BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIX, PAGES 1419-1429 (1977)

Concentration of Antibiotics by Reverse Osmosis R. DATTA, L. FRIES, and G. T. WILDMAN, Merck Sharp & Dohme Research Laboratories Division, Merck & Co., Innc., Rahway, New Jersey 07065

Summary Reverse osmosis is shown to be a viable process for the concentration of highly labile antibiotic solutions. Mathematical models of antibiotic inactivation by chemical and biological contamination are proposed and correlated with experimental data. Procedures for obtaining high antibiotic recoveries are discussed.

INTRODUCTION Reverse osmosis membrane separation, extensively studied for water desalination,’ has more recently been developed as a concentration technique. Sourirajan2 derived the general equations of proccss design for batchwise reverse osmosis concentration and later extended the validity of the correlations de~eloped.~Matasuura and Sourirajan4 studied the reverse osmosis separation of certain organic solutes like glucose, maltose, and glycols in aqueous solution. Separation of alcohols and/or hydrocarbons by reverse osmosis has been found fea~ible.~Recovery of chocolate wastes from waste water streams is one of the recent reverse osmosis applications in the area of pollution abatement.6 Applications of membrane separation techniques in the processing of natural products in the pharmaceutical industry have not been extensive in spite of the inherent advantage offered by these techniques for heat sensitive products. Biological contamination resulting in membrane clogging7 and product degradation are major problems which often need to be overcome. Loeb and Manjikian8 maintained reverse osmosis membrane performance for six months by periodic treatment with the quaternary ammonium bacteriocide salt, “Arquad S-50.” Aqueous solutions of antibiotics are routinely concentrated via vacuum evaporation during isolation and purification. Antibiotic losses occur in varying degrees during low temperature evaporation 1419

01977 by John Wiley & Sons, Inc,

DATTA, FRIES, AND WILDMAN

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depending on the thermal stability of the antibiotic and the evaporator design. An alternative concentration technique utilizing reverse osmosis membrane separation has been evaluated on the pilot plant scale with an extremely labile antibiotic and determined to be feasible. Problems relating to chemical and microbial antibiotic inactivation were resolved satisfactorily.

EQUIPMENT Initial experiments to assess the feasibility of reverse osmosis for the concentration of the antibiotic-containing solution were conducted using the Havens International Osmotik Test System. The Havens reverse osmosis module consists of a bundle of 18, 4 in. porous fiber glass tubes coated on the inside with a cellulose acetate membrane. The membrane area for the test system is 1 f t 2 . Havens membranes Nos. 310 and 520, which reject 75 and 95% sodium chloride, respectively, were evaluated. The recirculating concentrate was cooled by passage through a stainless steel coil immersed in an ice bath. The pilot plant reverse osmosis equipment, which consists of a Havens “Osmotik Processor” and auxiliary equipment, is shown schematically in Figure 1. Pilot plant runs utilized ten No. 310 production modules in series, a total membrane area of 167 ft2. MOWLE INLET PRESSURE GAUGE

M O M X E OU L E T PRESSURE GAUGE

8

8 BACK PRESSURE REGULATOR

I

I

HIGHPRESSURE W L T l STAGE CENTRIFUGAL PUMPS ( 2 I N SERIES)

9

4

PERMEATE (10SEWER)

LOW FLOW SWITCH

CONCENTRATE RETURN

Fig. 1. Schematic of pilot plant reverse osmosis system.

CONCENTRATION OF ANTIBIOTICS BY OSMOSIS

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Two Could high pressure centrifugal pumps were used to recirculate the concentrate through the modules. Recirculating antibiotic solution was cooled by 0°C chilled water in a Heliflow heat enchanger. In all cases, the terms “concentrate” and “recirculating antibiotic solution” refer to the membrane-retained solutions.

EXPERIMENTAL PROCEDURE Laboratory Laboratory studies with the Osmotik test unit were carried out strictly for feasibility since the desired volume reductions and time cycles could not be achieved. Antibiotic concentrations were determined by disc assay on Streptococcus aureus (culture MB-108) plates and/or high pressure liquid chromatography. Samples were also assayed for total solids which, with the antibiotic assay, were used to determine specific activity (gram antibiotic per gram solids) and permeation and retention of nonactive solids. Two or 3 liter of precooled antibiotic feed were recirculated through the membrane module and heat exchanger. Inlet pressure of 400500 psig, inlet temperature 10-20°C, and pH of 6-7 were maintained. Permeate and concentrate stream samples were collected at 10 min intervals and diluted in O.lM, pH 7 phosphate buffer for assays to establish antibiotic concentration . profiles. Concentrate and corn bined permeate samples were also collected and assayed to determine the percent antibiotic retention and mass balance. A typical laboratory run would last 1 hr and achieve a twofold concent ration.

Pilot Plant Experimental procedures in the pilot plant were similar to that in the laboratory. Typical feed volumes were 120-190 gal of aqueous antibiotic solution which contained 1-1.5% (v/v) pyridine. The batches were concentrated using the No. 310 membrane modules at 500 psig inlet pressure, pH 6.5-7.0, and temperature range of 10-20°C. Typical concentration time was 3 4 hr for a 6-10-fold volume reduction. Water permeation rate was about 40 gal/hr.

RESULTS Laboratory The antibiotic was fairly stable under the operating conditions of pH, temperature, and time. The results are summarized in Table I.

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DATTA, FRIES, AND WILDMAN TABLE I Laboratory Evaluation of Membrane Nos. 310 and 520

Pressure Feed pH Flux Temperature Antibiotic lost in permeate Antibiotic retained

Havens No. 310

Havens No. 520

400 psig 6.7 0.32 gal/hr/ft* 25°C 8.9% 91%

500 psig 6.5 0.26 gal/hr/ft* 20°C 0.16% 99.3%

Antibiotic retention was higher (>99%) for the No. 520 membrane than for the No. 310 membrane (>91yo). The flux, however, was lower for the tighter No. 520 membrane. The total solids data indicated that there was no purification of the antibiotic by reverse osmosis-the antibiotic, as well as the inactive components of the solution, were equally retained by the membrane.

Pilot Plant Antibiotic recoveries in the first two reverse osmosis concentration runs in the pilot plant were significantly lower than laboratory experience predicted. The percent antibiotic remaining in the concentrate versus concentration time for runs 1 and 2 is depicted in Figure 2. A rapid loss of antibiotic began to occur 1 to 2 hr after the start of concentration. Since the antibiotic levels in the permeate were at the expected level, the observed losses in the concentrate had to be due to antibiotic degradation. This degradation or inactivation had not been observed in the 1 hr laboratory runs.

Antibiotic Inactivation by Pyridine Pyridine, present in feed at concentrations of 1-1.5y0 (v/v), was suspected to be an antibiotic inactivating agent. Gas chromatographic assays for pyridine in the concentrate and permeate showed that pyridine was freely permeable through the membrane and that there was no buildup of pyridine in the concentrate. The suspicion of antibiotic inactivation by pyridine was confirmed in separate experiments in which the antibiotic stability was measured at several pyridine concentration levels. Pseudofirst-order kinetics were then used to calculate the pyridine inactiva-

CONCENTRATION OF ANTIBIOTICS BY OSMOSIS

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t TIM€ t

IHAS.1

Fig. 2. Reverse osmosis:pilot plant runs. ( 0 )Run I ; (+) run 2; ( A ) run 3.

tion rate constant, since the pyridine concentration was much greater than that of the antibiotic. The experimentally determined rate constants are tabulated in Table 11. It is apparent from these values that the drastic antibiotic degradation observed in pilot plant runs 1 and 2 could not be due solely to inactivation by pyridine. Antibiotic Inactivation by Microorganisms

Samples of concentrate were streaked on nutrient agar plates and found to be heavily contaminated by microorganisms despite membrane sanitization with a bacteriostat (alkyl benzyl ammonium TABLE I1 Antibiotic Inactivation Rate Constants Thermal inactivation Temp. ("C)

(PH = 7) krn (hr-9

3 28 30

0.0017 0.0252 0.0249

Pyridine

+ thermal

1.8% ' pyridine pH = 7

k p G Obr-9 0.0213 0.073 0.074

+

Pyridine thermal 1 90pyridine pH = 7 k p G 0 W1) 0.0126 0.0518 0.0522

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DATTA, FRIES, AND WILDMAN

chloride) specified by the Havens Corporation. It was apparent that a stronger bacteriocide was necessary to eliminate microbial decomposition of the antibiotic. The membranes were then treated with a 2% (v/v) formaldehyde solution prior to run 3. Antibiotic recovery rose to 89% (Fig. 2), indicating that microbial contamination was the major cause of antibiotic loss during reverse osmosis concentration and that 2% (v/v) formaldehyde is an effective membrane sanitizing reagent.

DISCUSSION Antibiotic Inactivation Models Mathematical modeling of antibiotic inactivation by pyridine and microorganisms led to a better understanding and subsequent solution of the problem. In modeling the antibiotic degradation the concentrate is chosen as the system whose volume changes continuously and wherein the antibiotic inactivation occurs as a chemical reaction. Antibiotic permeation losses also occur continuously. The concentrate volume is given by the following equation:

v

=

vo - Pt

(1)

The concentration of the antibiotic in the permeate, Cap,has been measured and found to be essentially constant. Hence, Capis not a strong function of C,. Therefore, without any degradation the antibiotic recovery would be :

cav = ca,v,- Ca,Pt

(2)

Case 1 : Antibiotic recovery with permeation and thermal degradation losses. The antibiotic balance is given by the equation:

This equation can be solved for a changing volume [eq. (l)]and initial conditions :

The fraction of antibiotic remaining in the concentrate, F , is plotted for the experimental conditions where kth g 0.006 hr-I (at 15°C and pH 7), 6 = P / V O 0.25 hr-I, and Cap/Cao‘v 0.05 (Fig. 3, case 1).

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Fig. 3. Reverse osmosis: theoretical curves. Case 1: Thermal degradation and permeation losses only. Case 2 : Pyridine and thermal inactivation and permeation losses, 1% pyridine. Case 3: Microbial inactivation losses: (a) j3 = 0.03 hr-l; (b) (3 = 0.04 hr-l.

Case 2 : Antibiotic recovery with permeation, pyridine, and thermal degradation losses. The pyridine balance, assuming negligible amounts of pyridine are consumed in the degradation reaction and that C, >> Ca, is given by :

c, = cpo(i - etp-1

(4)

where CY = C,,/C, determines the retention of pyridine in the membrane. From gas chromatographic data C,, II C, and hence CY = 1 and the pyridine concentration does not change, i.e., C, = C,,. The antibiotic balance is given by the equation :

Substituting C,

=

C,,, eq. (I), and the initial conditions:

For a 1% (v/v) pyridine solution k,C,,,

which accounts for pyridine

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DATTA, FRIES, AND WILDMAN

and thermal inactivation, has been experiment,ally determined to be 0.021 hr-1 (Table 11). The inactivation curve is plotted for 0 = 0.25 hr-l, Cap/Ca0= 0.05, and kpCpocv 0.021 hr-I (Fig. 3, case 2 ) .

Case 3 : Antibiotic recovery with microbial degradation losses. For the case of antibiotic degradation by microbial contamination, the microbial population is assumed to increase exponentially :

vcb =

VoCboekbt

(6)

Hence cb

=

VoCboekb'/(Vo- Pt)

@a>

For a medium which is low in sugar and is at low temperature, kb is small (assumed to be between 0.05 and 0.1 hr-l). The maximum value of t is 4 hr and hence k b < t-' or kd < 1 . Hence, eq. (6a) can be linearized to give: cb

=

vOcbo(l -k

kbt)/(T/'O

-

Pt)

(7)

The antibiotic balance equation for a second order inactivation reaction between the antibiotic and the microorganisms is given by :

Substituting cb from eq. (7) :

For the case where Cap

Concentration of antibiotics by reverse osmosis.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIX, PAGES 1419-1429 (1977) Concentration of Antibiotics by Reverse Osmosis R. DATTA, L. FRIES, and G. T. WILD...
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