R&D AREA

3. R&D AREA

3.1. Sterilization Engineering Sterilization engineering personnel primarily focus their

efforts on determining whether a new parenteral formulation packaged in a particular container configuration can be ster- ilized in a current sterilization cycle or whether a new cycle must be developed. Sterilization feasibility studies usually occur preliminarily to ascertain the physical effects of the cycle on a container and its emulsion. Once the basic

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engineering parameters (temperature, time, F 0 ) are estab- lished, engineering thermal container mapping studies are performed (2). F 0 is the integrated lethality or equivalent minutes at 121.1

C for the hottest and coldest thermocouple containers. Product attributes that can be affected by a steam steri- lization cycle include:

product sterility; closure integrity; emulsion potency; pH, color; shelf-life stability (potency of emulsion); visible and subvisible particulates.

3.2. Thermal Mapping Studies An R&D vessel is smaller than a production vessel but can

simulate the sterilization cycles conducted in the larger pro- duction vessels. Container thermal mapping studies are performed in an R&D vessel.

To locate the coldest zone or area inside a container. To determine the cold zone in an R&D vessel and the relationship to the location monitored by the produc- tion thermocouple. The data generated are used during the setting of pro- duction sterilization control parameters.

When conducting thermal mapping studies, there are various factors to be considered, and these are dependent upon the

type of container (flexible or rigid); shaking =static solutions; viscosity; autoclave trays =design=surface contact; autoclave spray patterns =water flow.

Engineering map data obtained for lipid emulsions con- tained within a 1000 mL glass container are shown in Tables 1 and 2 .

Case Study: A Lipid Emulsion—Sterilization 397

Table 1 Heat Input (F 0 units)

Run CLHK00.049 Run CLHK01.050 Tc Number bt1 1

bt1 2 Avg (Std Dev) 1, 12

bt1 2

bt1 1

7.91 C7.28 8.13 C7.36 7.67 (0.415) 2, 13(PC)

C7.46 7.40 C7.71 7.47 C7.51 (0.137) 4, 15

H16.09 H16.10 (0.856) 11, 22

0.33 0.21 0.31 0.28 0.28 (0.053) Note: H denotes hottest TC location; C denotes coldest TC location; PC denotes

approximate location of the Production Profile TC; Data from TC#9 used with a post- calibration variance þ0.25

C at 100 C; All heat input values are calibration corrected.

The following summarizes typical heat map data obtained from a 1000 mL glass Abbovac intravenous container filled with 1035 mL of lipid emulsion:

1. Heat input in F 0 units.

2. Emulsion heat rates in minutes.

3. Figure 1 depicts the thermocouple locations

4. Figure 2 contains the average heat input (F 0 ) at various locations.

Thermocouple probes (Copper Constantan, type T, 0.005 in diameter) were used to monitor 11 emulsion locations within the 1000 mL container. The thermocouple probes were positioned at various distances (in inches) as depicted in Fig. 1. Each container was filled with 1035 mL of the lipid emulsion, evacuated to 20 in of mercury, and sealed with an aluminum overseal.

A flat perforated rack on a reciprocating shaker cart was used in the autoclave. The cycle’s target temperature was

398 Berger Table 2 Solution Heat Rates (minutes)

Run CLHK00.049 Run CLHK01.050 btl 1

btl 2 Avg (Std Dev) Coldest location

btl 2

btl 1

Thermocouple no 3 12 3 12 — Time to 100 C 19.0 19.0 19.0 19.0 19.00 (0.000) Time

C 21.0 21.0 21.0 21.0 21.00 (0.000) Time

C 4.0 3.0 4.0 3.0 3.50 (0.577) Time 120–100 C 4.0 5.0 4.0 5.0 4.50 (0.577)

Max temp ( C) 120.82

120.77 120.82 (0.071) Heat input (F 0 )

7.46 7.28 7.71 7.36 7.45 (0.187) Production profile TC location

Thermocouple no 2 13 2 13 — Time to 100 C 19.0 19.0 19.0 19.0 19.00 (0.000) Time

C 22.0 21.0 22.0 21.0 21.50 (0.577) Time

C 4.0 3.0 4.0 3.0 3.50 (0.577) Time 120–100 C 5.0 5.0 5.0 5.0 5.00 (0.000)

Max temp ( C) 120.91

120.92 120.89 (0.047) Heat input (F 0 )

7.79 7.49 8.02 7.64 7.74 (0.226) Note: H denotes hottest TC location; C denotes coldest TC location; PC denotes

approximate location of the Production Profile TC; Data from TC#9 used with a postcalibration variance þ0.25

C at 100 C; All heat input values are calibration corrected.

Figure 1 The numbers inside the 1000 mL glass emulsion bottle are the thermocouple locations for duplicate bottles from two sepa- rate runs. The numbers outside the bottle are distances in inches.

Case Study: A Lipid Emulsion—Sterilization 399

Figure 2 The numbers inside the 1000 mL glass emulsion bottle are the average heat input (F 0 ) at the various thermocouple locations.

C, recirculating water spray cycle with 70 rpm of axial agi- tation, 30 psig (pounds per square inch) of air over-pressure and a minimum requirement of 6.0F 0 units in the coldest location. When the sterilization cycle was controlled to give a heat input of approximately 7.5F 0 units in the coldest emulsion area, the average coldest emulsion area was measured by thermocouple number (TC#) 3,14. The average hottest emul- sion area was measured by TC# 10,21. The difference between the hottest and coldest emulsion areas ranged from

7.5 to 10.0F 0 units with an average of 8.6F 0 units. Therefore, when the coldest emulsion area registered 7.5F 0 units, the hottest emulsion area would average 16.1F 0 units. The emulsion area approximating the production profile thermocouple location was measured by TC#2,13 and aver- aged 7.7F 0 units when the coldest emulsion was approxi- mately 7.5F 0 units. (Refer to Figs. 1 and 2.)

3.3 Emulsion: Moist Heat Resistance Analysis

A BIER vessel is an acronym for a biological indicator (BI) evaluator resistometer vessel that meets specific performance requirements for the assessment of BIs as per American National Standards developed and published by AAMI (Asso- ciation for the Advancement of Medical Instrumentation) (3).

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One important requirement for a BIER steam vessel as used in our studies is the capability of monitoring a square wave heating profile.

Figure 3 is a schematic of the steam BIER vessel used to generate the D and z-value data. D-value is the time in min- utes required for a one log or 90% reduction in microbial population. The z-value is the number of degrees of tempera- ture required for a 10-fold change in the D-value.

The family category of lipid emulsions and their respec- tive D 121 C and z-values as well as classification in terms of microbial resistance are shown in Table 3 . A categorization of parenteral formulations with associated D 121 C and z-values and their potential impact on microbial resistance using the BI, Clostridium sporogenes, were previously reported (4). In addition, the methodologies used for D and z-value analyses were likewise cited (4). The data in Table 3 indicate that 20% emulsion (List 4336) is at the top of the list, since it affords the most microbial moist heat resistance. It is therefore the emulsion that should be microchallenged

Figure 3 This is a schematic of the steam BIER unit used to gen- erate spore crop (BI) and product D and z-values. Nine 5-mL glass ampules filled with various emulsion formulations can be tested at one time in the represented sample chamber.

Case Study: A Lipid Emulsion—Sterilization 401 Table 3

IV Lipid Emulsion Ranking Predicted spore

List # Solution D 121 c z-Value log reduction 4336

20% Emulsion 0.7 10.6 7.1 0720

10% Emulsion w =increased linolenate

10% Emulsion w =100% soybean oil

20% Emulsion w =100% soybean oil

20% Emulsion w =increased linolenate

10% Emulsion w =50% safflower & 50% soybean oil

20% Emulsion w =50% safflower & 50% soybean oil

0.6 12.7 9.5 The columns represent the list number of the product, the emulsion or product name,

its average D 121 C value and z-value and finally the PSLR value.

(inoculated with spores) as part of the emulsion validation scheme. D and z-value data have been reported for other BIs such as Bacillus stearothermophilus (5,6) and Bacillus subtilis 5230 (7). There are many factors that can affect moist heat resistance including a BI’s age, the sporulation media used, as well as the particular spore strain employed (8).

3.4. Closure Microbial Inactivation Studies In lieu of using the large type steam sterilizers in the produc-

tion environment, microbial inactivation at the closure =bottle interface of an emulsion container can be assessed in a devel- opmental R&D sterilizer. The closure microbial inactivation (kinetic) studies can address how the size of the container, type of closure compound used as well as closure pre- paratory processes (leaching, washing, siliconing, autoclav- ing, etc.) influence microbial inactivation. Kinetic studies are conducted at various time intervals in a given sterilization cycle. Surviving organisms are ascertained by direct plate

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(DP) count or fraction-negative (F =N) methodologies. Various BIs can be used and test data have been generated demon- strating the value of using both a moist heat organism (C. sporogenes) and a dry heat organism (B. subtilis) BIs for the sterilization validation of closure systems (9).

3.5. Lipid Emulsion PSLR Values Lipid emulsion moist heat resistance values (D 121 C and

z-values) were generated in the steam BIER vessel using the BI C. sporogenes as shown in Table 3 . The columns in Table 3 represent the list number of the product, the emulsion or pro-

duct name, its average D 121 C value and z-value and finally the PSLR value. Those parenteral formulations with the lowest PSLR value(s) are those that should be microbially vali- dated at subprocess conditions, since these provide the most microbial resistance.

3.6. Accumulated F(Bio) for Lipid Emulsions Accumulated F(Bio) by list number and z-values ( Table 4 ) was

used to construct the PSLR ranking for lipid emulsions as previously discussed for Table 3. The F(Bio) is the heat input for the biological solution based on the emulsion’s moist heat

D and z-values. By inputting the sterilizer temperatures from the coldest thermocouple of an engineering run for a particular container =sterilization cycle, the emulsion can be

ranked according to PSLR values. The combined D 121 C and z-value allows comparison of moist heat rankings between emulsions.

The data in Table 4 demonstrate that L. 4336, a 20% emulsion, has the lowest PSLR (7.105), thereby affording the highest moist heat resistance upon inoculation. Generation of this table allows prediction of which emulsion to microbio- logically challenge as part of validation in the production sterilizer.

IV emulsions were inoculated in the oil phase after emul-

sification and filtration ( Tables 5 and 6 ).

Case Table 4 Accumulated F(BIO) by List Number and z

Study: Solution

L 9789 L 4335 A Temp ( C) Time (min.)

F (PHY)

z ¼ 10.1 z ¼ 12.8 z ¼ 10.6 z ¼ 10.7 z ¼ 12.7 z ¼ 11.1 Lipid 105.4

z ¼ 10.0

z ¼ 10.6 z ¼ 11.4

0.0579 0.0384 Emulsion—Sterilization 110.1

0.0296 0.0178 Total F

5.7044 5.1573 D value

9.507 12.893 The data demonstrate that L. 4336, 20% emulsion, is the emulsion that has the lowest PSLR (7.105) thereby affording the highest moist

heat resistance upon inoculation. 403

© 2005 by Taylor & Francis Group, LLC

404 Berger Table 5 Plate Count Results of the IV Lipid Emulsion Inoculated

with the BI, C. sporogenes, ATCC 7955, and Spores in the Oil Phase 5 pass emulsion

Initial count of emulsion 3 mL

After pass #5

3 mL

1 mL 15 pass emulsion

After 0.8 UM filtration

Initial count of emulsion 3 mL

After pass #15

4 mL

<10=mL Based on the data, one would not have to routinely inoculate the BI in the oil phase

After 0.8 UM filtration

prior to performing an emulsion microbial validation since the bacterial population count does not change significantly upon multiple emulsion processing steps.

3.7. R&D Sterilization Validation of IV Emulsion Inoculated in the Oil Phase after Emulsification and Filtration

Since the bacterial population count does not change signifi- cantly upon multiple processing steps (5 pass vs. 15 pass), it is not necessary to routinely inoculate the BI in the oil phase prior to performing a microbial validation.

3.8. Maintenance of Sterility Studies The maintenance of sterility (MOS) studies are run on all

moist heat terminally sterilized products with closure or componentry systems in order to demonstrate that the

Table 6 Plate Count Results of the IV Lipid Emulsion Inoculated with BI, B. stearothermophilus, ATCC 7953, and Spores in the Oil Phase

5 pass emulsion (lipid Initial count of emulsion 4 mL emulsion with emulphor)

After pass #5

4 mL After 0.8 UM filtration

<10=mL 15 pass emulsion (lipid

Initial count of emulsion 4 mL emulsion with emulphor)

After pass #15

4 mL After 0.8 UM filtration

50 =mL Based on the data, one would not have to routinely inoculate the BI in the oil phase

prior to performing an emulsion microbial validation since the bacterial population count does not change significantly upon multiple emulsion processing steps.

Case Study: A Lipid Emulsion—Sterilization 405

closure or componentry system is capable of maintaining the emulsion and fluid path in a sterile condition throughout the shelf life of the product

In an MOS study, the product container is sterilized at a temperature which is higher than the upper temperature limit of the chosen sterilization cycle and for a time that is greater than the maximum time limit for the cycle or producing an F subzero level greater than the maximum F subzero level for the cycle. The rationale for the selection of the maximum tem- perature and heat input level for the pre-challenge sterilization is that rubber and plastic closures are subjected to thermal stresses during sterilization and those stresses are maximized at the highest temperature and the longest time allowed.

3.9. Endotoxin Studies Endotoxins are lipopolysaccharides from the outer cell mem-

brane of Gram-negative bacteria. Endotoxins can be detected by the manual gel-clot method known as the limulus amebocyte lysate test (LAL). There are also various quantitative methods (turbidimetric and chromogenic) which use more rapid auto- mated methodologies. All final product formulations are required to be tested for endotoxins and the method must be validated using three different lots of the final product. Emul- sion formulations, if colored or opaque, cannot be tested by the turbidimetric method and therefore must use the LAL test.