PROCESS OPTIMIZATION

3. PROCESS OPTIMIZATION

3.1. Premix Formation Having established a viable formulation, developmental efforts

for this PFC emulsion next focused on process optimization. As shown in Fig. 2 , formation of a coarse oil-in-water dispersion or premix containing all excipients precedes high pressure homo- genization. At the premix stage, relative size homogeneity of oil droplets is critical to producing a high quality finished product with a minimum number of large (i.e., >5 mm) droplets (10). We found several good methods to monitor premix formation and optimize high shear process time. The simplest is optical phase-contrast microscopy on in-process samples. Examples of photomicrographs

Fig. 5 . Under tested conditions, 10 min of processing time (at 20,500 rpm) appears sufficient to produce a very uniform disper- sion. Since these parameters are highly correlated with the specific formulation, equipment type, and even batch size, pro- cess optimization must be an ongoing, project-specific activity.

Since microscopy is subjective and not very quantitative, we evaluated two other methods. The first is large particle counting using the Coulter ZM with a 100 mm orifice. Samples are diluted in 0.9% saline prior to counting. These data, sum- marized in Fig. 6 , suggest a continuous formation of smaller particles (2–10 mm) with a parallel disappearance of larger droplets during premix processing. Since this analytical method is labor-intensive and failed to identify a process endpoint, we evaluated a third procedure using the same

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Figure 5 Evaluation of in-process premix samples by phase- contrast microscopy (1 mm ¼ 2.3 mm). The preferred endpoint during premix formation is a relatively homogeneous drop size distribution.

capillary flow viscometer described above. As shown in Fig. 7 , flow viscosity drops dramatically during the first 10 min of premix processing, with only small changes thereafter. From studies such as these, we were able to optimize the premix process time at each production scale. For other similar pro- jects, our methods of choice for premix evaluation remain phase-contrast microscopy and flow viscosity.

3.2. High Pressure Homogenization Earlier pilot studies had shown that better quality emulsions

were prepared using the highest available homogenizer

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Figure 6 Evaluation of in-process premix samples by electrozone sensing. Shifts in particle size distribution during processing may

be monitored by large particle counting using the Coulter ZM with a 100 mm orifice. This is a quantitative analysis of particle counts per milliliter, unlike microscopy or laser light scattering measure- ments.

pressure, 10,000 psig, with 15% of this value (1500 psig) cho- sen as the second stage back-pressure. Earlier studies also indicated that homogenization at lower temperatures, e.g., 5–10

C, resulted in poor quality emulsions while higher tem- peratures, e.g., above 60

C, resulted in extensive losses of volatile PFC. Intermediate temperatures, e.g., 35–40

C, pro- duced the best results and this range was selected for future batches.

Our next challenge was to optimize the number of homo- genizer passes through the spring-loaded valve system. Mul- tipassing is known to narrow the drop size distribution, but to have relatively little effect on particle mean diameter. This phe- nomenon is shown in Fig. 8 . However, an excessive number of passes may produce large PFC droplets due to over-processing and may promote more degradation of the unsaturated phos- pholipid emulsifier due to hydrolysis and oxidation (11). In addition, over-processing may result in significant loss of

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Figure 7 Evaluation of in-process premix samples by flow viscosity. A glass capillary viscometer was used to demonstrate a dramatic reduction in flow viscosity in premix samples as continued processing reduces drop size distribution.

volatile PFC raw material and will extend production time, adding to costs. For all these reasons, we sought a reliable method to optimize homogenizer passes.

During homogenization, in-process ‘‘pass samples’’ are removed and analyzed for large particle counts by the Coulter ZM as described above. Three size classes are monitored, and typical results are summarized in Fig. 9 . We observe a rapid initial drop in count rate, followed by a slower but continuous reduction during further processing. However, based on these data, we were not able to establish an optimum process endpoint.

A second parameter used to evaluate the homogenization process is phospholipid binding (12). For this test, aliquots of non-sterile pass samples or sterile final product are centrifuged

=E centrifuge with JA-20 fixed angle rotor). Aliquots of resulting supernatant or of whole emulsion are vortex-mixed with ethanol (1:2 v =v).

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Figure 8 Evaluation of homogenizer multipassing by laser light scattering. Homogenizer multipassing narrows the emulsion drop size distribution but has relatively little effect on the mean dia- meter. This oil-in-water emulsion was prepared using a laboratory- size Gaulin homogenizer model 15MR. Particle size distributions were determined by laser light scattering measurements.

Samples are then evaporated in a vacuum oven (3 hr at 60 C) and phospholipid concentration determined gravimetrically. Phospholipid bound to the PFC fraction is estimated as the dif- ference between total phospholipid in the whole emulsion minus the unbound fraction in the supernatant. Figure 10 shows that phospholipid binding to PFC droplets is essentially complete by pass 12, and a reciprocal drop in supernatant (free) phospholipid is observed during the process time-course. We also note some loss in binding during terminal heat steriliza- tion (typically down to 40–50%). This loss parallels an observed shift in population size distribution towards larger sizes with less total surface area. For example, mean diameter for a

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Figure 9 Evaluation of homogenizer multipassing by electrozone sensing. Large particle counts decline with processing time as sub- micron particles are being created but these submicron sizes are not detected by the Coulter ZM.

typical batch may shift from about 190 to 285 nm during autoclaving, with relatively small further changes occurring during prolonged storage. We believe that small amounts of phospholipid are sloughed from the surface of emulsified PFC droplets during autoclaving. This phospholipid then folds in upon itself in the aqueous phase. As a result, empty spherical vesicles (liposomes) are formed with phospholipids arranged in one or more bilayers (13).

A third in-process measurement is based on our obser- vation that PFC emulsion turbidity, as measured by light transmittance at 410 nm, is closely correlated with unbound (supernatant) phospholipid concentration. This wavelength was chosen to give strong light scattering with minimum absorbance from the unsaturated phospholipids. Figure 11 shows that turbidity declines dramatically at 10,000 psig

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Figure 10 Evaluation of homogenizer multipassing by monitoring phospholipid binding. Homogenizer multipassing reduces the number of larger particles thereby increasing the total interfacial surface area per unit volume of emulsion. Phospholipids are recruited continuously from the aqueous phase and bind to newly created interfaces between PFC and water. When the ratio between bound and free (aqueous) phospholipid becomes constant, proces- sing at the chosen conditions of temperature and pressure is complete.

during the first 4–6 passes at 35–40

C, but at 5–10

C, over

12 passes are required. We observed no evidence of over- processing under these conditions. Initially, measurements were made on pass samples using a UV =VIS spectro- photometer with sipper attachment (Beckman DU 640). Later, in-line spectrophotometric monitoring was accomplished

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Figure 11 Evaluation of homogenizer multipassing by monitoring turbidity. Homogenizer multipassing reduces the concentration of unbound phospholipid that is most responsible for visible light scatter- ing. Therefore, in-line measurement of light transmittance is a simple way to monitor the effect of continued processing on emulsion quality. This method is especially useful here since the refractive index of the dispersed phase (PFC) is close to that of the continuous phase (water).

by means of a probe colorimeter (Brinkmann Instruments; Westbury, NY; Model PC800) fitted with a 420 nm filter. A

1 cm path length fiber optic probe is inserted into the product stream with an in-line stainless steel T-fitting. A strip chart recorder gives us a permanent record of product turbidity during processing. Homogenization is continued until a

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pre-determined percentage of transmittance is attained (usually after 8–10 passes) (14,15).