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15 appendices

| 15.1                                                                                                                                                                                       | APPENDIX A: PERFORMANCE STATUS CRITERIA ECOG Performance Status Scale   | Karnofsky Performance Scale                                  |             |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------|--------------------------------------------------------------|-------------|
| Grade                                                                                                                                                                                      | Descriptions                                                            | Percent                                                      | Description |
| 100                                                                                                                                                                                        | Normal, no complaints, no  evidence of disease.                         |                                                              |             |
| Normal activity. Fully active,                                                                                                                                                             |                                                                         |                                                              |             |
| 0                                                                                                                                                                                          | able to carry on all pre-disease  performance without restriction.      | Able to carry on normal activity;                            |             |
| 90                                                                                                                                                                                         | minor signs or symptoms of  disease. Normal activity with effort;       |                                                              |             |
| 80                                                                                                                                                                                         | some signs or symptoms of  disease.                                     |                                                              |             |
| Symptoms, but ambulatory.  Restricted in physically strenuous  activity, but ambulatory and able  to carry out work of a light or  sedentary nature (e.g., light  housework, office work). |                                                                         |                                                              |             |
| 1                                                                                                                                                                                          | Cares for self, unable to carry on                                      |                                                              |             |
| 70                                                                                                                                                                                         | normal activity or to do active  work.                                  |                                                              |             |
| In bed <50% of the time.  Ambulatory and capable of all  self-care, but unable to carry out  any work activities. Up and  about more than 50% of waking  hours.                            | Requires occasional assistance,                                         |                                                              |             |
| 60                                                                                                                                                                                         | but is able to care for most of  his/her needs.                         |                                                              |             |
| 2                                                                                                                                                                                          | 50                                                                      | Requires considerable assistance  and frequent medical care. |             |
| 40                                                                                                                                                                                         | Disabled, requires special care  and assistance.                        |                                                              |             |
| In bed >50% of the time.  Capable of only limited self-care,  confined to bed or chair more  than 50% of waking hours.                                                                     |                                                                         |                                                              |             |
| 3                                                                                                                                                                                          | Severely disabled,                                                      |                                                              |             |
| 30                                                                                                                                                                                         | hospitalization indicated. Death  not imminent.                         |                                                              |             |
| 20                                                                                                                                                                                         | Very sick, hospitalization  indicated. Death not imminent.              |                                                              |             |
| 100% bedridden. Completely  disabled. Cannot carry on any  self-care. Totally confined to bed  or chair.                                                                                   |                                                                         |                                                              |             |
| 4                                                                                                                                                                                          | 10                                                                      | Moribund, fatal processes  progressing rapidly.              |             |
| 5                                                                                                                                                                                          | Dead.                                                                   | 0                                                            | Dead.       |
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15.2 appendix b dtm standard

1. PRODUCT MANUFACTURING - COMPONENTS AND M**ATERIALS**
1. Cells Patients will undergo up to 15 liter PBMNC apheresis (autologous), as estimated by weight and target cell dose in the Dowling Apheresis Clinic of the DTM, per standard operating procedure. Bilateral peripheral venous access will be used whenever possible. Alternatively, a temporary central venous catheter (CVL) may be placed for collection. The CVL will be inserted by Critical Care or Interventional Radiology staff with the assistance of anesthesiology as indicated. Cells will be processed for further manufacturing in CPS/DTM according to SOPs (see Product Manufacturing section 2: Procedures).
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a. donor screening and testing

Cells manufactured in the current protocol are of AUTOLOGOUS nature and donor eligibility determinations are not required (21 CFR 1271.90(a)(1)). However, eligibility requirements for product handling in CPS manufacturing facility require that subjects be HIV seronegative, hepatitis C seronegative and negative for Hepatitis B surface antigen. In addition, on the day of product collection, peripheral blood is submitted for testing: HIV 1 and 2 (antibody and NAT), HTLV I and II (antibody), Hepatitis C (antibody and NAT), Hepatitis B (surface antigen), West Nile Virus (NAT), T cruzi (antibody), and Treponema pallidum (antibody). Should any of these results be positive, DTM isolation procedures as defined in BB-DMF 11054 will be followed.
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b. cell bank system

No feeder cells or cell lines were used in this protocol.  
2. Reagents 1. Tabulation of Reagents Used in Manufacture: (see Table 5)
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3. excipients

a. Final product is infused freshly and contains monocytes and Peginterferon alfa-2b
(Sylatron) and Interferon gamma-1b (Actimmune) suspended in Plasmalyte-A, 4% human serum albumin.  
b. The concentration, source and qualifying documents of all excipients are listed in Table 1 and Appendix 3.
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4. additional considerations (none)

Product Name Trade Name Vendor NDC Qualification Use Regulatory  
Status  
Product  
Manufacture  
| 0944- 0493- 02   | Package                | Buffer additive                   | FDA   |
|------------------|------------------------|-----------------------------------|-------|
| insert           |                        |                                   |       |
| NA               | Certificate of         | Buffer additive,                  |       |
| Analysis         | elutriation  procedure |                                   |       |
| 0264- 7800- 00   | Package                | Buffer additive,                  |       |
| insert           | elutriation  procedure |                                   |       |
| Package          | Prevent cell           |                                   |       |
| 6030-3           | insert                 | clumping,  elutriation  procedure |       |
| 100-40           | Package                | Prevent cell                      |       |
| insert           | clumping,  storage     |                                   |       |
| 0338- 0221- 03   | Package                | Infusion                          |       |
| insert           | Solution               |                                   |       |
| 4348- 01         | Package                | Activate                          |       |
| insert           | monocytes              |                                   |       |
| 42238-           | Package                | Activate                          |       |
| 111-01           | insert                 | monocytes                         |       |  
Dornase alpha (DNAse)  
Pulmozyme Genentech, Inc. 50242100-40 Package  
insert  
Prevent cell clumping,  
storage  
Interferon gamma-1b  
ACTIMMUNE® Horizon Pharma,  
Inc.  
42238111-01  
Package insert  
Activate monocytes  
FDA approved  
Final product preparation  
Package  
insert  
Buffer additive FDA  
approved  
Final product  
preparation  
Tricitrasol Citra Labs, LLC 237316030-3  
Package insert  
Prevent cell clumping,  
elutriation procedure  
Package  
insert  
Infusion  
Solution  
FDA  
approved  
Final product  
preparation  
Package insert  
Activate monocytes  
FDA approved  
Final product preparation  
| Human Serum                                              | Flexbumin 25%           | Baxter Healthcare               |        |
|----------------------------------------------------------|-------------------------|---------------------------------|--------|
| Albumin                                                  | Corp.                   |                                 |        |
| Hanks Buffered                                           | HBSS                    | American Pharmaceutical         |        |
| Salt Solution                                            | partners, Schaumbur, IL |                                 |        |
| Sodium Chloride  Injections USP                          | Saline                  | B.Braun Medical  Inc. Irvine CA |        |
| 0.9% sodium  chloride Sodium Citrate  Concentrate  46.7% | Tricitrasol             | Citra Labs, LLC                 | 23731- |
| Pulmozyme                                                | Genentech, Inc.         | 50242-                          |        |
| Dornase alpha  (DNAse) PLASMA-LYTEA                                                          | PLASMA-LYTE-A           | Baxter Healthcare  Corp.        |        |
| injection pH 7.4                                         |                         |                                 |        |
| Peginterferon                                            | SYLATRONTM              | Schering Corp.                  | 0085-  |
| alfa-2b Interferon                                       | ACTIMMUNE®              | Horizon Pharma,                 |        |
| gamma-1b                                                 | Inc.                    |                                 |        |  
Table 5: Reagents used for the manufacture of **autologous monocytes with Interferon (IFN)**  
| Product                       |                                    |
|-------------------------------|------------------------------------|
| Status                        | Manufacture Final product          |
| approved                      | preparation                        |
| GMP grade  ancillary  reagent | Intermediate  product, elutriation |
| FDA                           | Intermediate                       |
| approved                      | product, elutriation               |
| FDA                           | Intermediate                       |
| approved                      | product, elutriation               |
| FDA                           | Intermediate                       |
| approved                      | product, elutriation               |
| FDA                           | Final product                      |
| approved                      | preparation                        |
| FDA                           | Final product                      |
| approved                      | preparation                        |
| FDA                           | Final product                      |
| approved                      | preparation                        |
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2. product manufacturing - procedures

Figure 5. Manufacture of Autologous Monocytes in combination with SylatronTM **and** Actimmune®  
  
Figure 5. **Manufacturing process schema.** (Day 0) Autologous Peripheral Blood Mononuclear Cells (PBMNCs) will be collected by apheresis in DTM Dowling Clinic and transported to the Cell Processing Section (CPS) of DTM. The PBMNC product will undergo counter flow centrifugation elutriation (CCE) to enrich for monocytes in the product. After elutriation, all fractions except the monocyte fraction will be issued to PI laboratory. The monocytes will be washed and re-suspended at the appropriate concentration with 1-3 aliquots cryopreserved for future infusions. On the day of infusion, the monocytes will be washed, filtered, counted and resuspended at dose level with Peginterferon alfa-2b (SylatronTM) and Interferon gamma-1b
(Actimmune®) as described below. Assays for safety, purity, and identity will be performed before the product is issued for infusion.  
- Preparation of AUTOLOGOUS cells 1. Method of cell collection/Processing 1. Day 0 - Mononuclear cells will be collected by apheresis (up to 15 L) to reach a target cell number of monocytes. Apheresis will be performed in the Dowling Apheresis Clinic, Department of Transfusion Medicine (DTM) using a Cobe Spectra or an equivalent cell separator device using current standard operating procedures.  
2. Counter flow centrifugation elutriation (CCE) will be performed using the Elutra device.  
3. All products, intermediate and final, will be contained within a closed system using bags and sterile connection devices.  
4. The monocytes are contained in the rotor off (RO) fraction from elutriation. The monocytes will be stored overnight at refrigerated temperature before further processing on day of infusion.  
5. 1-3 aliquots will be cryopreserved for future infusions 6. Day 1 - the monocytes are prepared for infusion, with final testing including FACS to assess percent of CD14 (cell purity), cell counts, viability, and sterility testing, including Gram stain.  
2. Irradiation Not Applicable 3. Day of infusion 7. On Day 1, day of infusion, cells are taken from refrigerated storage (4°C), mixed, and allowed to warm to room temperature for 30 minutes. Cells are filtered, washed, and re-suspended in infusion solution.  
8. For cryopreserved monocytes, cells will be washed twice and resuspended at 8 x10e6 CD14+/16+/mL in Plasmalyte A+ 4% Albumin (Human), USP (HSA) and 10mcg/mL DNAse 9. Samples are taken for cell count, viability and FACS to determine subset components. 10. Cells are filtered and diluted in Plasmalyte A+ 4% Albumin (Human), USP (HSA) at a concentration in accordance with Dose Level according to SOP:
11. Dose level 1 and 3b: No cells will be given.  
12. Dose level 2: cells are diluted to 0.3x106/mL ( 20%). 13. Dose levels 3 and 4: cells are diluted to 3x106cells/mL ( 20%).  
14. Peginterferon alfa-2b (Sylatron™) and Interferon gamma-1b (Actimmune®) are added in quantities according to SOP to achieve the doses specified in the treatment protocol.  
15. Dose levels 1, 2, and 3 will contain 25 mcg Peginterferon alfa-2b (Sylatron) and 5 mcg Interferon gamma-1b (Actimmune);
16. Dose level 4 and 3b will contain 250 mcg Peginterferon alfa-2b (Sylatron) and 50 mcg Interferon gamma-1b (Actimmune).  
17. Monocyte cell suspension, in Plasmalyte-A and 4% HSA, with Peginterferon alfa-2b and Interferon gamma-1b, is issued for intraperitoneal infusion.  
- Process Timing and Intermediate Product Storage
(See **Table 6**)
- Final Formulation Final product is autologous monocytes suspended in Plasmalyte-A + 4% HSA + Peginterferon alfa-2b (Sylatron) + Interferon gamma-1b (Actimmune).  
| Da                                        | Time                    |                             |                                                                       |                                      |                         |                                          |    |
|-------------------------------------------|-------------------------|-----------------------------|-----------------------------------------------------------------------|--------------------------------------|-------------------------|------------------------------------------|----|
| y                                         | require d (hours)       | Process                     | Product (or                                                           |                                      |                         |                                          |    |
| Step                                      | Intermediat e Fraction) | Sample Designati on         | Sample Type (Cells/Su p)                                              | Assay                                | Lot  release  (Yes/N o) |                                          |    |
| 0                                         | NA                      | Patient                     | Patient PreApheresis                                                                       | Patient PB                           | CBC, TTD1 ,             |                                          |    |
| sampling                                  | ABO/Rh                  | Yes (TTD)                   |                                                                       |                                      |                         |                                          |    |
| 0                                         | NA                      | Patient                     | Patient PostApheresis                                                                       | Patient PB                           | CBC                     | No                                       |    |
| sampling                                  |                         |                             |                                                                       |                                      |                         |                                          |    |
| 0                                         | 1                       | Apheresis  product  receipt | PBMNC                                                                 | Bag                                  | Cells + Sup             | Cell  count, volume, Flow  differenti al | No |
| product                                   |                         |                             |                                                                       |                                      |                         |                                          |    |
| 0                                         | 2-3                     | Elutriation                 | PostElutriation                                                                       | RO Fraction                          | Cells+Sup               | Cell  count, Volume, Flow phenotyp e     | No |
| Supplemente                               | Supplement              |                             |                                                                       |                                      |                         |                                          |    |
| 0                                         | 1                       | d bag                       | ed monocyte  bag                                                      | D0 (RO tube)                         | Cells+Sup               | Cell  count,  viability                  | No |
|                                           | Stored bag              | Stored                      |                                                                       |                                      |                         |                                          |    |
| 1                                         | 2                       | monocyte  bag               | Cells+Sup                                                             | Cell  count,  viability Volume, FACs | Yes (FACs)              |                                          |    |
| D1 or PT (post  thaw)                     |                         |                             |                                                                       |                                      |                         |                                          |    |
| Final product                             | FN                      | Cells+Sup                   | Cell  count,  viability,  FACS,  gram  stain,  sterility,  endotoxi n | Yes                                  |                         |                                          |    |
| 1                                         | 2                       | Final  resuspensio n        |                                                                       |                                      |                         |                                          |    |
| 1 Transfusion Transmitted Disease Testing |                         |                             |                                                                       |                                      |                         |                                          |    |  
Table 6. Timeline and Lot Release Criteria for the manufacture of **autologous monocytes**
with Peginterferon alfa-2b (SylatronTM) and Interferon gamma-1b (**Actimmune**®).
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b. producttesting 1. microbiological testing

Samples for sterility testing will be taken from Final product (FN) (see **Figure 5**).  
The sterility sample will be prepared per CPS-SOP-5008 for bacterial and fungal testing which is performed by the Department of Laboratory Medicine, Clinical Center, NIH (See Appendix 4 for applicable CPS SOPs). Sterility tests comply with 21 CFR 610.12. In addition to samples inoculated into the Bactec bottles, a sample is submitted for fungal testing, consisting of plating onto solid media, for incubation for 14 days.
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2. identity

Identity will be tested on Final product (FN) by flow phenotype and cell count. The final release criteria on the final product will include Trypan blue viability: ≥ 70%, CD14+of viable: ≥ 40%
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3. potency

CD14 will be used as a surrogate marker for potency since the addition of monocytes to IFN therapy correlates with a reduction in tumor size in animal models with xenografted human ovarian tumors (Nakashima et al. 2012).
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4. other - general safety

General action plans for dealing with positive tests for sterility testing are presented in our Master File BB-MF-11054. See Cross reference letter for BB-MF-11054 contained in Section I.
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- viability

Viability testing will be performed on the Final product (FN). Trypan blue viability on the final product must be ≥ 70%.
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- cell number/dose

The cell number target will be based on the following dose levels. Dose level 1 and 3b: No cells will be given Dose level 2: 75 x 106 ( 20%) viable CD14+ monocytes total Dose levels 3 and 4: 750 x 106 ( 20%) viable CD14+ monocytes total
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c. final product release criteriatesting

Product Release Criteria: final product testing is performed on the day of infusion (Day 1). On day of infusion, samples for trypan blue viability, STAT gram stain, and FACS will be submitted and results used for release criteria. Sterility culture will also be  
| Acceptable                                                    |              |                       |
|---------------------------------------------------------------|--------------|-----------------------|
| Test                                                          | Method       | Limit Normal - milky; |
| Appearance                                                    | Visual check | no aggregates         |
| % Viable monocytes  (CD14+)                                   | FACs         | ≥ 40%                 |
| % Viability                                                   | Trypan Blue  | ≥ 70%                 |
| Sterility on final product                                    | Bactec       | *                     |
| Gram stain                                                    | Gram stain   | No organism seen      |
| Donor eligibility                                             | NA           | Auto                  |
| *sent on the day of infusion. Result available 14 days later. |              |                       |  
performed on the final product but results will not be available at the time of infusion. These results will be reported on the Certificate of Analysis listed below.
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d. product stability

The final product will be delivered to the patient care unit immediately after required assays for release. Expiration time is 4 hours post final product sampling.
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e. other issues product tracking

Manufacturers of cellular therapy products are required to maintain traceability of the products received, processed and stored in the facility through the final disposition of the product. CPS-SOP-5107 describes the procedures for receipt of products in the Cell Processing Section (see Drug Master File: 11054). CPS-SOP-5105 outlines the requirements for distribution and documentation of products to administration sites, research or discard (see Drug Master File: 11054). At final disposition, the product is assigned a status of infused, shipped, allocated to research or discarded.
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product labeling

The following is an example of the product label for final product, autologous monocyte cell suspension, in Plasmalyte-A and 4% HSA, with Peginterferon alfa-2b and Interferon gamma-1b
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container/closure

The starting apheresis material, intermediate products, and final product will be contained within a closed system using sterile transfer pack containers. All connections will be made using a Sterile Tube Welder (TerumoBCT).
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environmental impact

The Women's Malignancies Branch of the National Cancer Institute, National Institutes of Health requests a claim for categorical exclusion for this proposed clinical trial as provided for in 21 CFR 25.31(e), in that the agents studied under this IND are intended to be used in clinical trials in which the amount of waste expected to enter the environment may reasonably be nontoxic. Furthermore, it is understood that the clinical supplies of the investigational agents are to be used under the direction of qualified investigators (physicians, pharmacists and nurses) knowledgeable in the use and handling of investigational agents, including the professional staff in the Department of Transfusion, Cell Processing Section, NIH. All waste is disposed in accordance with the environmental protection laws, regulations and guidelines of the Federal Government and the State of Maryland.  
➢ QUALIFICATION OF THE MANUFACTURING PROCESS:
Validation Summary:
Preparation of monocytes in combination with Peginterferon alfa-2b (SYLATRONTM) and Interferon gamma-1b (ACTIMMUNE®)
The NCI protocol entitled 'Phase I study of intraperitoneal infusion of autologous monocytes with Sylatron (Interferon alpha 2b) and Actimmune (Interferon gamma) in women with recurrent or refractory ovarian cancer, fallopian tube cancer, or primary peritoneal cancer' (17-C-0011), utilizes an elutriated apheresis product to obtain monocytes which are mixed with varying doses of interferons before being administered to patients. Here, patients undergo an apheresis procedure each time they are admitted, which can be up to every 4 weeks. In the following study, we designed and validated a new process that would allow us to prepare one initial fresh dose of monocytes and cryopreserve the remaining cells so that they may be thawed and infused at a later date. This new modification would reduce unnecessary stress to the patient due to having to undergo multiple apheresis and it will also relieve the burden of performing many of these aphereses altogether by Dowling Clinic. Three separate samples were tested independently, two samples from healthy donors and one sample taken from a patient with ovarian cancer. Each sample was processed as a fresh and as a cryopreserved specimen as they would be during clinical manufacturing. Results below compare multiple attributes of each of these specimens. First, we examined cell recovery following cryopreservation to ensure the cells were capable of surviving the cryopreservation process. Cells from all three donors were cryopreserved in aliquots >330x106 TNC (or >260x106 CD14/CD16 monocytes) (**Table 7**). These aliquots represent the typical cell number that would be cryopreserved for dose level 2 and one-third of dose level 3 and 4. Upon thaw, cells were counted and recovery was calculated. TNC recovery from all three samples was >88% (>91% for CD14/CD16 monocytes). We conclude that there is no detrimental effect of the cryopreservation procedure on the TNC or monocyte recovery.  
| PD17060149 B   | 3.50E+08   | 3.50E+08    | 100.0%   | 2.85E+08   | 2.94E+08   | 103.3%   |
|----------------|------------|-------------|----------|------------|------------|----------|
| PD17060190 B   | 4.22E+08   | 3.97E+08    | 94.1%    | 3.16E+08   | 3.12E+08   | 98.6%    |
| PD17060193 B   | 3.33E+08   | 2.93E+08    | 88.0%    | 2.60E+08   | 2.37E+08   | 91.3%    |
| Sample         | TNC        | CD14+/CD16+ |          |            |            |          |  
Next, we compared the frequency of CD14/CD16 cells that were in the starting product (bag), fresh sample, and cryopreserved sample. After elutriation, we observed a consistent increase in the frequency of monocytes that were within the RO fraction. The frequency ranged from 1443% in the starting bag to >75% post elutriation (**Table 8**). There was <5% difference when comparing the frequency of monocytes between SM and day 1 of fresh sample and cryopreserved sample. Similarly, there was <9% difference when comparing the frequency of monocytes between FN samples of fresh vs. cryopreserved samples. Together, this data indicate that cryopreservation of the intermediate product did not significantly affect the monocyte frequency.  
| PD17060149   | 15.30%           | 81.40%          | 79.10%   | 84.10%   | 87.60%   |
|--------------|------------------|-----------------|----------|----------|----------|
| PD17060190   | 14.00%           | 74.80%          | 79.10%   | 78.40%   | 74.80%   |
| Bag          | SM               | FN              | Day 1    | FN       |          |
| PD17060193   | 43.50%           | 78.10%          | 76.80%   | 81.00%   | 80.60%   |
| Sample       | Fresh sample (A) | Cryo sample (B) |          |          |          |  
Loss of CD14/CD16 monocytes that occurred during processing from day 1 until the FN was packaged was minimal. Fresh sample recovery during this time point was >93% and cryopreserved sample recovery was >86% (**Table 9**).  
| Day 1      | FN               | Recovery        | Day 1   | FN       | Recovery   |        |
|------------|------------------|-----------------|---------|----------|------------|--------|
| PD17060149 | 7.50E+07         | 7.55E+07        | 100.67% | 2.45E+08 | 2.25E+08   | 91.84% |
| PD17060190 | 2.16E+08         | 2.20E+08        | 101.85% | 2.80E+08 | 2.43E+08   | 86.82% |
| PD17060193 | 7.50E+07         | 7.03E+07        | 93.73%  | 2.61E+08 | 2.36E+08   | 90.34% |
| Sample     | Fresh sample (A) | Cryo sample (B) |         |          |            |        |  
Functional assays were performed in Dr. Annunziata's laboratory using the final product from both the fresh and cryopreserved samples. Monocytes plus IFN's (per protocol) were mixed with ovarian cancer cells as outlined in the validation plan and allowed to incubate together for 3 days. Monocyte and IFN cytotoxicity is measured as percent survival compared to untreated controls. Percent killing is calculated as (100-% survival). There were no significant differences in % killing between fresh and cryopreserved samples. All samples were highly efficient in their ability to lyse target tumor cells. The percent killing from all samples was >94%, and the difference of percent killing between individual fresh vs. cryopreserved samples was <2%
(**Table 10**). This data indicate that there was no loss of function that occurred within the cryopreserved samples.  
| PD17060149   | 94.1%                            | 95.8%   |
|--------------|----------------------------------|---------|
| Sample       | Fresh sample (A) Cryo sample (B) |         |
| PD17060190   | 96.1%                            | 94.7%   |
| PD17060193   | 95.7%                            | 95.3%   |  
Monocyte viability pre and post cryopreservation was examined next. Samples were taken at day 1 and in the final product after resuspension with cytokines and assessed for viability using AO/PI uptake on the cellometer instrument. The viability of all samples >92% (**Table 11**). In addition, we examined the stability of the monocytes post thaw at 2 hour and 4 hour time points after final packaging with cytokines to ensure that they remained stable while they are transferred to the clinic for administration to the patient. We found that the viability at these time points was not significantly different than the viability at the 0 hour time point (all >93%) and conclude that the monocytes would remain stable for up to 4 hours (**Table 12**).  
Day 1 FN **Day 1** FN  
PD17060149 99.0% 98.0% 99.6% 98.9%  
PD17060190 97.0% 94.5% 92.8% 93.8%  
PD17060193 99.0% 99.0% 96.4% 98.8%  
Sample Fresh sample (A) Cryo sample (B)  
Table 12 **Stability of Cryopreserved Monocytes**  
Sample FN (0hr) FN (2hr) FN (4hr)  
PD17060149 B 98.9% 98.6% 99.0%  
PD17060190 B 93.8% 94.8% 93.6%  
PD17060193 B 98.8% 98.3% 98.2%  
Conclusion: We validated a new manufacturing process for the preparation of cryopreserved monocytes and found that the cell product was extremely consistent between cryopreserved fraction vs. fresh fraction and also consistent among the three different donors tested here. All samples tested passed the validation criteria set forth in the validation plan, which also included product safety testing (sterility, endotoxin). We therefore approve the implementation of this process within DTM Cell Processing Section.
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f. quality assurance practices

For a complete description of Quality Assurance and Quality Control practices in CPS DTM refer to BB-MF 11054.
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16.1 appendix c list of

Patients receiving any medications or substances that are strong inhibitors or inducers of CYP1A2 and CYP2D6 are ineligible. Because the lists of these agents are constantly changing, it is important to regularly consult a frequently-updated list. We will refer to http://medicine.iupui.edu/clinpharm/ddis/; medical reference texts such as the Physicians' Desk Reference may also be consulted. As part of the enrollment/informed consent procedures, the patient will be counseled on the risk of interactions with other agents, and what to do if new medications need to be prescribed or if the patient is considering a new over-the-counter medicine or herbal product.
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16.2 appendix d standard operating

If patient has existing intraperitoneal catheter, this will be used. If not, then a tunneled 6F or 9F catheter will be placed in interventional radiology.  
1) Access Port-a-Cath or peritoneal infusion catheter 2) Place patient on complete bed rest in semi-Fowler's position throughout administration of IP infusion. Head of bed must be no higher than 30 degrees to prevent dislocation of port needle or kinking of infusion catheter, and to allow IP fluid to flow freely. A flat position during infusion may increase pressure on diaphragm, causing respiratory compromise/GI upset in patients receiving IP infusions. If any significant movement of patient occurs during IP infusion, the nurse must assure proper needle positioning is maintained. 3) In this protocol, 0.9% Sodium Chloride Injection is administered warmed or at room temperature per patient preference. If warming is preferred, place 500 mL 0.9% Sodium Chloride Injection in warm water bath (keeping outer manufacturer wrapping intact) for approximately 15 minutes. RATIONALE: Warmed fluid is more comfortable for patient during infusion and decreases the incidence of cramping associated with IP infusions. 4) Prime IV tubing with attached Y port with 0.9% Sodium Chloride Injection, either warmed or room temperature per patient preference. Attach to port or peritoneal infusion catheter. 5) Infuse 200-250 mL of 0.9% Sodium Chloride Injection, as rapidly as possible via gravity. 6) Observe catheter site for swelling, leakage, or redness. Observe entire abdominal surface for unusual local swelling. Observe patient for complaints of pain, SOB, dyspnea, respiratory distress, and cramping. Stop infusion and notify physician if any of above conditions occurs. RATIONALE: Migration of catheters or dislodging of right angled needle may occur. 7) If no adverse effects noted after completion of 0.9% Sodium Chloride infusion, attach primed Monocyte infusion agent to free Y connector. Clamp 0.9% Sodium Chloride infusion line and infuse monocytes as rapidly as possible, via gravity. Monocytes should be infused at room temperature. NOTE: IP monocyte infusion may take as little as 30 minutes or as long as 1 hour (+/- 10 min) to infuse. If infusion takes longer than 1 hour, 10 min (70 min), RN should notify physician for troubleshooting. 8) After infusion of monocytes is complete, clamp monocyte tubing and open 0.9% Sodium Chloride tubing. Infuse an additional 50-100 mL of 0.9% Sodium Chloride Injection as rapidly as possible by gravity. 9) Flush port needle or catheter with 10 mL of low strength heparinized (10 Units/mL) 0.9% Sodium Chloride. De-access Port-a-Cath or clamp catheter. 10) After de-accessing needle or clamping catheter, begin turning protocol: reposition patient every 15 minutes clockwise, (i.e.: from side, to back, to other side, if possible prone (abdomen), Fowler's, Trendelenburg and knee chest) for a total of two hours. RATIONALE: Repositioning disperses fluid throughout the peritoneal cavity. 11) Patient may ambulate after turning protocol is complete.  
12) Document chemotherapy administration according to institutional policy
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16.3 appendix e sample chemotherapy/biotherapy

Paracentesis will be done prior to treatment regimen and repeated after at least 24 hours following treatment have elapsed. DOSE LEVEL 1 or 3B (Put in dose depending on dose level at asterisks):
➢ 200-250 mL warmed 0.9% Sodium Chloride Injection (or room temperature per patient preference) will be given intraperitoneally first followed by:
➢ Peginterferon alfa-2b ** mcg Interferon gamma-1b ** mcg The drug will be mixed with PLASMA-LYTE-A, HSA, and IFN in a total quantity of 248 ±2 mL Infuse by gravity over 30-60 min
➢ Infuse an additional 50-100 mL of 0.9% Sodium Chloride Injection, flush port with heparin flush of 10 Units/mL (10 mL)
➢ The patient will then be repositioned for a total of 2 hours to allow for maximal distribution of the treatment.  
DOSE LEVEL 2, 3, or 4 (Put in dose depending on dose level at asterisks):
- 200-250 mL warmed 0.9% Sodium Chloride Injection (or room temperature per patient preference) will be given intraperitoneally first followed by:
- Monocyte cell suspension (**) in PLASMA-LYTE-A + 4% HSA and will be mixed with:
Peginterferon alfa-2b **mcg Interferon gamma-1b **mcg The infusion will be in a total quantity of 248 ±2 mL
Infuse by gravity over 30-60 min
- Infuse an additional 50-100 mL of 0.9% Sodium Chloride Injection, flush port with heparin flush of 10 Units/mL (10 mL)
- The patient will then be repositioned for a total of 2 hours to allow for maximal distribution of the treatment.