Viral Clearance Processes for Plasma Derivatives

 

 

GUIDE TO INSPECTIONS OF VIRAL CLEARANCE PROCESSES

 

FOR PLASMA DERIVATIVES TABLE OF CONTENTS

 

Scope and Intent . . Pg 1

 

Introduction . . Pg 1

 

Background . . Pg 2

 

Scope of the Risks . . Pg 2

 

Methods of Viral Clearance . . Pg 3

 

Validation of Viral Clearance . . Pg 4

 

Operations and Inspections . . Pg 6

 

Removal Methods . . Pg 7

 

Inactivation Methods. . .Pg 7

 

Additional Considerations. . .Pg 7

 

Conclusions. . .Pg 8

 

 

SCOPE AND INTENT

 

The intended audience for this document is

 

the ORA field investigator. The document's

 

origins stem from the transfer of primary

 

responsibility for GMP inspections of

 

fractionators from CBER to ORA in the latter

 

part of 1996. While FDA field investigators

 

are well versed in the application of GMP to

 

the drug industry, this document is intended to

 

communicate differences in the application of

 

these regulations occasioned by the uniqueness

 

of plasma derivative products.

 

In particular, GMP requires and ORA field

 

investigators determine through inspections,

 

that significant aspects of drug manufacturing

 

operations have been validated. Although the

 

fractionation industry shares many of the same

 

process validation concerns with the larger

 

sterile drug manufacturing community, the

 

validation of viral clearance processes presents

 

issues with which the field investigator may not

 

be familiar.

 

This document, which has been prepared

 

jointly by the Office of Regulatory Affairs

 

(ORA) and the Center for Biologics Evaluation

 

and Research (CBER), attempts to provide

 

basic information on viral clearance validation

 

concepts, to enable the investigator to

 

distinguish those areas of viral clearance

 

processes that are appropriate for GMP

 

inspectional coverage. It is not an

 

all-encompassing technical guide on viral

 

inactivation processes, but rather a document

 

that seeks to assist field investigators in

 

conducting fractionator inspections. If the need

 

arises, this document may be amended or

 

expanded to include other issues of interest to

 

the field investigator concerning viral clearance

 

processes.

 

This guidance represents the Agency's

 

current thinking regarding inspectional

 

considerations of viral clearance processes for

 

plasma derivatives.

 

 

INTRODUCTION

 

Plasma-derived proteins are important

 

therapeutics for many patients in the U.S. every

 

year. Because they are manufactured from

 

human plasma, special precautions must be

 

taken during the production of these proteins

 

to assure against the possibility of the products

 

transmitting infectious diseases to the

 

recipients.

 

This document focuses on the possibility

 

that a small proportion of the individual units of

 

plasma used as source material may be

 

contaminated with human viruses and the

 

processes used to remove or inactivate those

 

viruses. Additionally, as is the case for any

 

pharmaceutical, there is a possibility of

 

introducing adventitious pathogens during

 

production. This risk is one specifically

 

addressed by adherence to current Good

 

Manufacturing Practices (GMP) and for this

 

reason it is a natural focus of any facility

 

inspection. This document is to be used in

 

conjunction with the Compliance Program for

 

the inspection of Plasma Derivatives of Human

 

Origin (7342.006), the Guideline on the

 

General Principles of Process Validation (May

 

1987), the Investigations Operations Manual

 

(IOM), and the Code of Federal Regulations,

 

Title 21 (21 CFR).

 

 

BACKGROUND

 

Addressing the risks that may be associated

 

with plasma derivatives begins with the plasma

 

from which the products are manufactured. A

 

number of precautions are taken to prevent

 

contaminated units from being collected and to

 

eliminate contaminated units when they are

 

collected. These now familiar precautions

 

include:

 

1. Screening donors by medical and behavioral

 

risk history;

 

2. Maintaining donor deferral registries to

 

prevent the use of units from unsuitable

 

donors;

 

3. Testing blood and plasma donations for

 

markers of disease;

 

4. Quarantining blood and plasma until tests

 

and control procedures establish its safety;

 

and,

 

5. Monitoring and investigating adverse

 

incidents to ensure that deficiencies are

 

corrected.

 

Many investigators will be familiar with these

 

procedures from their inspections of blood

 

banks and plasma collection facilities. When

 

the plasma is used as source material for the

 

manufacture of plasma derivatives, an additional

 

layer of safety may be achieved by including

 

effective viral clearance step(s) in the

 

manufacturing process.

 

 

SCOPE OF THE RISKS

 

The viruses of greatest concern for safety of

 

plasma derivatives include the hepatitis viruses

 

(hepatitis B and C, abbreviated HBV and HCV,

 

respectively) and the human immunodeficiency

 

viruses Types 1 and 2 (collectively HIV; the

 

causative agent of AIDS). Another virus,

 

human parvovirus B19 (B19), is a common

 

contaminant of plasma. Hepatitis A virus (HAV)

 

contaminates plasma products less frequently.

 

B19 and HAV pose some unique problems

 

because they are small (15-30 nm) and do not

 

possess an outer envelope composed of lipids

 

(both are termed "non-enveloped" viruses for

 

this reason). Both B19 and HAV are

 

extraordinarily resistant to heat and chemical

 

treatment and are difficult to remove by

 

nanofiltration. Furthermore, no licensed

 

screening kits exist in the U.S. for either virus.

 

Fortunately, the diseases caused by B19 and

 

HAV are less serious than for other viruses of

 

concern, although they may cause serious

 

disease in susceptable populations.

 

Nevertheless, there are intense efforts

 

underway to develop new methods for

 

removing or inactivating these viruses and some

 

of these methods may begin to appear in

 

manufacturing processes over the next few

 

years.

 

The most clinically significant viruses, HIV,

 

HBV and HCV, are all "enveloped" viruses,

 

which renders them more susceptible to

 

inactivation methods based on chemical

 

treatments or heat. These viruses are also

 

larger and more effectively removed by

 

nanofiltration. Nearly all plasma derivative

 

manufacturing processes now include effective

 

methods for their inactivation and/or removal.

 

It should be noted that certain viruses which

 

may pose serious risks when associated with

 

blood components (human T-lymphotropic

 

virus (HTLV-I & -II) or cytomegalovirus (CMV))

 

are not considered to be significant risks with

 

respect to plasma derivatives. These viruses

 

infect white cells and therefore are associated

 

with the cellular components of blood which

 

are mostly absent from plasma. The viruses

 

also tend to lose their viability over periods of

 

refrigerated storage and they do not tolerate

 

freezing and thawing. For this reason, HTLV-I

 

& -II and CMV are not risk factors for plasma

 

derivatives.

 

It should also be noted that the possibility

 

that bacteria or protozoa may contaminate a

 

unit of plasma or whole blood is not

 

considered a significant risk to recipients of

 

plasma derivatives. Manufacturing under GMP

 

should remove or inactivate any of these

 

endogenous agents, and any exogenous agents

 

introduced during manufacture of the drug

 

substance. Therefore, properly processed

 

plasma derivatives pose no additional risk of

 

bacterial or protozoal contamination beyond

 

those associated with any aseptically processed

 

parenteral. However, the processes must be

 

carefully evaluated to insure that they

 

consistently produce sterile products ( see

 

Compliance Program 7342.006 and the

 

"Guideline on Sterile Drug Products Produced

 

by Aseptic Processing" 1987).

 

This document also does not cover possible

 

risks posed by the poorly characterized agents

 

thought to be responsible for transmissible

 

spongiform encephalopathies. There are no

 

known effective methods for screening out

 

plasma units that contain the infectious agent,

 

and no known manufacturing methods that

 

would be effective in removing or inactivating

 

the infectious agent.

 

Finally, this document applies to human

 

plasma derived products only. Products

 

derived from animal plasma pose different risks

 

than human plasma derivatives. Bovine plasma,

 

for example, may not be sourced from

 

countries in which the disease, bovine

 

spongiform encephalopathy (BSE) exists (FR

 

94-21279). Otherwise, no formal

 

recommendations regarding manufacturing

 

safeguards for TSEs have been developed at this

 

time.

 

 

METHODS OF VIRAL CLEARANCE

 

A number of different types of

 

manufacturing steps are capable of removing or

 

inactivating viruses that may be present in

 

plasma pools from source or recovered plasma

 

donations. These steps may be divided into

 

two broad categories: removal, or partitioning,

 

is the physical separation of the virus or viral

 

particles from the therapeutic component; and

 

inactivation, which destroys the virus so that

 

the remaining viral fragments lack the structure

 

and components needed to infect an individual

 

receiving the product.

 

Removal processes include filtration, affinity

 

chromatography, ion exchange chromatography,

 

and polyethylene glycol fractionation. Heating

 

and solvent detergent treatments are examples

 

of processes that inactivate viruses.

 

Additionally, some processes, such as ethanol

 

fractionation, both remove and inactivate

 

viruses. Finally, a number of new methods are

 

under development, such as irradiation,

 

photoinactivation and treatment with a variety

 

of chemicals. Several of these novel techniques

 

have been incorporated into the production of

 

investigational products. In order to be

 

effective, viral inactivation techniques must

 

destroy at least one viral element essential to

 

replication. Photosensitizing techniques use

 

light-activated dyes that are irradiated, causing

 

the dyes to convert to molecules that can alter

 

DNA or membrane lipoproteins. Heat

 

treatment denatures viral proteins and nucleic

 

acids, rendering viruses incapable of replication.

 

Irradiation processes may destroy viral nucleic

 

acids by inducing breaks and linkages. Solvent

 

detergent techniques destroy the viral envelope

 

in lipid-enveloped viruses.

 

 

VALIDATION OF VIRAL CLEARANCE

 

All processes which claim to remove virus

 

must be fully validated by the manufacturer, as

 

there are no methods codified in the applicable

 

regulations. All of these methods are employed

 

during production of the drug substance.

 

A specific viral inactivation method that is

 

employed on the drug product in final

 

containers, is heat-treatment at an attained

 

temperature of 60 C ñ 0.5 C for 10-11

 

(continuous) hours. This process is mandated

 

for two products: Albumin (Human) and Plasma

 

Protein Fraction (Human) in 21 CFR 640.81

 

and 640.91, respectively. Because the

 

conditions of treatment are specified by

 

regulation, manufacturers are not required to

 

validate the effectiveness of the treatment itself,

 

but must nevertheless demonstrate that the

 

process operating parameters are met

 

consistently during production.

 

Some other products are heat treated, in

 

bulk tanks or containers, using similar

 

methodology (e.g. Antihemophilic Factor

 

(Human), Intravenous Immune Globulin

 

(Human)); alternative methods may also be

 

used, such as vapor heat treatment of

 

lyophilized intermediates. Another inactivation

 

method that may be encountered (e.g. Immune

 

Globulin Intravenous (Human) is treatment of

 

in-process material with solvent and detergent,

 

for a given time at a given temperature, and

 

subsequent removal of these additives. This

 

removal is generally accomplished by extraction

 

and/or the use of chromatographic methods. In

 

all of these cases, it is the responsibility of the

 

manufacturer to validate the effectiveness of

 

the method, as well as demonstrate that the

 

validated operating parameters are met

 

consistently during production.

 

Validation studies are performed when a

 

new product is under development or when a

 

manufacturer wishes to introduce a new step

 

into a pre-existing manufacturing process. In

 

these cases, the validation is designed along

 

with the process which may be optimized for

 

viral clearance. It is necessary to consider

 

scale-up problems and the possibility that

 

validation studies at the pilot scale may not be

 

relevant to the final manufacturing process. If

 

scale-up involves changes in the manufacturing

 

process that could adversely affect the viral

 

clearance validated at the pilot stage, then viral

 

clearance would have to be revalidated for the

 

altered process.

 

It is also possible that a manufacturer, with a

 

product made by a well established process,

 

identifies a step in that process which may be

 

effective in removing or inactivating viruses.

 

The manufacturer may then attempt to validate

 

this step without changing it. In this case, the

 

manufacturer should take the process as it

 

exists, without attempting to optimize it for

 

viral clearance, in order to avoid additional

 

studies to demonstrate that the product has

 

not been adversely affected. If the process has

 

changed, the manufacturer should demonstrate

 

that the final product has not been adversely

 

affected.

 

Regardless of the setting in which the

 

validation is performed, all viral clearance

 

validation studies share certain common

 

features. These can be summarized as follows.

 

1. Validation is performed on scaled down

 

laboratory models of the production

 

process. It is an unacceptable hazard to

 

introduce viruses into a production area in

 

order to validate a viral clearance step. It is

 

also impossible in most cases to produce

 

sufficient amounts of a virus to validate its

 

removal at the full manufacturing scale. The

 

necessity of working with scaled down

 

systems presents discreet challenges to

 

validation of viral clearance processes:

 

a. The scaled down model should

 

accurately represent the full scale

 

manufacturing process. This is insured

 

by controlling parameters such as

 

temperature, volume, flow rates, contact

 

times, relative geometries, and load. The

 

most important measure is the actual

 

performance of the scaled down process,

 

measured by parameters such as

 

capacity, yield and purification if

 

applicable.

 

b. The starting material (process

 

intermediate) should accurately reflect

 

that of the full scale manufacturing

 

process. This is often accomplished by

 

sampling process material from a full

 

scale batch.

 

c. Even though scaled down systems are

 

used for validation, the studies should be

 

designed to represent the worst case

 

conditions that could be encountered

 

during full-scale production, i.e., those

 

conditions under which the removal or

 

inactivation of viruses would be expected

 

to be the least effective. The viral

 

titrations should be designed with

 

adequate replicates to assure a

 

scientifically and statistically sound result.

 

Validation of the scale-down model

 

should involve multiple runs of the

 

model, the results of which are

 

compared to those from the full-scale

 

process.

 

d. Appropriate operational and

 

performance qualification with reference

 

to the critical operating parameters

 

should be performed for the full-scale

 

manufacturing system. Qualification

 

should include acquiring sufficient data to

 

verify that the full-scale system

 

consistently delivers all critical

 

performance conditions as was specified

 

in the successful validation studies with

 

the representative scale-down system.

 

e. Subsequent changes in the full scale

 

manufacturing process can affect the

 

validity of prior viral clearance studies.

 

Whenever a process and/or equipment

 

change is made, the possible effect of

 

that change on viral clearance should be

 

considered and evaluated, and the viral

 

clearance should be revalidated to the

 

extent necessary.

 

2. Viral clearance validation studies usually take

 

the form of "spiking" experiments, in which

 

large amounts of a virus is added to the test

 

article. It should be noted that the viral

 

load is far in excess of what would be

 

expected in a "contaminated" plasma pool.

 

The reduction in the amount of added virus

 

by the manufacturing step in question is then

 

measured. Appropriate controls are

 

included to insure that the measurement of

 

the amounts (titers) of the virus does not

 

change the performance of the scaled-down

 

manufacturing step. Additional controls are

 

included to confirm viability of the indicator

 

cells and the infectivity of the virus.

 

3. In vitro analyses are most commonly used to

 

quantify virus levels in the course of a

 

validation study. These may take the form

 

of "plaque assays" or assays that measure

 

"cytopathic effect", both of which are

 

performed in tissue culture. These assays

 

measure infectivity of the virus used in the

 

study. Biochemical assays may also be

 

encountered, such as antigen-based or

 

nucleic acid assays (e.g., PCR). These are

 

acceptable when predictive of infectivity.

 

Finally, some studies may use animal models

 

such as primates or ducks, but these have

 

become less frequent because they are

 

expensive and adequate alternative methods

 

are available.

 

4. Most validation studies are performed with

 

model viruses sharing characteristics of the

 

relevant human viruses. The selection of

 

appropriate models is of critical importance.

 

Among the human viruses of concern, only

 

HIV and HAV have appropriate in vitro

 

systems with which their titers can be

 

measured. The clearance of HBV and HCV,

 

by some manufacturing processes, has been

 

validated in primate models, but these

 

studies are not common today.

 

5. The extent of validation with respect to the

 

number of steps validated and the number of

 

different viruses studied varies greatly from

 

product to product and from manufacturer

 

to manufacturer. With the exception of

 

some IGIM products (see footnote 4, page

 

8), all plasma derivatives are subjected to at

 

least one viral clearance step, in addition to

 

the fractionation process itself which has

 

been shown to reduce viral load for some

 

agents.

 

6. A validation study may be acceptable even if

 

some detectable virus is found, as

 

these studies should be designed to add

 

many times more virus to the test article

 

than would be encountered in actual

 

practice. Large amounts of virus ("high

 

titers") permit more precise quantification

 

and provide safety margins to the

 

manufacturing process.

 

7. When more than one manufacturing step in

 

a process has been validated to clear a

 

particular virus, the overall clearance of the

 

process is the product of the two steps

 

(often calculated by adding log reduction

 

factors), provided that the two steps

 

operate by independent principles. For

 

instance, the results of a filtration step and a

 

heating step may be combined because they

 

operate by different mechanisms. On the

 

other hand, merely repeating a step would

 

not double the viral clearance because there

 

would be no second mechanism involved; an

 

additive effect cannot be presumed.

 

8. Validation of the process or processes

 

serves to establish that the operating

 

parameters, used during normal production,

 

are appropriate. Changes may be made

 

based on the validation study. It is therefore

 

important that the parameters established

 

during the study are those used in actual

 

manufacture.

 

 

OPERATIONS AND INSPECTIONS

 

The validation of viral clearance methods is a

 

highly sophisticated and scientifically complex

 

undertaking. From time to time, it is expected

 

that technical questions will arise during the

 

inspection. The investigator should keep in

 

mind that the scientific resources of the Agency

 

are at his/her disposal. Questions may be

 

referred at any time during the inspection to

 

the Inspections Task Force (ITF) in CBER's

 

Office of Compliance (301-827-6191). The ITF

 

representative will contact the appropriate

 

reviewer to provide information.

 

The following elements are suggested as

 

important aspects of inspecting a manufacturing

 

process from the point of view of viral safety.

 

1. In most cases, the validation studies have

 

undergone scientific review, or are in the

 

process of being reviewed by CBER staff, as

 

part of the license application. As such, a

 

detailed technical review by the

 

investigatorof the findings is generally not

 

necessary. However, the manufacturer

 

should be following the process exactly, as

 

approved or submitted for approval, and the

 

investigators should familiarize themselves

 

with the approved and pending procedures

 

in applications and supplements. As an

 

alternative, studies which support claims of

 

viral safety, either approved by or pending

 

approval by CBER should be available for

 

review in the manufacturer's license files,

 

on-site, for the product(s) in question.

 

2. Validation studies may have been conducted

 

by the manufacturer, but often the studies

 

are performed by a contract firm that

 

specializes in such studies. As such, the raw

 

data for the viral assays may not be available

 

at the licensed site. Available records may

 

consist of reports from the contractor.

 

Audits of these contract sites should be

 

performed periodically to review and verify

 

raw data generated during these, and other,

 

studies. Data available at the licensed site

 

should also be reviewed for integrity.

 

3. Examine the full-scale manufacturing step

 

itself to insure that it consistently meets the

 

critical validated operating parameters that

 

were defined in the viral validation study. In

 

addition, manufacturing records should be

 

reviewed to insure that the process is

 

carried out as intended. Some examples

 

follow:

 

Removal Methods:

 

a) Removal methods involving nanofiltration

 

are fairly new in the industry. The

 

membranes are generally single use

 

involving tangential flow. Insure that:

 

i. validated filtration parameters are

 

being followed, e.g. pressure and

 

flow rates; and

 

ii. the membrane is post-production

 

integrity tested. (NOTE: unlike

 

membranes used for microbial

 

retention, for some of these

 

membranes this is accomplished

 

using colloids such as dextran.

 

These tests may be destructive.

 

Forward flow and bubble point

 

methods are not applicable with

 

these membranes).

 

b) Chromatographic methods (e.g. affinity,

 

ion exchange) are used in purification

 

of many plasma derivatives. These

 

methods may also be validated for their

 

viral removal capabilities. Insure that:

 

i. validated parameters (e.g. flow

 

rates, column height, protein load)

 

are being consistently followed;

 

ii. if a validated number of uses has

 

been established for the column

 

resin(s), the firm is consistently

 

using the resin(s) for no more than

 

the validated number of uses

 

(NOTE: Often, concurrent

 

validation is allowed to establish an

 

acceptable number of times for use

 

of the resin. If this is the case,

 

in-process parameters should be

 

consistently monitored to insure

 

column performance. In addition,

 

the resins continued ability to

 

remove validated levels of virus

 

should be evaluated, periodically,

 

during the concurrent study); and

 

iii. regeneration procedures are

 

followed between each lot of

 

product.

 

Inactivation Methods:

 

a) Heat treatment processes, either

 

codified in the regulations or validated

 

by the manufacturer, should be

 

performed according to pre-established

 

parameters to insure effectiveness.

 

Insure that:

 

i. the proper time and temperature

 

parameters are met consistently;

 

ii. the equipment used is qualified,

 

maintained and monitored to

 

insure that these parameters are

 

met; and

 

iii. if problems occur, e.g., the process

 

is interrupted, the manufacturer

 

should not "repeat" the process

 

unless such repetition has been

 

validated and approved under the

 

license to demonstrate that the

 

additional heat treatment does not

 

adversely affect the properties of

 

the product.

 

b) Manufacturers who use

 

Solvent/Detergent methods must

 

demonstrate that the validated

 

parameters are met both during the

 

treatment and the subsequent removal

 

of the additives used. Insure that:

 

i. the proper concentrations of

 

additives are consistently used, and

 

treatment occurs for the validated

 

time.

 

ii. removal procedures are being

 

properly followed (e.g. If a column

 

is used, it is employed for the

 

validated number of uses or

 

monitored for performance (see

NOTE section 3 (b)(ii)); and

 

iii. the in-process drug substance is

 

tested to insure that the additives

 

have been removed to validated

 

levels.

 

Additional Considerations:

 

1. Inspect the Adverse Event Report/complaint

 

files and the procedures the company

 

has developed for handling them. Companies

 

are required to report incidents of

 

suspected transmission of viruses to FDA in

 

30 day periodic reports.

 

2. Establish that the firm has not changed steps

 

upstream of the validated viral removal step

 

without the proper reporting to FDA. This

 

may significantly change the characteristics of

 

the intermediate entering the viral

 

removal/inactivation step(s) and should be

 

submitted to the Agency per 21 CFR 601.12.

 

3. Insure that there has been no change in the

 

ordering of steps in the manufacturing

 

process. This may affect the effectiveness of

 

the viral inactivation/removal steps. These

 

changes also should be reported as specified

 

in 21 CFR 601.12.

 

For your information, sanitizing agents

 

(bacteriocidal and/or sporicidal) used in the

 

cleaning of the facility and equipment generally

 

serve to inactivate the viruses of concern for

 

plasma derivatives (refer to the Compliance

 

Program 7342.006 for further instruction on

 

inspection of cleaning of facilities and

 

equipment).

 

 

CONCLUSIONS

 

The establishment inspection provides a

 

critical element in assuring the viral safety of

 

plasma derivatives. Although scientifically

 

sound methods for removing or inactivating

 

viral contaminants may have been designed into

 

a production process, it is essential that these

 

methods be executed consistently and reliably

 

on a day-to-day and lot-to-lot basis in order to

 

provide assurance that the product is safe for

 

use. The establishment inspection is the

 

principle setting in which the operational

 

validity of viral clearance procedures may be

 

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