PROCESS SCALE-UP

4. PROCESS SCALE-UP

4.1. Nonemulsified Perfluorocarbon Previous process development work was performed at the 4–8 L

scale, filling 100 mL bottles. At the 50-L scale and in 500 mL bottles, process conditions such as temperature, pressure, and passes were revisited and re-optimized using the techni- ques described above. However, a new problem emerged.

sediment taken from the bottom of a sterilized 500 mL bottle after standing for 1 week. In these containers, we observed large numbers of rapidly sedimenting, poorly emulsified PFC droplets (‘‘fish eggs’’) with diameters in the 50–200 nm range. The presence of free or poorly emulsified PFC in this product was quantified by extraction with a lower molecular weight perfluorocarbon liquid, fluorodimethycyclohexane (F-DMCH),

Figure 12 Poorly emulsified droplets of perfluorocarbon. These dense droplets sediment rapidly to form a layer at the bottom of

this layer reveals PFC droplets in the 50–200 nm range with a ‘‘fish egg’’ appearance.

388 Lyons

followed by gas chromatographic analysis. Earlier tests demonstrated that F-DMCH could serve as an extraction sol- vent since it removed only non-emulsified (free) perfluorodi- methyladamantane.

Briefly, an exact volume (e.g., 3–5 mL) of F-DMCH is injected by syringe directly into each bottle which is then sha- ken vigorously by hand for about 30 sec. Bottles are inverted and allowed to stand at room temperature for 1 hr, after which time a portion of F-DMCH is removed by syringe nee- dle through the stopper. An aliquot of this extract is then injected into a gas chromatograph (Hewlett Packard 3388) equipped with an automatic sampler (7672A), a Supelcowax

10 column (30 m length, 0.32 mm ID, 0.25 mm film thickness), and a flame ionization detector. Operating conditions were: oven temperature 60

C, detector temperature 300

C, injector temperature 275

C, column head pressure 13 psi, with helium carrier gas at 0.70 mL =min. Retention time for F-DMCH is about 1.5 min compared to 2.1 min for extracted PFC, allowing simple quantitation of the latter.

We suspected that refluxing was occurring in the bottle during terminal heat sterilization at temperatures up to 121

C. During cooling, PFC vapors would condense and settle to the bottom as large, poorly emulsified droplets. When the mass of recovered (free) PFC was plotted vs. the square of headspace volume, we observed a linear relationship, shown in Fig. 13 . We found that a minimal headspace volume (here, 7% of 500 mL) is essential to minimize the formation of non-emulsified PFC in sterile product.

4.2. Product Uniformity The density of liquid perfluorodimethyladamantane is 2.025

C. For this reason, emulsified PFC droplets tend to sediment fairly rapidly, and the potential exists for non-uniformity in PFC content from bottle to bottle. This is especially true during scale-up, since the bottle filling operation time increases with the batch size, e.g., up to

g =mL at 20

2 hr for a 50 L batch. Any emulsion sedimentation in the holding tank would result in a gradual increase in product

Case Study: Injectable Perfluorocarbon Emulsion 389

Figure 13 Effect of headspace volume on free PFC formation in autoclaved 100 mL bottles. During autoclaving at 121

C, PFC vapors form in the nitrogen headspace and then condense upon cooling to form small, non-emulsified oil droplets. This effect can almost be eliminated by minimizing headspace volume during filling.

PFC concentration across the fill series. In order to avoid this, we established a continuous recirculation of product in and out of the holding tank through a 1 =4-in stainless steel tubing using a centrifugal pump (Eastern). Unfortu- nately, rapid recirculation caused a progressive increase in Coulter large particle counts. Typical data are shown in Fig. 14 . We now believe that shear forces generated by high flow velocity through the narrow tubing was responsible for continuous de-emulsification and particle coalescence (16). Eventually, this problem was solved by means of a low shear,

390 Lyons

Figure 14 Evaluation of product recirculation during filling by electrozone sensing. Large particle counts increase steadily when the PFC emulsion is recirculated too rapidly during the filling operation. Data were generated using the Coulter ZM instrument with a 100 mm aperture and 0.9% saline as diluent.

positive displacement pump (Waukesha Pumps; Waukesha, WI) coupled to larger ID tubing to reduce flow velocity. Bot- tle-to-bottle uniformity was verified by analyzing every 25th bottle in an entire 50 L batch for both PFC concentration and drop size distribution.

5. CONCLUSIONS The development of an injectable PFC emulsion is compli-

cated by formidable challenges of both a biological and physi- cochemical nature. We found that the early establishment of several reproducible and relevant biological screens is

Case Study: Injectable Perfluorocarbon Emulsion 391

essential during formulation optimization and process devel- opment.

One inherent problem relates to the immiscibility of PFC liquid and phospholipid emulsifier. Raising the ionic strength in the aqueous phase may help by forcing more phospholipid to the oil–water interface. In addition, process conditions must be carefully optimized to avoid both over-processing and high shear conditions after homogenization, e.g., during final filtration. These adverse treatments can strip phospho- lipid from the interface and induce de-emulsification.

PFCs are selected for high vapor pressure to facilitate excretion via the lungs. However, this high vapor pressure means that careful temperature control during processing is critical. It also means conditions leading to vaporization and condensation must be avoided, e.g., autoclaving bottles with large headspace volumes.

During scale-up production of perfluorocarbon emul- sions, extra care must be taken to avoid sedimentation and stratification of heavy emulsified droplets. For this reason, bottle-to-bottle uniformity must be verified in the finished product, especially with regard to PFC content and drop size distribution.

An injectable product very similar to the one described in this review was manufactured in multiple 50 L batches at our Clayton, North Carolina facility. This product exhibited satisfactory storage stability and was well-tolerated in an animal model. Unfortunately, this project was halted for non-technical, business reasons by our collaborating partner. We can only hope that ‘‘lessons learned’’ will benefit and expedite future projects of this type, both at our facility and perhaps at yours.

REFERENCES 1. Biro GP, Blais P. Perfluorocarbon blood substitutes. CRC Crit

Rev Oncology =Hematology 1987; 6(4):310–371. 2. Tremper KK, ed. Perfluorochemical Oxygen Transport.

Vol. 23. Boston: Little, Brown and Company, 1985.

392 Lyons 3. Geyer RP. Review of perfluorochemical-type blood substitutes.

In: Proceedings of the Tenth International Congress for Nutri- tion, Kyoto, Japan, 1975:3–19.

4. Collins-Gold LC, Lyons RT, Bartholow LC. Parenteral emul- sions for drug delivery. Adv Drug Delivery Rev 1990; 5:189.

5. Brecher G, Bessis M. Present status of spiculed red cells and their relationship to the discocyte-echinocyte transformation:

a critical review. Blood 1972; 40:333. 6. Hulman G, et al. Agglutination of Intralipid by sera of acutely

ill patients. Lancet 1982; 2:1426. 7. Lindh A, et al. Agglutinate formation in serum samples mixed

with intravenous fat emulsions. Crit Care Med 1985, 13:151. 8. Hamai H, et al. Viscosimetric study of fluorocarbon emulsions

and of their mixtures with blood. J Fluorine Chem 1987; 35:259. 9. Lyons R. Inhibiting aggregation in fluorocarbon emulsions. US

Patent No. 5,073,383 (1991). 10. Pandolfe WD. Effect of premix condition, surfactant concentra-

tion, and oil level on the formation of oil-in-water emulsions by homogenization. J Dispersion Sci Tech 1995; 16:633.

11. Herman CJ, Groves MJ. The influence of free fatty acid formation on the pH of phospholipid-stabilized triglyceride emulsions. Pharmaceut Res 1993; 10:774.

12. Groves MJ, et al. The presence of liposomal material in phos- phatide stabilized emulsions. J Dispersion Sci Tech 1985; 6:237.

13. Lasic DD. The mechanism of vesicle formation. Biochem J 1988; 256:1.

14. Deackoff LP, Rees LH. Testing homogenization efficiency by light transmission. APV Gaulin Technical Bulletin No. 63, 1981.

15. Lashmar UT, Richardson JP, Erbod A. Correlation of physical parameters of an oil-in-water emulsion with manufacturing procedures and stability. Int’l J Pharmaceutic 1995:315–325.

16. Han J, Washington C, Melia CD. A concentric cylinder shear device for the study of stability in intravenous emulsions. Eur J Pharm Sciences 2004; 23:253–260.

Case Study: A Lipid Emulsion—Sterilization

THOMAS BERGER Pharmaceutical Research & Development,

Hospira, Inc., Lake Forest, Illinois, U.S.A.

1. OUTLINE The following information outlines a case study pertaining to

the engineering and microbiological activities required for an emulsion product in order to gain FDA approval prior to man- ufacture. Minimum details for each research, development, and production activity are discussed to demonstrate the sup- porting documentation that may be required in order to file a New Drug Application with the FDA.

An organized sequential flow of activities must occur as a new parenteral formulation is developed in an industrial rese- arch and development (R&D) environment, and subsequently

394 Berger

processed in a manufacturing facility. Sterilization of pharma- ceutical emulsions must be established and verified through a series of activities that confirm the product has been rendered free of any living microorganisms. In the case of moist heat ster- ilization, which is discussed here, the R&D phase activities must include sterilization developmental engineering consist- ing of sterilization cycle development; container thermal map- ping; microbial closure studies; parenteral formulation microbial growth; D-value analysis; container maintenance of sterility (mos) studies; and final formulation stability studies.

Production phase activities for moist heat sterilization must include an initial sterilization vessel certification, which demonstrates that the vessel will deliver the defined steriliza- tion process in a consistent and reproducible manner. Emulsion and container closure microbial validation studies must be conducted at subprocess production sterilization conditions employing heat resistant microorganisms. Equipment valida- tion, filtration studies and assessment of the bioburden on com- ponent parts, and in the environment, must also be ascertained.

The developmental and production phase sterilization technology activities must be included in the documentation submitted as part of a New Drug Application. The procedures must follow the FDA guideline requirements for products that are either terminally sterilized or aseptically processed. These studies allow the establishment with a high level of steriliza-

tion assurance, the correct sterilization cycle F 0 (equivalent sterilization time related to the temperature of 121

C and a z-value of 10 C), temperature, and product time above 100 C to be used for the sterilization of a specific parenteral formula- tion in a particular container =closure system.

2. INTRODUCTION This section will address sterilization and associated activ-

ities that occur in the R&D and production areas.

2.1. R&D Area

1. Sterilization engineering.

2. Thermal mapping studies.

Case Study: A Lipid Emulsion—Sterilization 395

3. Emulsion: microbial moist heat resistance analysis.

4. Closure microbial inactivation studies.

5. Lipid emulsion predicted spore logarithmic reduc- tion (PSLR) values.

6. Accumulated F(Bio) (biologically derived steriliza- tion value) for lipid emulsions.

7. R&D emulsion oil phase studies.

8. Maintenance of sterility studies.

9. Bacterial endotoxin.

2.2. Production Environment

1. Engineering penetration and distribution (P&D) studies.

2. Sterilization cycles.

3. Sterilizer microbial emulsion subprocess validations.

4. Sterilizer microbial closure subprocess validation.

5. Production environment bioburden screening pro- gram.

Refer to Young’s detailed discussion of sterilization by moist heat processing (1).

2.3. Regulatory Submission Checklist for aseptically processed and terminally sterilized

products.