filters were ssembled either inline at. 1:1 Alte area rato or cecoupled using the labscale POD format of 270.m’ for DOHC. For the various fe trates, the capacity of each depth fer grade was evaluated using the Fr, constant flow sizing method [10] Briefly, the feed solution ‘was pumped through the various test depth filters at a constant filtrate fx of 95-138 mh). The differential pressure, curula tive fate volume and turbidity were recorded as a function of process time. The depth filtrate quality was assessed by measuring the capacity of 2 sterlizing grade filter (0.5/0.2 um Millipore Express® SHC, 35cm? filter area, Merck KG2A, Darmstadt, Germany) at constant pressure of 5 psi (0.4 bar. The sizing of these filters was performed according to the flow dscay Vi ‘method (11) 24, Analytical asays The tuiidity was measured using the 2100P 150 portable turidimeter (Hach, Berlin, Germany). The product concentration was measured with an IgG assay ublizing the COBAS INTEGRA 400 plus (COBAS® INTEGRA4OO plus Roche Diagnostics, Germany) (CHO-Host Cell Protein (HCP) levels were assessed using a set of ant-CHOP reagents, including CHO HCP standard and proprietary Aanti-cHO HCP antibodies. Analysis was. performed applying ELISA-based assay. The HCP clearance was calculated as lgarth- ‘mic removal factor (LRV}: ng HCP mi feed ng HCP, mi irate Residual host cell DNA. was detected by quantitative PCR (aPCR}-Similarty, the DNA clearance was calculated using the fol Towing equation: RV = log, a) LRV = lo, a pg DNA/m filtrate ‘The particle size distribution of untreated and flocculated fed expressing mAb3 was measured using a coulter counter (CASY “Model T7C analyzer, Roche Diagnostics GmbH) which has a detec- tion range from 3.2 to 120 um, The amount of residual PDADMAC was determined by surface plasmon resonance technology (SPR) sing the Biacore T200 enhanced sensitivity (GE Healthcare) according to polymer’s manufacture. The samples obtained after filtration were diluted 1:10 before being subjected to SPR. The ‘nial binding rates were potted against the pDADMAC concentra- ‘ion resulting into a regression cuve with a correlation coeficient R=0981 ae chem of HEA he hep raed ute Toe ipernity 12015) 29-275. ™ 3. Results and diseussion 311, Fccuttion optimization study Inorderto characterize the effect ofthe polymer concentration call culture fluids containing monoclonal antibody mAbI or mab2 ‘were mixed with inereasng amounts of PDADMAC. For either feed stream a decrease in turbidity was observed as compared to that of| the untreated feed stream (Fig. 2) This indicates that pDADMAC is effective in focculatng cells and cellular debris. The polyationie polymer presumably adsorbs onthe surface of negatively charged Patile by electrostatic interaction, Thereby i induces the forma- tion of larger-size patces that sediment faster by applying cen- tsiagal force at low accelerations. At higher polymer dases the turbidity of the centrate increases again. Ths turbidity increase Is not caused by the polymer alone as it shows no turbidity increase over concentration range used (data not shown), and the ‘acceleration rate was relatively ow to have a significant impact fon the floes, but cannot be completely excluded. It seems that the turbidity is rather allected by floc properties. tis assumed that the locculant dosage depends on the ell density, as the portion of| {otal biomass (cells colloids) increases wit increasing cel density. “Turbidity is significantly reduced ever a wie range of pDADMAC concentrations indicating that one can have a robust process from bateh-to-bateh. The lowest centate turbidity was achieved With (005% wt pDADMAC, This final concentration was taken as the appropriate polymer dosage for the clarification of mabt and Aba, No product loss was determined for all flocculant conditions tested, implying the high specificity of pDADMAC for negatively charged particles The protein concentration analysis of filtered Supernatants revealed recoveries of >S8% compared to uncreated feed streams. An unusual increase of product recovery. was ‘observed following pDADMAC flocculation with more than 005% ‘wt, Tis may be de to interference frm relatively higher level (of residual polymers in these samples impacting the reliability of the Ig6 quantification assay. This effect has noc been observed when the samples vere measured with an analytical Protein A ‘Chromatography method (data not shown). 22. fect of the polymer onthe parle ize dstibuion ‘The article size distribution of the cll culture harvest before and after flocculation was investigated, AS the feeds streams Tut TU) a a a OADMAG tha concnraton 2. Fcuaton imzato study whites ADADMAC ans ed pst cent Tabily (an eave (SB oes are shown containing mADI or mAb2 were not analyzed for particle sizes, an aliquot of mab3 harvested cell culture uid was submited for particle size analysis (Fig. 3} The mean particle diameter in the lntreate cel culture suspension was determined to particle size of about 22 um. It is assumed that a similar behavior for mMAbt and mA? expressing cell cultures would be obtained [12] Upon 3 cascade of electostati interactions, the particle size distribution is shifted towards larger and likely denser flocs. Interestingly. the higher dose of pDADMAC has ot rested in the formation of big set particles °72 um) or higher volume ofthe particles with a ‘ean diameter of 22 um, The analysis rather revealed that the Sverage size decreared (to about 20 jum) with the highest dose of [PDADMAC used. Furthermore, the volume of the particles in the ‘ange of 10 um (“unflcculate peak") increased with pDADMAC concentration. This all supports the hypothesis that a generous polymer addition destablizes larger size clusters and corelates| ‘withthe inrease in centrate turbidity upon flocculation of high polymer dose. ‘he second, smaller particle size peak in the untreated feed stveam represents a poplation that might be hard to remove evea ‘with traditional separation techniques. Kempken et al. (13) assessed the clarification efficiency of mammalian cell cultures by using a disk stack centrifuge and determined that a substantial cello oad below um was present in the liquid phase after cell, Separation, Notably, there isa decrease in Volume of particles with 2 mean diameter of 4-5 ym. This population was likely stablized jn pDADMAC-mediatedflocculates, 33. Filter capacity throughput and protein recovery ‘A depth tration train consisting of DOHC followed by XOHC is targeted for traditional clarification of biopharmaceuticals pro- dduced in mammalian cell culture. The summary of filtration perfor ‘mance resuits on MAT and mAD2 feed streams is shown in Table 1. The endpoint throughputs and pressure drop values are reported, in most cases filtration experiments were run uncil a pressure drop of >15 psi wat reached, The tration tan consisting, ‘f DOHCIKOHC in seres ata 1:1 fier area cao was evaluated for lunreated broths resulting in depth filter loadings of 118 and 112 Um? for mAbt and mA respectively. The higher endpoint pressure for DOHC in this two-stage depth filtration set-up ind fates that this grade predominately contributes to the cell and ct oid retention. The very low turbidity of fna rates compared to that of bulk harvests (1000 NTU) implies vey good retention by +f tf # F OADMAG ta concowaton [DOADMAC concent fr Ab!) sa MAGE eB} (ee mean ae erences ents Mp gd ee eed oe we mm Toma parton od Pfeil 1 (2015) 28-25, volume Parise damater um) 3 Fate se cso a tepesenatve CHO cl cue ed sa {rap tat wat ceed and abated for abe Dat sve som for Shested hg Me) ans pOADMAC eed lected stam or Seren ofthe aren tocol hs gr eer he rede reed ‘oihewed vesbon sare) oe € e s rod f. the fter media. The throughput result obtained from the uncou- pled assembly suggest that the capacity of the secondary filter may exceed 500 Lm? anda ratio of 3:1 of 4:1 (DOHC to XOHC) may be ‘more adapted In comparison, the experimental assays performed on the bulk harvest that was flocculated with a pDADMIAC dose of 05% wt and subsequently filtered through pretreatment adapted dept l- {ers provided promising capacities. ll pretreatment adapted depth Alters demonstrated trial capacities of >350 Lm while the pool {rbidity was similar to tha of the control run (untreated feed pro- «essed using DOHCIXOHC filtration tain}. No increase inthe tur- bidity ofall Altres was observed 12h post depth tration (@ata rot shown), implying tht flocculation was complete at the time of filtration. 20M and 40S filters performed with higher capac- tes compared to 6OHX, te acer is designed to retain particles of| pretreated feed streams of 60 jum. Therefore data suggest thatthe fetention was not solely achieved by sieving. but adsorption may also contribute to it Furthermore, the depth filters are nominally ‘ated and process conditions could have an effect on the retention efficiency ofthe filters. There was lite diference in depth fta- tion performance of 20MS and 40MS for mAb with a 105 higher 40M filter capacity compared to 20MS for mAb2 "Among other factors, the recovery of antibodies was set as ci- terion for 2 successful clarification. The analysis revealed that >9T% and >92% of mAbI and mAb2, respectively, were recovered DA clerace (LAV) - LOL OES 4 ces efrmane in ems tbe impure HC? (tan A (ght entane shown oY (hog ection var mb (hg ie) and mab (ack el dariterettan of he elena hth owe oem the reader redo he web vero tt ale) ‘iuen penne obtained fer depth and eile es in the ication of monconl anism ad AB? Ug eet the wana (uaueute oe Tren Recast conden Dep ie nb Unwed DonepoHe 275 8 nested Done 3a 1 ‘oss pomomnc ins 1S So Mose poate ws 28 on fase poamnc Goes 2 maka Unread oucrowc zp aa fost ponomnc ens tas = Mos panne st oe ose poate ote 1D = ‘abe Ap p=) Pasha a) oak bi TU) Pra ace Sere er Heap 20 2 10m Fy a Som Tome pron a Pro rey 2 (2015) 20-275 2» Response (AU) -feERERE Time see) Response (RU) Tie 20) i 5, Resa pOADMAC eet sn PR specosay. esas eves YDADNAC in sandal (ean sales (ah ae Sbowe in Sensgrans, whee de ‘Ser abtien ster doph anon wee ates ED tee feng re Rang te: Pa inthe filtrates compared to untreated feed streams. Thus. loccula- tion with pDADMAC and subsequent filtration through pretreat- ‘ment adapted depth filters were efficient i canifying mADI and ‘maba, ‘The sterile ter capacities for each filtrate are also shown in Table 1 trates denoted with Vina values >1000D1m di noe show significa flux decay within the experimental range and hence are considered to be non-pugzing fluids Tn summary, the floceulaton of the cell culture harvest with [BDADMAC and subsequent filtration using retreatment adapted 4epeh tration simplifies te clarfiation of mammalian eet cl “ue broths expressing monoclonal antibodies compared to depth filtration without prior flocculation. The clarification throughput ‘oud be increased versus the standard clarification trai by flter- ing occulated broths through pretreatment adapted depth filters Gesigned to capture larger particles. Throughput is likely depen- ‘dent on the total cell density and viability Nevertheless, the use ‘of a flocculant in combination with pretreatment adapted depth filtration provides robust performance with the potential of redc- ing lication to a one-stage filtration process. Other pretreat ‘ment approaches showed comparable simplification [12.14] Ths Compressed and simplified process can be applied to various ‘mAb-containing cell culture harvests with effective removal of cells and ell debs, 34, Removal of soluble impure from mab feed streams ‘The clarification experiments with mAb and mAb2 demon- strated an efficient removal of cells and cll debris. At this stage. ‘ny adcltiona clearance of soluble contaminants euch as hos ell, [DNA ad proteins) wil hkely have positive impact on the down- seam unit operations, although purification performance might be euler dependent [15]. The removal of host cell proteins (HCP) and nucleic acids (DNA) during the casifiation process of ‘ADI and mAb? has been investigated in this study and compared to the coresponding untreated cell culture harvest Fig. 4. Follow ing filtration of the untreated cell culture harvest with DOHC) .OHC, the level of HCP remains high compared tothe feed material, although for some harvests a more effective HCP clearance is seen ‘The removal of HCP coud not be significantly improved by Noceu- lation and us of pretreatment adapted depth tes. The DNA level was not significantly reduced when the untreated feed streams were filtered through DOHC/XOHC depth filters. As the residence {ims of the harvests within the depth ters were similar for cou- pled and decoupled assemblies, he -fld higher loadings on XOHC In the decoupled mode obviously lowered DNA clearance in the secondary filtrate poo. By contrat, abi and mAR2 Mocculated feed streams that were processed using pretreatment adapted depth filters demonstrated >3 login and >5 logo clearance of (strongly negatively charged) DNA. 20M and 40MS ters showed highest removal of host ell DNA greater than 5 logs. These results| demonstrate good host cell DNA clearance asa result of the loccu- Iacin precess with pDADMAC, 5.5 Detection of residual focewant ‘As polycationic polymers are Becoming more and more attrac- tive in biopharmacetical applications, they have been investi- gated for their cytotoxicity to minimize the risk of any harm for patents [16,17], Previous reports based on in vivo assays have shown that polycationic polymers can change cell morphology and induce hemolysis depending on the polymer propery. dose {and incubation time. Plyethylenimine isthe most cytotoxic pely- ‘er tested, whereas the results on pDADMAC-mediated hemolysis suggest tha upto a dose of 10 ppm no disturbance ofthe red blood cell membranes was observed [16 In the present study, we have analyze for residual pDADMAC ater depth and stele fltation appiving SPR spectroscopy. The Sensograms of pure pDADMAC-standards and pDADMAC- containing samples clariying mAbl are shown in Fig. 5. The response signal of standard increased with increasing pDADMAC ‘i ) Flvaton train ie. catatonia er A fe eth (DANE, XE, 205 stan ane cl Se er queens Moe oes 3 0 iterate recede we a Teme at prt fenton Teta 1 (2015) 28-25, rece O ner] & | Hy ev rep. o> > > SEED Pelpeep@e pes ser | | |S | aS 1g 7. me ders te actin chim by POADMAC Nee rgd pres uc calls (bp). acc as (els) ad at CHO prota (hte pre) re cated y ieee ment ef pale POADINC arin mane hee ples exes psitie cage 98 oe aie Stace ss ano tbe epi rs ofan oppose

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