Page 1985 - Hematology_ Basic Principles and Practice ( PDFDrive )

C H A P T E R 116

PREPARATION OF PLASMA-DERIVED AND RECOMBINANT HUMAN

PLASMA PROTEINS

David B. Clark

The development of large-scale methods for the preparation of starting materials for further purification. Fig. 116.1 shows a sche-
human plasma proteins began more than 70 years ago, soon after the matic of the ethanol process.
outbreak of World War II. The US Armed Forces issued an urgent The first precipitate, fraction I at 8% alcohol, contains factor VIII,
request to the medical community for 300,000 units of human whole fibrinogen, and other poorly soluble proteins. Fractions II and III are
blood or plasma, which appeared to be an impossibly large amount precipitated together and contain the immunoglobulins, which are
at the time. Thinking that albumin could be used instead of plasma, separated in a subsequent series of precipitations to produce fraction
Dr. Edwin J. Cohn of Harvard Medical School drew together a task II, essentially pure immunoglobulins. Because many of the fraction
force of investigators who developed methods for the fractionation I proteins are removed in the cryoprecipitate, some manufacturers do
of plasma based on differential precipitation of various proteins with not produce a separate fraction I. Instead they collect a combined
ethanol. Although albumin was the only product distributed during fraction I + II + III. Fraction IV, produced from the supernatant of
the war, the remaining plasma fractions were carefully preserved, and fraction (I +) II + III, is sometimes produced in two subfractions.
other preparations, including fibrinogen and immunoglobulins, were Fraction IV-1 contains the vitamin K–dependent (VKD) clotting
soon developed. This was the beginning of the plasma fractionation factors, AT and α 1 -proteinase inhibitor (API), whereas fraction IV-4
industry. contains transferrin, haptoglobin, and some of the albumin. Fraction
V is almost pure albumin.

PLASMA FRACTIONATION
PRODUCT SAFETY
Beginning in the post-World War II era and continuing to the
present, major improvements have occurred in the preparation of Ensuring the safety of plasma products depends on a complex system
human plasma protein products. Most large-scale manufacture of that starts with donor selection and carries all the way through to the
plasma-derived products is still based on modifications of the original patient receiving the product. The system is highly redundant so that
method developed by Cohn’s group supplemented by more selective a failure in one area may be compensated for by another.
purification techniques to produce a wide variety of products. In
addition, genetic engineering technology has allowed recombinant
human plasma proteins to be produced in cell culture systems and Donor Selection, Screening, and Testing
transgenic animals. This chapter describes current methods and
future directions for the preparation of plasma-derived and recombi- Gone are the days when anyone could be a donor, with prisons and
nant human plasma proteins for clinical use, primarily for products mental hospitals providing much of the country’s plasma. Plasma
available in the United States. collection centers screen their potential donors rigorously, both for
Plasma is estimated to contain approximately 10,000 different medical history and any social behaviors that might put them at risk
proteins, most of which have yet to be identified. One of the unique of infection. Plasma products licensed in the United States are only
features of plasma fractionation is the ability to produce multiple produced from plasma collected from US donors in US Food and
products from a single raw material. Plasma for fractionation is Drug Administration (FDA)–licensed establishments.
derived from two sources, either directly by plasmapheresis, termed Every donation is tested for a number of different viral diseases
source plasma, or as a byproduct of whole blood donation, termed by a battery of tests, again with redundancy. Sensitive antigen and
recovered plasma. The plasma is usually shipped frozen as individual antibody tests are followed by NAT testing (nuclear amplification or
units from local blood or plasma collection centers to a central pro- nucleic acid testing, a form of polymerase chain reaction), which can
cessing plant. detect extremely small numbers of virus particles. Because of the
At the plant, sufficient units to produce typically 2000–3000-L “window period” between the time a donor is infected and the time
pools are thawed slowly at 1° to 5°C to produce cryoprecipitate, a antibodies or viruses can be detected in his or her plasma, most
cold-insoluble fraction that contains significant amounts of factor manufacturers also hold donations for at least 60 days until a donor
VIII, von Willebrand factor (vWF), fibrinogen, fibronectin, and has returned for a repeat donation. If the repeat donation still tests
factor XIII, along with a number of other proteins present in smaller negative, there is a high likelihood that the first donation is safe. In
quantities. The cryoprecipitate is usually recovered by centrifugation. addition to FDA oversight, many manufacturers and collection agen-
The cryo-supernatant or cryo-poor plasma may be treated with a cies belong to the Plasma Protein Therapeutic Association (PPTA),
chromatographic media to capture the factor IX complex or anti- which has strict quality regulations to help ensure the safety of
thrombin (AT) before it enters the Cohn process. There it goes donated plasma.
through a series of precipitations as the ethanol concentration is
increased in steps from 8% to 40% at specific combinations of pH,
ionic strength, protein concentration, and cold temperature. The Viral Inactivation and Removal Processes
precipitates and supernatants are separated either by the traditional
continuous-flow centrifugation or in large-scale filter presses. The Maximizing the safety of the incoming plasma is only the first step.
method provides both relatively pure fractions containing albumin Viral inactivation and removal methods are now incorporated into
and immunoglobulins, which need minimal additional processing, as all purification processes. One of the early methods, still used today,
well as fractions enriched in other proteins, which are used as the is pasteurization of albumin; otherwise, except for donor screening

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