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High-Resolution Flow-Zonal Centrifuge System

Flow-zonal centrifuge rotors, which are available commercially, offer various combinations of path lengths and gravitational forces at the gradient interface. There is no rotor, however, that combines development of the high gravitational force with the long sedimentation path length required to effect good separation of virus particles (such as those of the herpes virus and myxovirus families) from unwanted debris. To fulfill this need, we have developed a modified core that accommodates a Beckman CF-32 flow rotor and provides a greatly increased path length in an adequate force field. Data relating to the attributes of the modified rotor and its performance are presented here.

Fig. 1.

Top view of modified rotor core (all dimensions in in.).

Biotechnology and Bioengineering, Vol. XXI, Pp. 317-321 (1979) OOO6-3592/79/0021-03 17$01.00 @ 1979 John Wiley & Sons, Inc.

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BIOTECHNOLOGY AND BIOENGINEERING, VOL. XXI (1979) DESCRIPTION

Figures 1-3 are mechanical drawings of the modified rotor core that is fabricated from Noryl plastic. This change, together with the addition of an “0”ring (Universal “0” ring, No. 122) that is affixed to the 1.328 in. diam groove at the top of the core, comprises the total alteration needed to effect the greatly altered performance characteristics of the rotor. All loading and unloading procedures are the same as outlined in the Beckman CF-32 Instruction Manual. Table I gives the specifications of the standard and modified CF-32 rotors and compares their attributes with those of two other commercially available flow rotors. viz.. B-16 (Spinco, Palo Alto, Calif.) and RK-5 (Electro-Nucleonics. Caldwell. N.J.). The gravitational force at the interface in the modified CF-32 rotor was only 61% that of the standard CF-32 rotor, but the sedimentation path was more than tripled in length. The lesser gravitational force was readily compensated for by reduced flow rate. The B-16 rotor, commonly employed for viral separation, has a very short sedimentation path, and the RK-5 rotor with a long sedimentation path developed only 29,000 ,q at the interface. The modified CF-32 rotor provided a useful compromise in giving a long sedimentation path, an acceptable force field, and a reasonable rotor volume. FINDINGS IN SEPARATION EXPERIMENTS Influenza virus from the harvested allantoic fluid of embryonated hens’ eggs infected with BiMassachusetts strain influenza virus was purified by centrifugation

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Fig. 2.

Bottom view of modified rotor core (all dimensions in in.).

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in the modified CF-32 rotor and the B-16 rotor. The B-16 rotor has been the basic rotor for small-batch flow-zonal separation for several years and was the precursor of the KII Electro-Nucleonics large-scale separator. To test the modified CF-32 rotor, a three-step gradient was prepared by loading 400 ml phosphate-buffered saline solution into the rotor followed by 700 ml 30% sucrose and 300 ml 60% sucrose. The rotor was accelerated to 28,000 rpm. and 9 liter clarified allantoic fluid infected with BiMassili71 influenza virus were passed at the rate of 2 literihr. For the test of the 9-16 rotor, a two-step gradient. consisting of 200 ml 60% sucrose and 500 ml phosphate-buffered saline, was prepared. The rotor was accelerated to 40.000 rpm, and 10 liter clarified allantoic fluid infected with BiMassi3166 influenza virus were passed at a rate of 5 literihr. Figure 4 presents superimposed plots of the ultraviolet ( U V ) absorbance measured in the sucrose gradient samples obtained from the two centrifuge rotors. An ISCO ultraviolet light monitor. set at 254 nm UV. was used to determine absorbance. and the percentage of sucrose was measured using an Anacon flow refractometer. It is seen that the influenza virus was separated in

LRAOIUS ALL C O S ~ . I C ~ ~ S

Fig. 3.

Cross section of modified rotor core (all dimensions in in.).

BIOTECHNOLOGY AND BIOENGINEERING, VOL. XXI (1979)

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200

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500

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600

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GRADIENT VOLUME IN rnl

Fig. 4. Comparison of purification of influenza B virus using the (-) CF-32 flow rotor and the (---) B-16 flow rotor.

modified

the modified CF-32 rotor in a discrete peak widely separated from that of the debris By contrast, the virus was incompletely separated from the debris in the B-16 rotor that had only a I cm long sedimentation path. More than 150 virus purification runs have been carried out in our laboratories during the past three years employing the modified CF-32 rotor. The system performed without difficulty. The modified rotor has also performed kffectively in separating herpes simplex viruses from cell culture fluids when passed at a flow rate of 2 to 3 litedhr.

SUMMARY A modified CF-32 Beckman flow centrifuge rotor has been developed that provides a long sedimentation path length with high gravitational force at the gradient sample TABLE I Comparison of Specifications of the Modified CF-32 Flow with those of the Standard CF-32 Rotor and Two Other Commercially Available Rotors Attributes

Rotor CF-32 modified CF-32 standard B-16 RK-5 * Sample: gradient.

Sedimentation path length (cm) 4.5 1.4 I .o 4.5

Gravitational force ( x g ) maximum 11 II 9 9

x 104 x 104 x 104 x 104

at interface” 55 x 90 x 73 x 29 x

103 103 103

103

Rotor capacity (ml) 1400 400 700 4200

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interface. The modified rotor exhibits excellent separative capability and extraction efficiency when applied to purification of human influenza B and herpes simplex viruses. WILLIAMJ. MCALEER WILLIAMHURNI EDWARD WASMUTH MAURICER. HILLEMAN Division of Virus and Cell Biology Research Merck Institute for Therapeutic Research West Point, Pennsylvania 19486 Accepted for Publication May 16, 1978