Application, Sterilization, and Decontamination of Ultrafiltration

2 Application, Sterilization, and Decontamination of Ultrafiltration Systems for Large-Scale Production of Biologicals

Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch002

R. T. RICKETTS, W. B. LEBHERZ III, F. KLEIN1, MARK E. GUSTAFSON2, and M. C. FLICKINGER National Cancer Institute, Frederick Cancer Research Facility, Fermentation Program, Frederick, MD 21701 Several ultrafiltration (UF) membrane systems have been evaluated for large-scale (20-300 liter) recovery of lymphokines, virus and monoclonal antibodies from eukaryotic cells and for concentration of a microbially produced cytotoxic chromoprotein, largomycin F - l l . Membrane concentration and recycle of viable eukaryotic cells has been studied in order to produce high concentrations of antibodies and lymphokines. Fetal bovine serum loads (0-15%) have been studied. Some parallel flow UF systems have limitations in flow rate/membrane area, shear forces, membrane reusability (cost), cleanability and potential for scale-up to processing of larger volumes. These applications require the integration of UF into the process without contamination of concentrated supernatant or recycled cells. Hypochlorite, azide and mild base have been found to be effective for chemical membrane sterilization, cleaning and restoration. Effective in situ steam sterilization requires staging. Membrane performance is directly related to preparation, cleaning, and handling procedures. This brief overview describes some experiences using tangential-flow and dead-end ultrafiltration techniques for concentration of eukaryotic cells, proteins and virus. The data and conclusions presented here have been drawn from process development work employing available apparatus and should be considered preliminary, rather than definitive or exhaustive. Previous ultrafiltration systems have been described (1-14) for both bench and pilot scale separations of proteins and virus. This paper primarily summarizes work on cartridge and sheet filter systems and their application to processes requiring sterilizable and contained systems. Current address: Cell-Max Corporation, Hagerstown, MD 21740. Current address: Monsanto Company, St. Louis, MO 63167.

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0097-6156/ 85/ 0271-0021 $08.00/ 0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PURIFICATION OF FERMENTATION PRODUCTS


Eukaryotic C e l l Concentration and C e l l Recycling Cartridge Systems. Cartridge systems have been used to remove a variety of particulates from suspensions, but very l i t t l e has been done on their a p p l i c a b i l i t y to eukaryotic c e l l culture harvest or recycling systems. Bench-scale studies were undertaken to explore readily scalable s t e r i l i z a b l e and r e l a t i v e l y inexpensive cartridges for application to eukaryotic c e l l cultures. Figure 1 shows the schematic of the f i r s t experimental setup for bench-scale dead-end f i l t r a t i o n employing a 2 square foot (sq. f t . ) P a l l 0.2 micron pleated cartridge. A l l culture-contact components were s t e r i l i z e d by autoclaving. Low positive pressures were used to drive the f i l t r a t i o n . A positive displacement (ColeParmer Masterflex) p e r i s t a l t i c pump was used to control the rate of flow of f i l t r a t e . A pressure d i f f e r e n t i a l of 2 to 3 prounds per square inch (psi) was maintained as well as possible during this 6 l i t e r t r i a l . Maximum observed pressure d i f f e r e n t i a l (delta P) was 4 p s i . Culture volume was reduced to system minimum dead volume after which the pump was reversed to back-flush loosely adherent c e l l s from the f i l t e r surface. Twenty-five percent of the o r i g i n a l c e l l population was recovered i n the 10X concentrate provided by the back-flushed material. Figure 2 shows an experimental design which incorporates a modified f i l t e r housing providing a side arm to r e c i r c u l a t e c e l l culture retentate back to the culture vessel and to sweep c e l l s from the f i l t e r surface. Recirculation was by a double pump head (CP 7017) at the rate of approximately 1 l i t e r / m i n . F i l t r a t i o n rates of 50-200 mls/min. were employed i n various tests of this system. The pressure drop across the membrane did not exceed 2.5 to 3.0 p s i . Various specialized b a f f l e s were tested i n the annulus between the pleated cartridge and the housing including s i l i c o n e jackets and stainless steel s p i r a l s , i n e f f o r t s to increase the sweeping action of the r e c i r c u l a t i n g culture f l u i d s . Back-flushing with f i l t r a t e was done by reversing the f i l t r a t e pump. This alone did not s i g n i f i c a n t l y improve c e l l recovery. Fresh media entered the system from either of the supply vessels and was recirculated following the addition period. This r e c i r c u l a t i o n did improve c e l l recovery by 15-25% o v e r a l l . C e l l recoveries i n t h i s system were improved over the dead-end f i l t r a t i o n when done by retrograde flow or back-flush, but only to 40 to 50% of the o r i g i n a l c e l l population recovered. These techniques indicate that present torturous path cartridge systems may be quite suitable for eukaryotic c e l l harvest and/or harvest followed by semi-continuous growth of such cultures, as l i t t l e decrease i n recovered c e l l v i a b i l i t y was observed so long as shear forces were minimized and excessive pressure changes were avoided. C e l l regrowth subsequent to these concentration tests was superior to the i n i t i a l growth cycle, or to normal saturation densities. Low t o t a l recoveries indicate that these f i l t e r s are not suitable for concentrated c e l l culture techniques. The same cartridge f i l t e r s with 2-4 sq. f t . surface area were employed i n a p i l o t - s c a l e production f a c i l i t y for semi-continuous growth of a murine lymphokine-producing c e l l l i n e . This was done as dead-end f i l t r a t i o n s , harvesting 80 to 85% of the 20-40 l i t e r culture as c e l l - f r e e f i l t r a t e and retaining 15 to 20% for inoculum to be regrown after volume restoration with fresh medium. No attempts were made to r e c i r c u l a t e or back-flush the f i l t e r cartridges.

In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


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Ultrafiltration Systems

F i g u r e 1. C e l l c o n c e n t r a t i o n "dead end" t e s t .

Receiving Filtrate Vessel

"B" Media Supply

F i g u r e 2. C e l l c o n c e n t r a t i o n " r e c i r c u l a t i o n "

test.



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A l l components i n these product runs i n c l u d i n g f i l t e r s were s t e r i l i z e d i n s i t u by steam. C o o l i n g and d r y i n g was a c c o m p l i s h e d by s t e r i l e a i r . T a n g e n t i a l - F l o w U l t r a f i l t r a t i o n . F i l t r a t i o n systems designed t o i n c o r p o r a t e sweeping o f t h e membrane d u r i n g f i l t r a t i o n a r e l e s s s u s c e p t i b l e t o c l o g g i n g than s t a t i c systems. S t i r r e d - c e l l t e c h n i c s are e f f e c t i v e f o r some m a c r o s o l u t e s b u t may i n v o l v e e x c e s s i v e shear f o r c e f o r e u k a r y o t i c c e l l s . M o d i f i e d p l a t e - a n d - f r a m e f i l t e r systems i n c o r p o r a t i n g t a n g e n t i a l f l o w and r e c i r c u l a t i o n o f r e t e n t a t e s h o u l d p r o v i d e minimum shear w h i l e a v o i d i n g e x c e s s i v e f i l t e r c l o g g i n g . F i g u r e 3 shows a c r o s s s e c t i o n o f one such system ( M i l l i p o r e P e l l i c o n ) . Only r e t e n t a t e f l o w and m a n i f o l d s a r e i l l u s t r a t e d . I n t h i s system, p a i r s o f Durapore membranes a r e s e p a r a t e d by mesh ( F i g u r e 4A) o r s i l i c o n e l i n e a r c h a n n e l s (4C) w i t h r e t e n t a t e i n l e t t h r o u g h t h e openings a t one end o f t h e s c r e e n , e x i t a t t h e o p p o s i t e end. I n t h i s flow pattern, retentate t r a v e l s p a r a l l e l to the membrane s u r f a c e , t h e number o f r e t e n t a t e pathways employed v a r i e s w i t h desired f i l t e r surface area. Figure 3 represents the P e l l i c o n u n i t setup w i t h t h r e e membrane p a i r s , o r 1 1/2 s q . f t . o f membrane. F i g u r e 5 shows permeate o r f i l t r a t e f l o w paths from t h e same s e t u p , w i t h f i l t r a t e s p a s s i n g through t h e membrane and o u t o f t h e u n i t through e i t h e r o r b o t h f i l t r a t e m a n i f o l d s . Figure 6 i l l u s t r a t e s our m o d i f i c a t i o n f o r a s e r p e n t i n e - f l o w pathway c r e a t e d by b l o c k i n g r e t e n t a t e c h a n n e l s i n one end o f t h e f i l t r a t e s c r e e n s ( F i g u r e 4 ) . The c o n v e n t i o n a l f i l t r a t e s c r e e n , ( 4 B ) , has openings complementary to those o f t h e r e t e n t a t e s c r e e n s . B l o c k i n g the r e t e n t a t e channels a t one end i s performed by s i l i c o n e (4D). I n c o r p o r a t i o n o f two such m o d i f i e d s c r e e n s p r o v i d e s f o r s e r p e n t i n e - f l o w through t h e u n i t w i t h the e n t i r e r e t e n t a t e volume sweeping each membrane p a i r . T a b l e I compares these two t e c h n i q u e s . Table I . Comparison of P a r a l l e l and S e r p e n t i n e

Flow

P a r a l l e l - F l o w Pathway Advantage: Simplicity Disadvantage:

Serpentine-Flow General:

Doubling area.

f i l t e r area halves cross-flow per u n i t

Pathway System r e q u i r e s odd number o f f i l t e r sheet p a i r s .

Advantage:

C r o s s - f l o w p e r u n i t a r e a n o t as r a p i d l y reduced by i n c r e a s e d a r e a .

Disadvantage:

Only one l e f t - c h a n n e l b l o c k and one r i g h t - c h a n n e l b l o c k a l l o w e d p e r s t a c k , each n e a r e s t r e t e n t a t e ports. _ _

T h i s p a r a l l e l - f l o w system i s t h a t f o r w h i c h t h e u n i t was d e s i g n e d , but f o r c e l l h a r v e s t s o r r e c y c l i n g t e c h n i c s i t s u f f e r s by r e d u c i n g f i l t e r c r o s s - f l o w by h a l f f o r each d o u b l i n g o f f i l t e r a r e a - a



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F i g u r e 3. C o n v e n t i o n a l p a r a l l e l - f l o w pathway ( r e t e n t a t e f l o w o n l y shown). Key: 1, r e t e n t a t e i n l e t d i s t r i b u t i o n m a n i f o l d ; 2, r e t e n t a t e o u t l e t d i s t r i b u t i o n m a n i f o l d ; - — -, end g a s k e t ; - - - -, f i l t r a t e s c r e e n ; - - — , membrane f i l t e r ( D u r a p o r e ) ; • · — , r e t e n t a t e s c r e e n on l i n e a r path r e t e n t a t e c h a n n e l ; r e t e n t a t e p a r a l l e l - f l o w pathway.


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F i g u r e 5. F i l t r a t e f l o w -» end g a s k e t ; - f i l t e r (Durapore); ·· channel; filtrate

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p a t h s . Key: 1 and 2, f i l t r a t e m a n i f o l d s ; - -, f i l t r a t e s c r e e n ; - , membrane , r e t e n t a t e s c r e e n on l i n e a r path r e t e n t a t e f l o w pathway ( c o n v e n t i o n a l ) .



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^

^

~

^

F i g u r e 6. S e r p e n t i n e - f l o w pathway generated by m o d i f i c a t i o n of f i l t r a t e screens ( r e t e n t a t e f l o w o n l y shown). Key: 1, r e t e n t a t e i n l e t d i s t r i b u t i o n m a n i f o l d ; 2, r e t e n t a t e o u t l e t d i s t r i b u t i o n manifold; , end g a s k e t ; , f i l t r a t e screen; , membrane f i l t e r (Durapore); · · — , r e t e n t a t e screen on l i n e a r path r e t e n t a t e c h a n n e l ; r e t e n t a t e s e r p e n t i n e - f l o w pathway.



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s i t u a t i o n w h i c h has been found t o l e a d t o some membrane c l o g g i n g and w h i c h l i m i t s p r o c e s s i n g volume and a r e a o f f i l t e r employed. The 1 1/2 s q . f t . u n i t i s t h e maximum t h a t can be used w i t h p a r a l l e l f l o w and one l i t e r / m i n . r e c i r c u l a t i o n r a t e . E x c e e d i n g t h i s r a t e r e s u l t s i n reduced (50%) r e c o v e r y r a t h e r than 90 t o 95% c e l l popu l a t i o n r e c o v e r y a f t e r 1 0 - f o l d c o n c e n t r a t i o n . The 1 1/2 s q . f t . p a r a l l e l f l o w system ( F i g u r e 7) can p r o c e s s 6 t o 10 l i t e r s o f a p p r o x i m a t e l y 2 m i l l i o n vc/ml c e l l c u l t u r e t o 1 0 - f o l d c o n c e n t r a t i o n w i t h r e c i r c u l a t i o n r a t e s o f 1 l i t e r / m i n . and f i l t r a t i o n r a t e s o f a p p r o x i m a t e l y 100 ml/min. I t i s p o s s i b l e t o s h u t - o f f f i l t r a t i o n a t a p p r o x i m a t e l y 1 l i t e r f i l t r a t e i n t e r v a l s o r whenever d e l t a Ρ exceeds 2.5-3.0 p s i and a l l o w 3 t o 5 min. r e c i r c u l a t i o n w i t h o u t f i l t r a t i o n to sweep l o o s e l y adherent c e l l s f r e e o f t h e membrane s u r f a c e . F i l t r a t i o n i s c o n t i n u e d t o system dead volume, i . e . no c u l t u r e l e f t i n c u l t u r e f l a s k , a f t e r which the f i l t e r i s back-flushed w i t h f r e s h medium t o purge c o n c e n t r a t e d c e l l c u l t u r e . E x c e s s i v e membrane c l o g g i n g can be r e l i e v e d t o some degree by b a c k - f l u s h i n g w i t h f i l t r a t e d u r i n g r e c i r c u l a t i o n . T h i s may n o t be n e c e s s a r y where adequate c r o s s - f l o w i s m a i n t a i n e d . The s e r p e n t i n e - f l o w m o d i f i c a t i o n can be used w i t h 1 1/2 t o 4 1/2 s q . f t . o f membrane s u r f a c e , b u t does n o t r e a d i l y s c a l e beyond t h i s s i z e . The l a r g e r s u r f a c e a r e a s have n o t been t e s t e d i n s t e r i l e o p e r a t i o n t o date. One l i m i t i n g f a c t o r i s t h e 1/4 i n c h i n t e r i o r d i a m e t e r t u b i n g employed throughout the system. L a r g e r diameter t u b i n g and l a r g e r c a p a c i t y low-shear pumps, p o s s i b l y coupled w i t h r e d r i l l i n g o f t h e P e l l i c o n b l o c k r e t e n t a t e m a n i f o l d s , c o u l d a l l o w l a r g e r volume p r o c e s s i n g . I n a l l e u k a r y o t i c c e l l c o n c e n t r a t i o n and r e c y c l i n g p r o c e d u r e s , c e r t a i n l i m i t i n g f a c t o r s have been i d e n t i f i e d : i ) avoidance o f shear by m a i n t a i n i n g r e s t r i c t i o n - f r e e f l o w and by u s i n g minimum d e l t a Ρ t o d r i v e t h e f i l t r a t i o n ( f o r b e n c h - s c a l e c e l l r e c y c l i n g and c o n c e n t r a t i o n a maximum o f 3 t o 5 p s i i n l e t p r e s s u r e was employed, w i t h f i l t r a t e back p r e s s u r e m a i n t a i n e d by f i l t r a t e pump r a t e s , r e t e n t a t e back p r e s s u r e s were 0-2 p s i from system r e s i s t a n c e ) ; i i ) sweeping t h e membrane by r e c i r c u l a t i o n w i t h f i l t r a t e - f l o w stopped t o c l e a r l o o s e l y adherent c e l l s from t h e membrane whenever d e l t a Ρ exceeds 2.5 t o 3.0 p s i ; and i i i ) b a c k f l u s h i n g w i t h medium a t t h e t e r m i n a t i o n o f t h e r u n t o purge t h e membrane s u r f a c e s and channels o f concentrated c e l l c u l t u r e . A s i m i l a r e x p e r i m e n t a l setup i s used f o r b e n c h - s c a l e t e s t i n g , w i t h t h e P e l l i c o n u n i t r e p l a c e d by t h e M i n i t a n ( M i l l i p o r e ) s m a l l s c a l e s e r p e n t i n e f l o w system. F i g u r e 8 shows t h e M i n i t a n f i l t e r p a c k e t ( r i g h t ) and t h e l i n e a r channel r e t e n t a t e gasket ( l e f t ) . S e r p e n t i n e f l o w i s a c h i e v e d b y a l t e r n a t i n g l e f t - and r i g h t - h a n d e d p o s i t i o n s f o r t h e r e t e n t a t e p a t h . T h i s u n i t can h o l d 1/2 s q . f t . of f i l t e r a r e a i n 0.1 s q . f t . p a c k e t s , and i s u s e f u l f o r volumes t o a maximum o f 3-5 l i t e r s . Protein Purification Recovery o f Largomycin F - I I . Largomycin F - I I i s a c h r o m o p r o t e i n o f a p p r o x i m a t e l y 30,000 MW produced by Streptomyces p l u r i c o l o r e s c e n s . In a d d i t i o n t o largomycin F - I I , s e v e r a l other b i o l o g i c a l l y a c t i v e p r o t e i n s , r a n g i n g i n apparent m o l e c u l a r w e i g h t from l e s s than 1,000 to g r e a t e r than 400,000 a r e p r e s e n t i n t h e f e r m e n t a t i o n b r o t h . U s i n g t h e B i o l o g i c a l I n d u c t i o n Assay (BIA) and M i c r o c o c c u s l u t e u s




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Filtrate Cell Culture

F i g u r e 7.

M i l l i p o r e P e l l i c o n or Minitan operating

F i g u r e 8.

schematic.

M i n i t a n system.



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RICKETTS ET AL.


31

(ML) as measures f o r b i o l o g i c a l a c t i v i t y , these c o n t a m i n a t i n g a c t i v e p r i n c i p l e s must be removed from the l a r g o m y c i n F - I I d u r i n g processing. Largomycin F - I I may be i s o l a t e d from S_. p l u r i c o l o r e s c e n s f e r m e n t a t i o n b r o t h from e i t h e r the s u p e r n a t a n t o r the mycelium. The r e c o v e r y scheme f o r the cake p u r i f i c a t i o n p r o c e s s and the s u p e r n a t a n t r e c o v e r y p r o c e s s a r e shown i n F i g u r e s 9 and 10. The advantages o f the cake procedure i n c l u d e low p r o t e a s e a c t i v i t y i n i t i a l l y and h i g h e r F - I I s p e c i f i c a c t i v i t y . The s u p e r n a t a n t c o n t a i n s more l a r g o m y c i n F - I I (up t o 400 micrograms/ml o f b r o t h ) than does a cake e x t r a c t , but a l s o c o n t a i n s many more c o n t a m i n a t i n g p r o t e i n s d e r i v e d from the c u l t u r e medium and c o n s e q u e n t l y r e q u i r e s more p r o c e s s i n g s t e p s . Both procedures i n c l u d e s e v e r a l d i f f e r e n t u l t r a f i l t r a t i o n steps. One s t e p c o n s i s t e n t w i t h b o t h p r o c e s s i n g schemes i s the g e l permeation chromatography on Sephadex G-100. I n i t i a l l y , c o n s i d e r able d i f f i c u l t y occurred i n preparing a concentrated largomycin F-II r i c h s o l u t i o n i n a s u f f i c i e n t l y s m a l l volume (2L) h a v i n g a v i s c o s i t y low enough t o e f f i c i e n t l y and r a p i d l y pass through the G-100 s t a c k column. By p a s s i n g l a r g o m y c i n F - I I r i c h m a t e r i a l s through a 0.45 m i c r o n Durapore f i l t e r ( M i l l i p o r e , P e l l i c o n ) we were a b l e t o remove enough o f the h i g h v i s c o s i t y m a t e r i a l s to produce c o n c e n t r a t e d s o l u t i o n s o f l a r g o m y c i n F - I I (about 20,000 micrograms/ml) which were i d e a l f o r chromatography on the Sephadex s t a c k column. Passage through the Durapore had the added b e n e f i t o f removing e s s e n t i a l l y a l l o f the F-I f r a c t i o n from the F - I I , e l i m i n a t i n g the need f o r the ammonium s u l f a t e p r e c i p i t a t i o n s t e p . The f l o w c h a r t f o r a t y p i c a l experiment i s shown i n F i g u r e 11. HPLC t r a c i n g s f o r each o f t h e s e f r a c t i o n s ( F i g u r e 12) shows the improved s e p a r a t i o n w i t h each u l t r a f i l t r a t i o n step. I n the c o u r s e o f l a r g o m y c i n F - I I p r o c e s s development, t h e performance o f a v a r i e t y o f u l t r a f i l t r a t i o n methodology was e v a l u a t e d . M i l l i p o r e , Dorr O l i v e r and DDS sheet and Amicon h o l l o w f i b e r equipment were t e s t e d . The d a t a i s p r e s e n t e d i n T a b l e I I . The g r e a t e r s u c c e s s w i t h the Dorr O l i v e r system i s p r o b a b l y due t o the c e l l u l o s i c n a t u r e o f these membranes, as the l a r g o m y c i n F - I I s u p e r n a t a n t d i d c o n t a i n p o l y p r o p y l e n e - d e r i v e d antifoâming agents which are known t o suppress f l u x r a t e s a c r o s s p o l y s u l f o n e membranes. The f l u x r a t e s f o r a l l sheet systems were s u p e r i o r t o those observed w i t h the h o l l o w f i b e r system. Recovery o f M o n o c l o n a l A n t i b o d i e s . The systems used a t the benchs c a l e f o r c o n c e n t r a t i o n and p a r t i a l p u r i f i c a t i o n o f murine hybridoma monoclonal a n t i b o d y were e s s e n t i a l l y i d e n t i c a l t o those shown e a r l i e r f o r membrane ( P e l l i c o n ) p r o c e s s i n g o f c e l l c u l t u r e s . I n s t e a d o f the Durapore membranes employed f o r c e l l p r o c e s s i n g , p r o t e i n s were c o n c e n t r a t e d w i t h 100- 30- 10-K d a l t o n nominal m o l e c u l a r w e i g h t p o l y s u l f o n e membranes. The a n t i b o d y produced i s on the o r d e r o f 140,000 d a l t o n s . T a b l e I I I emphasizes the importance o f the term " n o m i n a l i n m o l e c u l a r weight c u t o f f s . The n o m i n a l weight i s determined by the vendor f o r s t a n d a r d g l o b u l a r p r o t e i n s i n s p e c i f i e d s o l u t i o n s . When the p r o t e i n o f i n t e r e s t o c c u r s i n s m a l l amounts i n h i g h l y complex p r o t e i n m i x t u r e s such as t i s s u e c u l t u r e medium; o r when the i o n i c s t r e n g t h i s a l t e r e d t o i n f l u e n c e p r o t e i n a g g r e g a t i o n , and p o s s i b l y the membrane i t s e l f , n


32


LM Fermentation Broth Supernatant

Filter press Cake Extract with V2 volume P 0 buffer, pH 7, 0.01 M Mix for several hours, filter in filter press 4

Spent cake

Rich Extract


Concentrate on WFE, approximately 8-fold Rich Concentrate Centrifuge, M-16s Clarified Concentrate Dialyze/concentrate on DC-50 with in-line GAF filtration Dialyzed Concentrate Pass through 0.45 μ Durapore filter Durapore Permeate Concentrate with 10K membrane on Pellicon Stack Starting Material Pass through Sephadex G-100 Pool F-II rich fractions Stack Product Pool Concentrate with 10K membrane on Pellicon Dialyze if necessary Chromatograph on HA, 0.1 m M to 20 m M gradient Pool F-II rich fractions, concentrate, lyophilize Final Product Largomycin F-II

F i g u r e 9.

Largomycin F - I I cake r e c o v e r y scheme.


2.

RICKETTS ET AL.

33


LM Fementation Broth Filter press

Cake

Filtered Supernatant 0.45 μ Filter


Cone. With Pellicon Door Oliver DC-50 DDS

Concentrate on WFE, approximately 5-fold WFE Concentrate Centrifuge, M-16s Rich Concentrate Dialyze/concentrate on DC-50 Dialyzed Concentrate Treat with equal volume sat. (NH ) S0 Mix, centrifuge M-16s 4

2

4

Clarified Concentrate Dialyze/concentrate on DC-50 Dialyzed Concentrate Pass through 0.45 μ Durapore filter Durapore Permeate Concentrate with 10K membrane on Pellicon Stack Starting Material Pass through Sephadex G-100 Pool F-II rich fractions Concentrate with 10K membrane on Pellicon Stack Column Product Chromatograph on HA, 10 m M Flash column Pool F-II rich fractions Concentrate with 10K membrane on Pellicon Flash HA Column Product Chromatograph on HA 0.1 m M to 20 m M gradient Pool F-II rich fractions, concentrate, lyophilize Final Product Largomycin F-II

F i g u r e 10. Largomycin

F - I I supernatant r e c o v e r y scheme.



LM 85-FD

(600 g, 6%-7% F-ll)

+ 4 liters H 0


2

LM 85-30'

(11.2 mg/ml, ca. 4 liters, 44.8 g F-ll)

F-ll Permeates

Durapore LM 85-31 (8.1 mg/ml, ca. 3.3 liters = 26.7 g) + 2 liters H 0 2

Durapore LM 85-33

(5.2 mg/ml, ca. 2 liters = 10.5 g)

LM 85-35

(2.95 mg/ml, ca. 2 liters 5.9 g) 43.1 g

+ 2 liters H 0 2

Durapore

LM 85-36

(2 mg/ml, ca. 2.7 liters, 5.4 g F-ll) Figure

11. LM 85-FD Durapore

processing.


RICKETTS ET AL.



F-ll

LM 85-30' Starting material

LM 85-33 Second filtrate Figure

12.

Retentate after second pass Durapore p r o c e s s i n g

LM 85-35 Third filtrate o f LM 85

LM 85-36 Final retentate supernatant.


36


Table I I .

C o n c e n t r a t i o n o f 0.45 μπι F i l t e r e d L a r g o m y c i n F - I I Supernatant w i t h U l t r a f i l t r a t i o n U n i t s Vendor

Parameter

Millipore

ft*

Amicon DC-50

DDS

5

1.44

50

2.34

10K sheet Polysulfone

10K sheet Cellulose

10K hollow fiber Polysulfone

20K sheet Polysulfone

9 - 2 liter 43 min

1 0 - 5 liter 80 min

19—4 liter 30 min

20-12 liter 150 min

12.13

50.00

3.87

8.10

PSI

13

43

25

130

I/O

0

22

15

73

3.0 liter/min

11.7 liter/min

24 liter/min

6.5 liter/min

Membrane

Volume/ Time


Door Oliver

GFDs

Recirculation Rate

All units operated s steady state with ~ constant flux rates.

Table I I I .

Titer (mg IgG/ml)

Protein (mg/ml)

Specific Activity (mg IgG/mg protein)

Recovery (%)

Orig. Susp. Cone. Filtrate

0.065 0.957 0

27 107

0.0024 0.0089

80

+ t


0.024 0.137 0.016

1.35 5.70 ND

0.0178 0.0240

20

-t


0.086 0.726 0.005

1.65 14.5 ND

0.0515 0.0372

63

+ t


0.020 0.098 0.016

1.38 4.20 1.58

0.0145 0.0233 0.0101

36

+ t,*


0.016 0.703 0

0.94 18.25 0

0.0169 0.0385

>100

MWCO

0.5 M NaCI

100K

+*

10K

D3 Immunoglobin C o n c e n t r a t i o n as R e l a t e d t o Membrane M o l e c u l a r Weight C u t o f f

Sample

•RPMI-1640 medium + 15% FBS fRPMI-1640 medium + 15% amniotic fluid + 2% FBS tReprocessed filtrate from 100K



2.

RICKETTS ET AL.


37

n o m i n a l m o l e c u l a r w e i g h t l i m i t o f a g i v e n membrane and m o l e c u l a r w e i g h t o f the d e s i r e d p r o t e i n p r o d u c t a r e o n l y g u i d e l i n e s . The c e l l f r e e s u s p e n s i o n s p r o c e s s e d i n the f i r s t f o u r examples a r e c o m p a r i sons between a h i g h p r o t e i n medium and low p r o t e i n medium. A n t i body t i t e r s a r e determined by t r i p l i c a t e samples u s i n g an ELISA assay system. The s t a n d a r d d e v i a t i o n o f r e p l i c a t e t i t e r s on c o n c e n t r a t e d m a t e r i a l s i s a p p r o x i m a t e l y 30%. C e l l - f r e e s u p e r n a t a n t t i t e r s show s t a n d a r d d e v i a t i o n s o f a p p r o x i m a t e l y 10%. A l t h o u g h o r i g i n a l t i t e r s v a r i e d , t h i s v a r i a t i o n i s not r e l a t e d t o p e r c e n t r e c o v e r y ( d a t a not i n c l u d e d on t h i s t a b l e ) . The two s i g n i f i c a n t v a r i a b l e s a r e p r o t e i n c o n c e n t r a t i o n and i o n i c s t r e n g t h . I n the f i r s t example, a p p r o x i m a t e l y 80% o f the t o t a l p r o t e i n s p r e s e n t passed through the 100K d a l t o n membrane, but no a n t i b o d y c o u l d be d e t e c t e d i n the f i l t r a t e . Low p r o t e i n s o l u t i o n s showed s i g n i f i c a n t l o s s e s even though no a d d i t i o n a l NaCl had been used. When such s o l u t i o n s were t e s t e d w i t h 0.5M NaCl r e c o v e r i e s were u n a c c e p t a b l e , and s i g n i f i c a n t t i t e r s o f immunoglobulin were seen i n the f i l t r a t e . The f i l t r a t e s from t h e s e two examples were p o o l e d and r e p r o c e s s e d over 10K d a l t o n membrane. Recovery was complete and no a c t i v i t y c o u l d be demonstrated i n the f i l t r a t e . T a b l e IV p r e s e n t s d a t a from runs i n w h i c h c e l l c u l t u r e s were grown i n a d e f i n e d serum-free medium w i t h r e l a t i v e l y low t o t a l p r o t e i n s . C e l l - f r e e c u l t u r e s u p e r n a t a n t s were p r o c e s s e d o v e r a 100K d a l t o n membrane w i t h o r w i t h o u t NaCl a d d i t i o n s . Both h i g h and low s a l t s o l u t i o n s showed some immunoglobulin i n the f i l t r a t e . These f i l t r a t e s were r e p r o c e s s e d o v e r a 10K d a l t o n membrane w i t h complete r e c o v e r y o f immunoglobulin. Another p a i r o f runs were p r o c e s s e d over a 30K d a l t o n membrane w i t h o u t immunoglobulin appeari n g i n the f i l t r a t e s , b u t w i t h r e l a t i v e l y h i g h r e t e n t i o n o f e x t r a n eous p r o t e i n s . The i m p l i c a t i o n from t h i s i s t h a t f o r t h i s p a r t i c u l a r p r o t e i n p r o d u c t , crude c u l t u r e s u p e r n a t a n t s w i t h p r o t e i n conc e n t r a t i o n g r e a t e r than 3.0 mg/ml w i l l g i v e h i g h r e c o v e r i e s a f t e r s a l t i n g and p r o c e s s i n g o v e r a r e l a t i v e l y h i g h (100K d a l t o n ) m o l e c u l a r w e i g h t membrane w i t h s i g n i f i c a n t removal o f e x t r a n e o u s s m a l l e r p r o t e i n s i n the f i l t r a t e s . The o p t i m a l parameters f o r each p r o t e i n must be determined e m p i r i c a l l y . Recovery o f Lymphokines. A number o f p i l o t - s c a l e p r o d u c t i o n runs were performed u s i n g a murine c e l l l i n e w h i c h i s a c o n s t i t u t i v e p r o d u c e r o f a lymphokine, i n t e r l e u k i n - 3 . T h i s m a t e r i a l i s a p p r o x i m a t e l y 28,000 d a l t o n s , and was produced i n low p r o t e i n medium, 1.7% t o t a l r e s i d u a l serum. These p r o d u c t i o n runs were p r o c e s s e d u s i n g the equipment shown s c h e m a t i c a l l y i n F i g u r e 13. These runs may be grouped i n t o s m a l l volume 16 t o 32 l i t e r b a t c h e s and l a r g e r volume o f 80 t o 140 l i t e r p r o d u c t i o n l o t s . S m a l l volumes were p r o c e s s e d employing the p l a s t i c P e l l i c o n c a s s e t t e w i t h 5 s q . f t . o f 10K membrane. L a r g e r volumes used the s t a i n l e s s s t e e l u n i t w i t h 20 s q . f t . o f the same membrane. F i g u r e 14 shows f l u x c u r v e s f o r 6 s m a l l volume and 10 l a r g e volume p r o d u c t i o n r u n s . P l u s o r minus two s t a n d a r d d e v i a t i o n s a r e shown f o r each c u r v e . These g i v e some measure o f the r e p r o d u c i b i l i t y o f f e r e d by t h i s p r o c e s s i n g system. T o t a l c o n c e n t r a t i o n a c h i e v e d ranged from 10 t o 30 f o l d depending upon s t a r t i n g volume. F i g u r e 15 d e t a i l s f i l t r a t i o n r a t e s f o r the 10 l a r g e volume r u n s , a l l o f w h i c h employed the same membranes. By the t h i r d r u n t h e r e was l o s s i n f i l t r a t i o n r a t e , so


38



T a b l e IV. D3 Immunoglobin C o n c e n t r a t i o n as R e l a t e d t o Membrane M o l e c u l a r Weight C u t o f f (RPMI-1640 Supplemented Serum-Free Medium)

MWCO 100K

10K

100K

0.5 M NaCl

Sample

Titer (mg IgG/ml)

Protein (mg/ml)

Specific Activity (mg IgG/mg protein)

Recovery (%)

+


0.027 0.446 0.005

2.2 14.4 0.9

0.0123 0.0310 0.0056

>100

+*


0.008 0.158 0

0.96 11.0 0.02

0.0083 0.0144

>100

-


0.027 0.641 0.011

2.1 12.9 0.9

0.0130 0.0501 0.0122

>100


0.009 0.147 0

0.82 9.5 0.02

0.0198 0.0155

>100

*

10K

—

—

30K

+


0.055 0.514 0

2.30 16.8 0

0.0239 0.0306

87

30K

-


0.011 0.275 0

3.0 12.4 0.1

0.0038 0.0221

>100

— —

* Reprocessed filtrate from 100K


2.

RICKETTS ET AL.

39



1 6

....Li. 4 5

List of Equipment for Batch Ultrafiltration Process A

Processing Tank Containing Cell-Free Supernatant

Aseptic Sample Port

9

c

14 15

Retentate Return

10

Pall Filter (0.1 μνη)

11

Contaminate Drain

8

Product Inlet

4

Refrigerated Water Jacket Inlet

1

D

12

Millipore Pellicon Cassette System

Retentate Filtrate Outlets

Refrigerated Water Jacket Outlet

13

Pump Outlet Side

3

Rotary Pump Inlet

2

Ε Tri-Clover Rotary Pump

Primary and HEPA Filters Cabinet Exhaust

Processing Tank Outlet

Β

Biological Cabinet (negative pressure)

Chiller (compressor)

F i g u r e 13. Batch u l t r a f i l t r a t i o n

process.


5 6, 7



800 γ

Time (min) F i g u r e 14. IL3 c o n c e n t r a t i o n c u r v e s , s m a l l and l a r g e volume f l u x r a t e s . Key: 1, s m a l l volume; 2, l a r g e volume. (Reproduced w i t h p e r m i s s i o n from Ref. 4. C o p y r i g h t 1984, American S o c i e t y for Microbiology.)



RICKETTS ET AL.


10

20 30 40 50 60

70 80 90 100 110 120 130

Time (min) F i g u r e 15. IL3 c o n c e n t r a t i o n c u r v e s , l a r g e volume f l u x r a t e s , i n d i v i d u a l runs. Key: , i n l e t p r e s s u r e 60 p s i ; - - - i n l e t p r e s s u r e 80 p s i .


42


subsequent runs were performed a t i n c r e a s e d i n l e t p r e s s u r e . Further v a r i a t i o n s i n the f l o w r a t e s a c h i e v e d r e f l e c t e d the c a r e w i t h w h i c h the membranes were c l e a n e d between r u n s . T h i s s e t o f membranes was s t i l l as u s e f u l a t the end o f t h i s p r o d u c t i o n s e r i e s as a t t h e b e g i n n i n g , and thus p r o v i d e s an e c o n o m i c a l as w e l l as e f f e c t i v e p r o c e s s i n g s t e p . R e c o v e r i e s tended t o be 80 t o more than 100% o f the o r i g i n a l lymphokine a c t i v i t y , w i t h no l o s s e s seen i n t h e filtrate.


Virus

Concentration

Membrane c o n c e n t r a t i o n o f v i r u s e s has been w i d e l y r e p o r t e d i n t h e l i t e r a t u r e . R e c e n t l y t h i s t e c h n i c has been s c a l e d - u p t o t h e p i l o t p l a n t s c a l e ( F i g u r e 16) f o r the c o n c e n t r a t i o n o f g r e a t e r than 180 l i t e r s o f v i r u s - r i c h c e l l - f r e e s u p e r n a t a n t by 15 s q . f t . o f 100K membrane. The u n i t i s assembled i n a containment room and s t e r i l i z e d i n s i t u by steam i n the c o n f i g u r a t i o n shown. The v i r u s i n v o l v e d i s human T - c e l l lymphoma v i r u s from the C10/MJ-2 c e l l l i n e , and may be a s i g n i f i c a n t b i o l o g i c a l h a z a r d . F i g u r e 17 compares t h e c o n c e n t r a t i o n p r o c e s s w i t h p24 v i r u s c o r e p r o t e i n . F l u x r a t e s averaged a p p r o x i m a t e l y 1.0 l i t e r / m i n . u s i n g a s t a r t i n g i n l e t p r e s sure o f 50 p s i and i n c r e a s i n g t o 80 p s i as f l u x r a t e d e c r e a s e d . R e t e n t a t e o u t l e t p r e s s u r e s were 10 t o 15 p s i . O v e r a l l c o n c e n t r a t i o n was 5.6 f o l d , w h i l e v i r u s core p r o t e i n (p24) c o n c e n t r a t i o n was 3.8 f o l d . No core p r o t e i n a c t i v i t y was seen i n the f i l t r a t e . Apparent l o s s e s f o r t h i s p r e l i m i n a r y run were a p p r o x i m a t e l y 32%. Whether t h i s l o s s was due t o e x c e s s i v e p r o t e a s e a c t i v i t y generated d u r i n g c e l l r e m o v a l , from v i r u s adherence t o the membrane, o r from e x c e s s i v e shear f o r c e s has not been d e t e r m i n e d . I t s h o u l d be p o i n t e d out t h a t i n t h i s p r e l i m i n a r y t r i a l , a l t h o u g h sweeping was used, n e i t h e r b a c k - f l u s h i n g w i t h f i l t r a t e nor b a c k - f l u s h i n g through r e t e n t a t e c h a n n e l s was i n c o r p o r a t e d . These a d d i t i o n s t o t h e t e c h n i c a r e under i n v e s t i g a t i o n . S t e r i l i z a t i o n and D e c o n t a m i n a t i o n Most p r o c e s s i n g equipment i s designed f o r open o p e r a t i o n , so some m o d i f i c a t i o n s o f equipment and p r o c e d u r e s a r e commonly r e q u i r e d t o employ these systems i n b i o l o g i c a l l a b o r a t o r i e s where r e s t r i c t i o n s a p p l y b o t h t o the p r o t e c t i o n o f c u l t u r e s and p r o d u c t s from e x t e r n a l c o n t a m i n a t i o n and t o the p r o t e c t i o n o f p e r s o n n e l from p o t e n t i a l l y hazardous m a t e r i a l . Chemical D e c o n t a m i n a t i o n . S t a n d a r d i z e d p r o c e d u r e s f o r c h e m i c a l " s t e r i l i z a t i o n " are g e n e r a l l y set f o r t h i n manufacturer's i n s t r u c t i o n s , b u t such p r o c e d u r e s a r e o f l i m i t e d v a l u e when used i n c o n t a c t w i t h c o n c e n t r a t e d b i o l o g i c a l f l u i d s . These p r o c e d u r e s , used w i t h new and c l e a n components, w i l l g i v e a p e r c e n t a g e o f s t e r i l e r u n s , b u t o v e r a l l a r e l a b o r i o u s and m o d e r a t e l y e x p e n s i v e i n q u a n t i t y o f m a t e r i a l s and time r e q u i r e d . The p r e s e n t M i l l i p o r e M i n i t a n system i n c o r p o r a t e d steam s t e r i l i z a b l e hardware, b u t t h e f i l t e r p a c k e t s themselves cannot w i t h s t a n d steam s t e r i l i z a t i o n . T h i s system has been t e s t e d under c h e m i c a l d e c o n t a m i n a t i o n succesf u l l y , o r i t can be a s e p t i c a l l y assembled a f t e r steam s t e r i l i z a t i o n of hardware and gas s t e r i l i z a t i o n o f f i l t e r p a c k e t s . Neither of



valve

S

Η ) π

tel

Γ

π

F i g u r e 16. G e n e r a l u l t r a f i l t r a t i o n (UF) scheme used i n HTLV p r o c e s s . Key: 1, s u p p l y / r e c y c l e ( r e t e n t a t e ) v e s s e l ; 2, s a n i t a r y pump; 3, membrane UF u n i t ; 4, b a c k - f l u s h loop and pump; 5, p e r meate v e s s e l ; 6, containment room; 7, e n c l o s e d g l o v e box f o r sampling r e t e n t a t e ; 8, e n c l o s e d g l o v e box f o r h a r v e s t i n g r e t e n t a t e and/or filtrate.

Magnahelic gauge

Tube clamp

(M)

Pressure gauge

Pump

Cp

®

^5"

Direction of flow

Legend

D+O

7


®

I

> r

Η

m

η

7*


F i g u r e 17. R e l a t i o n s h i p between volume c o n c e n t r a t i o n ( f o r m a t i o n of v i r u s - r i c h r e t e n t a t e ) and a c t u a l v i r u s t i t e r d u r i n g 100,000 MWCO membrane u l t r a f i l t r a t i o n ( U F ) . Supernatant was c o n c e n t r a t e d 5.6 times by t h i s method. V i r u s was c o n c e n t r a t e d 3.8 times. The permeate c o n t a i n e d no v i r u s a c t i v i t y .

Process Time (min)


Η

Ο α G η

δ

m

70

ο •η •η m

δ

π

2

a

2.

RICKETTS ET AL.

45


these p r o c e d u r e s i s o f s u f f i c i e n t l y h i g h r e l i a b i l i t y t o be employed routinely. R o u t i n e d e c o n t a m i n a t i o n and c l e a n i n g f o l l o w i n g manufacturer's suggestions, r e q u i r e forethought t o insure a p p l i c a b i l i t y , s a f e t y , and thoroughness. The system d e c o n t a m i n a t i o n f o l l o w i n g c o n c e n t r a t i o n o f t h e HTLV i s a case i n p o i n t . T h i s agent i s n o t o n l y i m p l i c a t e d i n human m a l i g n a n c y , b u t i s now under i n v e s t i g a t i o n as a p o s s i b l e agent i n a c q u i r e d immune d e f i c i e n c y syndrome (AIDS). F o l l o w i n g c o n c e n t r a t i o n o f HTLV over 100K membrane, t h e system i s p r o c e s s e d i n s i t u by t h e p r o c e d u r e d e t a i l e d i n T a b l e V.


Table V. Membrane System D e c o n t a m i n a t i o n Procedure In s i t u , a l l waste t o k i l l tank: 1. NaOH, 1M, 1 l i t e r / s q . f t . membrane, f i l t r a t e v a l v e s c l o s e d . 2. Repeat s t e p 1 w i t h f i l t r a t e v a l v e s open. 3. A l l o w i n g s t a n d i n g (30 min.) w i t h s o l u t i o n . 4. NaOCL, 525 ppm ( 1 % C h l o r o x ) , 1 l i t e r / s q . f t . membrane. 5. A l l o w s t a n d i n g , o v e r n i g h t , w i t h s o l u t i o n . 6. F l u s h w i t h R/0 w a t e r , 205 l i t e r s / s q . f t . membrane. Containment Room 1. Remove f i l t e r s t a c k . 2. P l a c e i n sodium a z i d e (0.1%) o v e r n i g h t . 3. S t o r e f i l t e r s a t 4°C. In

situ 1. R e s e a l u n i t . 2. Steam s t e r i l i z e system, 30 min. 121°C. 3. S t e r i l i z e k i l l tank.

Low h a z a r d p r o c e s s i n g systems, such as those d i s c u s s e d f o r use w i t h m o n o c l o n a l a n t i b o d y and lymphokines a r e a l s o c l e a n e d i n s i t u , but under l e s s r i g o r o u s containment c o n d i t i o n s , d e t a i l e d i n T a b l e V L Membrane c l e a n i n g i s e s s e n t i a l l y t h a t suggested by t h e v e n d o r , m o d i f i e d i n c e r t a i n a p p l i c a t i o n s . Durapore i s l e s s c h e m i c a l l y r e s i s t a n t than p o l y s u l f o n e , and sodium h y d r o x i d e i s no l o n g e r recommended, b u t i s used as noted i n s t e p 3, a p p r o x i m a t e l y 1%, w i t h o u t s i g n i f i c a n t damage. T h i s i s needed as t h e C h l o r o x s t e p i s o m i t t e d f o r Largomycin I I p r o c e s s e s due t o t h e extreme s e n s i t i v i t y of t h a t agent t o c h l o r i n e . F l u x r a t e s s h o u l d r e t u r n t o no l e s s than 80% o f pre-use v a l u e s . Table V I . Low Hazard Membrane C l e a n i n g

3.

Polysulfone PBS, 0.5-2.0 l i t e r / s q . f t . NaOCl 1%, 1.0 l i t e r / s q . f t . , Stand o v e r n i g h t . NaOH, 1M, 30 m i n .

4. 5. 6.

R/0 w a t e r , 2-5 l i t e r / s q . f t . Redetermine f l u x r a t e . S t o r e wet 4©C

1. 2.

1. 2. 3. 4. 5. 6.

Durapore Same. NaOCl 0.1%, 0.5 l i t e r / s q . f t . 30 min. t o 1 h r . Not recommended. NaOH, 0.025M, 30 min. Same. Same. Same.


46


Steam S t e r i l i z a t i o n . C a r t r i d g e f i l t e r systems d i s c u s s e d f o r c e l l h a r v e s t a p p l i c a t i o n s p r e s e n t no d i f f i c u l t i e s i n steam s t e r i l i z a t i o n . They may be s t e r i l i z e d by a u t o c l a v i n g , w i t h v e n t s open (and f i l t e r e d ) f o r 1 h r . a t 121QC. The same u n i t s c o u p l e d t o f e r m e n t a t i o n e q u i p ment may be s t e r i l i z e d i n s i t u f o r the same time. The t a n g e n t i a l and s e r p e n t i n e f l o w membrane f i l t e r u n i t s t e s t e d are a v a i l a b l e i n non-autoclavable a c r y l i c o r s t e r i l i z a b l e s t a i n l e s s s t e e l . As mentioned e a r l i e r , the f i l t e r p a c k e t s f o r the s e r p e n t i n e f l o w system w i l l not w i t h s t a n d steam s t e r i l i z a t i o n . The Durapore and p o l y s u l f o n e f i l t e r s h e e t s f o r the l a r g e r u n i t s w i t h s t a n d 1 h r . at 121°C w e l l , but must be t h o r o u g h l y w e t t e d p r i o r t o s t e r i l i z a t i o n . O r i g i n a l i n s t r u c t i o n s f o r s e p a r a t e l y a u t o c l a v i n g the s t e e l u n i t pre-assembled and w e t t e d , suggest " f i n g e r - t i g h t e n i n g o n l y o f t h e r e t a i n i n g n u t s p r i o r t o s t e r i l i z a t i o n . When t h i s was done i n o u r l a b o r a t o r i e s , c o n c e n t r a t i o n o f e u k a r y o t i c c e l l c u l t u r e s was much s l o w e r and c l o g g i n g a much g r e a t e r problem than had been a n t i c i p a t e d . E x a m i n a t i o n o f the f i l t e r s themselves showed t h a t s i g n i f i c a n t s h r i n k a g e had o c c u r r e d , r e s u l t i n g i n r e d u c t i o n o f r e t e n t a t e and f i l t r a t e manifolds to a f r a c t i o n of t h e i r o r i g i n a l s i z e . Polye t h y l e n e end gaskets were used i n t h i s assembly, and w h i l e these a r e not recommended f o r a u t o c l a v i n g , they are a p p a r e n t l y s u f f i c i e n t l y r e s t r a i n e d by the s t a i n l e s s s t e e l b l o c k s t h a t they do n o t change dimensions s i g n i f i c a n t l y , except f o r c u r l i n g beyond t h e margins o f the f i l t e r s t a c k , n o r do the f i l t e r s themselves change d i m e n s i o n s i g n i f i c a n t l y . The s c r e e n s , however, do s h r i n k s i g n i f i c a n t l y c a r r y i n g the f i l t e r s h e e t s , t o w h i c h they adhere, w i t h them. T h i s r e s u l t s i n some w r i n k l i n g o f the f i l t e r s u r f a c e , b u t t h i s does not seem t o a d v e r s e l y a f f e c t f i l t r a t i o n . The adverse e f f e c t comes from the s h r i n k a g e o f the s t a c k away from the openings i n the end g a s k e t s . To c o u n t e r a c t t h i s , u n i t s t o be a u t o c l a v e d s e p a r a t e l y s h o u l d be torqued t o the t e n s i o n a p p r o p r i a t e t o the membranes employed. T h i s procedure i s comparable t o the assembly f o r i n s i t u steam s t e r i l i z a t i o n ( F i g u r e s 18 & 19) where the pre-wetted u n i t must be f u l l y t i g h t e n e d t o r e t a i n steam. Steam l i n e s s h o u l d have s u f f i c i e n t upstream f i l t e r s t o p r o t e c t a g a i n s t l o a d i n g membrane s u r f a c e s w i t h pyrogenic m a t e r i a l s . For i n s i t u s t e r i l i z a t i o n , t e m p e r a t u r e - s e n s i t i v e p e n c i l s a r e a g a i n used t o i n s u r e s t e r i l i z a t i o n temperature i s a c h i e v e d f o r adequate t i m e . The a u t o c l a v e d u n i t i s b e s t c o o l e d t o ambient temperatures i n a p r o t e c t i v e environment such as a l a m i n a r - f l o w c a b i n e t , and brought back t o proper torque when c o o l e d . I n s i t u s t e r i l i z e d systems, i f not c o n t a i n e d , s h o u l d be r e - t o r q u e d s e v e r a l times d u r i n g c o o l i n g t o i n s u r e i n t e g r i t y .


n

Conclusions C a r t r i d g e and membrane f i l t e r systems t e s t e d i n p r e l i m i n a r y s t u d i e s i n our l a b o r a t o r i e s have proved t o be a p p l i c a b l e t o e u k a r y o t i c c e l l c u l t u r e p r o c e s s e s , i . e . c e l l removal, semi-continuous c u l t u r e growth, c e l l c u l t u r e c o n c e n t r a t i o n and r e c y c l i n g . P r e s e n t membrane systems are l i m i t e d i n p r o c e s s i n g volume; c a r t r i d g e s a r e s c a l a b l e , but have a n a r r o w e r range o f a p p l i c a t i o n . M a n i p u l a t i o n o f v i r u s , even more than r o u t i n e c e l l c u l t u r e s , i s l i k e l y t o r e q u i r e s t r i n g e n t containment t e c h n i q u e s w h i c h w i l l g r e a t l y c o m p l i c a t e the apparatus and f a c i l i t i e s n e c e s s a r y f o r p r o c e s s i n g . S u b s t a n t i a l f o r e t h o u g h t and e x t e n s i v e t e s t i n g a r e r e q u i r e d i n the d e s i g n and o p e r a t i o n of these p r o c e s s i n g systems.


R1CKETTS E T A L .



2.

American Chemical Society Library 1155 16th St. N. w. Washington. D. C. 20036 In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

47


48


Procedure: 1.

Install membranes and tighten UF unit to recommended levels. Open all valves on membrane UF unit.

2.

Turn on steam supplies 1 and 2 and open drain at supply 3. Do not open supply 3.

3.

Allow 60 min sterilization at 121 °C as measured by heat-sensitive pencil.

4.

Turn off steam and close drain. Open valve to 300-liter permeate vessel to charge lines with sterile air to maintain positive pressure while cooling.

5.

During cool-down, re-torque membrane UF unit to recommended levels.

6.

System can be operated when membrane UF unit and pump head are cooled to room temperature. F i g u r e 19. _In s i t u steam s t e r i l i z a t i o n o f the membrane u l t r a f i l t r a t i o n system.


2.

RICKETTS E T A L .


49

Most o f t h e t e c h n i c s mentioned h e r e a r e a p p l i c a b l e t o s t e r i l i z a t i o n , a s e p t i c p r o c e s s i n g and p o s t - u s e d e c o n t a m i n a t i o n , w i t h s i m i l a r r e s t r i c t i o n s t o those mentioned f o r v i r u s and c e l l c u l t u r e handling. P r o t e i n p r o c e s s i n g systems f o r c o n c e n t r a t i o n and/or p u r i f i c a t i o n o f f e r a v e r y b r o a d range o f a p p l i c a t i o n w h i c h may be t a i l o r e d t o a g i v e n p r o t e i n p r o d u c t p u r i f i c a t i o n scheme. The p a r t i c u l a r a p p a r a t u s and o p e r a t i n g parameters employed must be s e l e c t e d and r e f i n e d t o t h e unique r e q u i r e m e n t s o f any g i v e n p r o t e i n as w e l l as those o f t h e b r o t h i n which i t o c c u r s .

Literature Cited


1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

B e l l i n i , W.J., Trudgett, Α., and McFarlan, D.E. J . Gen. V i r o l . 1979, 43, 633-9. Berman, D., Rohr, Μ. Ε., and Safferman, R. S. Appl. Environ. Microbiol. 1980, 40, 426-8. Gangemi, J . D., Connell, Ε. V . , Mahlandt, B. G., and Eddy, G. A. Appl. Environ. Microbiol. 1977, 34, 330-2. Klein, F . , Ricketts, R. T., Rohrer, T. R., Jones, W. I., Clark, P. M., Flickinger, M. C. Appl. Environ. Microbiol. 1984, 47, 1023-6. Lee, J. C., Hapel, A. J., and Ihle, J . N. J . Immunol. 1982, 128, 2393-8. Mathes, L. E . , Yohn, D. S., and ulsen, R. G. J . Clin. Microbiol. 1977, 5, 372-4. Olsen, A. C. Proc. Biochem. 1977, 7, 333-43. Rosenberry, T. L., Chen, J . F . , Lee, Μ. Μ., Moulton, Τ. Α., and Onigman, P. J . Biochem. Biophys. Methods. 1981, 1, 39-48. Sekla, L . , Stackin, W., Kay, C., and VanBucken Hout, L. Can. J . Microbiol. 1980, 26, 518-23. Shant, J . L . , and Webster, D. W. Proc. Biochem. 1982, 17, 27-32. Shibley, G.P., Manousos, Μ., Munch, K., Zelljadt, I., Fisher, L . , Mayyasi, S., Harwood, Κ., Steven, R., and Jensen, Κ. E. Appl. Environ. Microbiol. 1980, 40, 1044-8. Van Reis, R., Stromberg, R. R., Friedman, L. I., Kern, J., and Franke, J . J . Interferon Res. 1982, 2, 533-41. Valeri, Α., Gazzei, G., Botti, R., P e l l e z r i n i , V . , Corradeschi, Α., and Goldateschi, D. Microbiology 1981, 4, 403-12. Zoon, K. C., Smith, M. E . , Bridgen, P. Α., Zurnedden, D., and Anfinsen, C. B. Proc. Natl. Acad. S c i . U.S.A. 1979, 26, 5601-5.

RECEIVED August 31, 1984


Application, Sterilization, and Decontamination of Ultrafiltration

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