Background Information for Allogeneic Islet Manufacture

**Purpose:** Provide background information and context for FDA\'s
regulatory concerns about different stages of the manufacturing process
for allogeneic islets, which encompass pancreas procurement, processing
and product characterization.

Specific FDA questions regarding manufacture of islets are provided at
the end of this background information.

[Pancreas Procurement]{.underline}

-   **Methods of harvesting and handling**

Historical data from the International Islet Transplant Registry (ITR)
and published sources indicate that certain practices in pancreas
procurement, handling and organ allotment, are detrimental for
preparation of suitable islet preparations. FDA is concerned that these
practices may result in the use of a \"substandard\" pancreata resulting
in \"substandard\" and inconsistent islet preparations. To ensure high
quality islet preparations can be consistently made, FDA believes only
the highest quality pancreata should be used.

[Pancreas Processing]{.underline}

-   **Control and consistency/reproducibility of manufacture**

Pancreas age, size, duration and conditions used for dissociation will
impact islet yield, size distribution and viability. If the
manufacturing process is not controlled, it will be difficult to
consistently produce an equivalent product from lot to lot and
consequently make it difficult to identify the critical parameters
necessary to ensure the desired clinical effect.

**Demonstration of control in islet processing**

Data available from the ITR clearly indicates that producing high
quality islet preparations consistently is a complex process which
requires considerable expertise. In such situations, FDA frequently
requests that sponsors of new INDs or new clinical manufacturing sites
provide data demonstrating that the therapeutic product can be
consistently prepared. This is usually accomplished by accumulating data
from several non-clinical production runs, which shows that the product
is of clinical grade; i.e. the manufacturing process is controlled,
consistent and meets specifications for release.

[Islet Characterization]{.underline}

-   **Identity, Purity, Potency, Viability, Others**

Like any cellular and tissue-based product, you must be able to
demonstrate that you can safely and reproducibly manufacture the
therapeutic product. This is typically done by characterizing the
product and establishing specifications for lot release. Lot release
specifications for cell and tissue-based therapies includes
demonstration of safety and assessments of several product
characteristics such as identity, purity, viability and potency as well
as others. This data is important in order to evaluate the manufacturing
process and consistency of the product lots.

**Identity**

An islet equivalent (IE) is defined based on both insulin content and
morphology/size. An insulin granule binding dye, such as
diphenylthiocarbazone (DTZ) is commonly used to identify beta cells.
Since beta cells are only one of several other cell types needed to
constitute an islet, a morphological assessment, based upon a mean
diameter of 150 um, is used in addition to staining by DTZ, to define an
islet equivalent. However, at least two other cell types are found in
islets; alpha cells, which secrete glucagon, and delta cells which
secrete somatostatin. Should measurements of these molecules also be
made? FDA recommends that lot release specifications for cellular and
tissue-based therapies for identity include assessments to identify the
specific *therapeutic* cells or tissues.

**Purity - composition of islet preparations**

Historical data in the ITR shows that functional transplants of islets
have ranged in purity from \<5% to \>95%. For cell and tissue-based
therapies, FDA recommends that lot release specifications for purity
encompass quantitative measurements of *therapeutic* cells or tissues
which are both viable and functional. It should also include
measurements of \"other\" cell types including, non-viable cells which
may be beneficial or detrimental to patients. For islet preparations,
one example of \"other\" cells are exocrine cells which secrete
hydrolytic enzymes. Most cell and tissue-based therapies, are complex
mixtures of both desirable cell types and other impurities, typically
other cells types which may be beneficial or detrimental to the patient.
Assuming these impurities are not harmful to the patient, FDA frequently
requests that investigators monitor the impurity profile as an
additional means of demonstrating that the clinical product can be
manufactured consistently from lot to lot. Significant variations in the
impurities, from one lot to the next, can serve as a useful indicator
that a manufacturing process is not controlled or consistent .

**Potency**

A suitable potency assay is one that demonstrates that the clinical
product possesses the specific ability to give the desired clinical
effect. To this end, investigators in islet transplantation use assays
to measure the function of islets that possibly could be developed into
lot release assays for potency. For example, in the glucose stimulated
insulin release assays, islets *in vitro* are exposed to glucose and any
insulin secreted as a result, is quantified. A more time-consuming
functional assay involves implanting islets into diabetic,
immunodeficient mice to effect a cure, which can take several weeks. If
investigators could validate these assays to demonstrate that a given
number of islet equivalents will release a given amount of insulin *in
vivo,* or some other suitable measure, one of these assays could
potentially serve as a potency assay

**Viability, number and size distribution of islets**

There is no historical data available in the International Islet
Transplant Registry (ITR) with regard to the viability of islet
preparations, though it is clearly an important parameter for any
cellular and tissue based therapy. For cell or tissue-based therapies,
FDA recommends that initial, lot release specifications for viability of
the *therapeutic* cells or tissues be established at 70%.

In addition, historical data in the ITR and published reports indicate
that a minimum of 6,000 IE/kg need to be transplanted to increase the
likelihood of a functional graft. There does not appear to be any
conclusive data correlating the number of transplanted islet equivalents
with those that actually engraft post-transplant. FDA recommends that
for cellular and tissue-based therapies, lot release specifications for
cell number include a specification for the minimum number of viable and
functional cells necessary to confer the desired therapeutic effect.

Also, as a result of differences between donor pancreata, measurements
of the size distribution of the islets obtained from each preparation
are often made, since it is likely that this will be variable and could
have an impact on engraftment, survival or function of the graft post
transplant

[Other issues]{.underline}

**Multi-donor islet transplants**

In general, FDA guidelines discourage the pooling of tissues from more
than one donor. This is due in part to concerns about increasing the
risk of transmission of adventitious agents to recipients, issues of
immunogenicity and manufacturing concerns such as differences in potency
and purity of islets from different pancreata. To minimize the concerns
mentioned above, FDA recommends that islets from each donor pancreas
should be processed apart from one another and meet all lot release
specifications, with the exception of minimum number of IE.

**Draft of Pancreatic Islet Cell CMC Questions ( 3-10-2000)**

1.  **Organ Quality - Source Material for Islets**

Based on data in the International Islet Transplant Registry (ITR), most
investigators recommend that in addition to standard infectious disease
screening, pancreata be excluded from use for clinical preparations of
islets based on the following:

> Donor age : \<14 or \> 60 years
>
> Warm ischemia \> 15 min
>
> Cold ischemia \> 8 hours
>
> Presence of infection or malignancy
>
> Methanol or carbon monoxide toxicity
>
> History of diabetes
>
> Serum lipase \>500

a.  Please discuss and provide recommendations regarding the
    appropriateness of each of these exclusion criteria. For example,
    are restrictions on minimum and maximum donor age appropriate?

b.  Are there other diseases or conditions that should also be
    exclusionary?

c.  Are there other appropriate serum markers in addition to, or as an
    alternative to serum lipase (serum amylase, for example)?

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2.  **Appropriate types of identity testing**

An islet equivalent (IE) is defined based on both insulin content and
morphology/size. However, a beta cell is only one of several other cell
types needed to constitute an islet.

For example, at least two other cell types are found in islets; alpha
cells, which secrete glucagon, and delta cells which secrete
somatostatin. Should measurements of these molecules also be made?

a.  Please discuss if additional assessments should be used to identify
    an islet equivalent.

b.  Please discuss whether assessment of these other cell types would be
    important in determining the quality of the product.

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3.  **Viability, Number and Size distribution of Islet Preparations**

There are no data available in the International Islet Transplant
Registry (ITR) with regard to the viability of islet preparations, those
this is a critical parameter for any cellular or tissue based therapy.
In addition, data in the ITR and published reports indicate that a
minimum of 6,000 IE/kg need to be transplanted to increase the
likelihood of a functional graft. Also, islets from different donor
pancreata may have different size distributions.

a.  Please discuss and provide recommendations for appropriate measures
    of viability for islets.

b.  Does the BRMAC recommend that in the absence of data supporting a
    lower viability specification, that an initial lot release
    specification for viability of 70% for islets is appropriate?

c.  For a single donor, allogeneic islet transplant, what
    recommendations does the BRMAC have regarding the minimum number
    viable and functional islet equivalents?

d.  Does the BRMAC have recommendations regarding the maximum dose of IE
    that should not be exceeded for portal vein infusions?

e.  Please discuss and provide recommendations about whether assessments
    should also include the size distribution of the IE to be infused?
    If so, is there a targeted standard size distribution for IE?

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4.  **Purity - Composition of Islet Preparations**

Historical data in the ITR reveals that functional transplants of islets
ranged in purity from \<5% to \>95%.

a.  Please discuss they types of assessment that should be performed to
    determine the purity of islet preparations.

b.  Does the BRMAC have recommendations for the minimum acceptable
    purity level for islet preparations for clinical use?

c.  Please discuss and provide recommendations for monitoring the
    impurity profiles of \"other\" cell types in islet preparations?

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5.  **Potency**

A suitable potency assay is one that demonstrates that the clinical
product possesses the specific ability to give the desired clinical
effect.

a.  Please discuss and provide recommendations for suitable potency
    assays that are predictive of functional islets in humans.

b.  Please discuss and provide recommendations for the types of potency
    assays that can performed prior to patient administration.

c.  Please discuss assays for measuring continued islet function in the
    patient, post-transplant.

d.  What recommendations does the BRMAC have for qualifying potency
    assays as they relate to clinical measures of islet function?

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6.  **Demonstration of Control in Islet Processing**

Data available from the ITR clearly indicate that producing high quality
islet preparations consistently is a complex process which requires
considerable expertise.

a.  Please discuss the need for investigators to demonstrate that high
    quality islet preparations can be consistently made prior to
    initiating clinical research studies in humans.

b.  Does the BRMAC have any recommendations about the types of data
    necessary for demonstration of adequate processing control of
    islets?

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7.  **Multi-donor islet transplants**

In general, FDA guidelines discourage the pooling of tissues from more
than one donor.

a.  Please discuss pooling of islets from multi-donors for use in a
    single recipient. For example, if a given pancreas failed to yield a
    sufficient number of viable, functional IE, would it be appropriate
    to pool preparations from one or more other preparations to obtain
    the desired number.

b.  Please discuss appropriate time frames for completion of all
    infusions in situations where a recipient may receive islets from
    multiple donors, sequentially.