CHAPTER 3

Essential Product and ProcessParameters in Summary

3.1 General Considerations

During the process development stage, a number of variables need to beconsidered; they include

� formulation details, i.e. solution composition and concentration� solution fill volume� type of container, method of container closure

The physics and chemistry of freezing show many complex and oftensurprising features, but the industrial freeze-drying process is limited tovery few operational degrees of freedom. Thus, for a given solutioncomposition and a given container and closure system, only three processvariables exist by which direct control over the drying cycle can bemaintained and which, therefore, determine the quality of the final driedproduct. They are

� shelf temperature� chamber pressure� time

On the other hand, the most important factor is of course the producttemperature and its change throughout the duration of the process. Itsmeasurement and control are discussed in more detail in several followingchapters.

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3.2 Formulation Parameters

In the manufacture of a therapeutic product, the initial stage requires thepurification of the biologically active component, the drug substance.Purification methods for ‘‘conventional’’ drugs are well established. Theyare based on synthetic organic chemistry and standard analytical tech-niques. The situation is more complex for biopharmaceutical products, inparticular those based on proteins. The drug substance may then be ofanimal, plant or microbial origin; it may be obtained from the naturalsource materials or by recombinant DNA (rDNA) methods. The extrac-tion and purification strategy depends to some extent on the source of theprotein. Possible impurities are many, as shown in Scheme 1. The down-stream processing always requires several stages and, depending on thesource material, may involve a combination of physical and chemicalmethods. A generalised flow sheet for the isolation and purification ofrDNA proteins (r-proteins) is shown in Scheme 2.The total yield of purified protein is related to the number of stages

required and their individual yields. For instance, in an isolation andpurification process consisting of n steps, each one of which can becarried out with a 90% efficiency (highly idealised!), the final percentageyield of product is given by

Yield (%) ¼ 9n � 10(2�n)

Thus, for a process consisting of five stages, each of which can beperformed with a 90% recovery, the yield of the final product cannot

Scheme 1 Types of impurities commonly found in crude protein extracts and that have tobe removed during downstream processing

21Essential Product and Process Parameters in Summary

Cytoplasmicsolubleinclusion bodyperiplasmic

Broth deactivated

Cell harvestCentrifugationMicrofiltration

Product realeaseMechanicalEnzymaticChemicalOsmotic shock

Inclusion bodyRelease & cleanup

Speparation of cellsfrom medium

SecretedPerfusionBatchEukaryoteProkaryote

ClarificationEnrichmentPrecipitationUltrafiltration

DenaturationUnfolding withurea, guanidine:reduction

RefoldingSH/SS exchange

Production concentrationChromatograpy

Membrane concentration

PurificationCation/anion exchangeAffinity chromatography

Hydrophobic chromatography

PolishingGel filtration

Crystallisation?

contained

Active drug toformulation/dosage form

STABILISATIONFreezingLyophilisation

Scheme 2 Typical downstream process flow sheet for r-proteins

22 Chapter 3

exceed 60%, which is reduced to 53% for a six-stage purificationprocedure. Since biopharmaceutical drug substances, whatever theirorigin, are usually expensive, a loss of 40% during the purificationprocess is a serious factor. A minimisation of the number of stages istherefore aimed at, but it has to be judged against other economicconsiderations and an acceptable degree of purity.In the manufacture of proteins for applications other than as thera-

peutic products, e.g. industrial enzymes, purity criteria are not as stringentas they are for parenteral products, in which case acceptable yields may beobtained with fewer fractionation and purification steps. For biochemicaland therapeutic uses, however, purity and long-term stability are theoverriding requirements. Fractionation, purification and stabilisation byfreeze-drying then generally account for 50% of the total productioncosts, but since such products command high premiums in the marketplace, production hardly figures in the cost equation. Examples of thetypical production cost breakdown for two product/process scenarios areshown in Table 1. The data illustrate that the cost contribution allocatedto freeze-drying is almost insignificant, compared to the cost of thepurified ‘‘raw’’ drug substance, assumed to be a protein.Before a purified protein, usually in a dilute aqueous solution, is

subjected to the final stabilisation procedure, e.g. by freeze-drying, it hasto undergo a process of compounding, loosely referred to as ‘‘formula-tion’’. Very few proteins can survive freeze-drying without the aid of theso-called lyoprotectant additives, which serve to ensure the recovery offull biological activity of the protein at the point of use, whether in thedry solid state or reconstituted in an aqueous medium. The science andtechnology of lyoprotection, as employed in freeze-drying, is onlygradually being put on a quantitative basis; several unresolved mysteriesremain (see Chapters 7 and 11).Apart from lyoprotectants, other substances are also commonly

added to the protein solution prior to drying. Some may also have been

Table 1 Economics of freeze-drying: production cost breakdown (excludinglabour) for two typical biopharmaceuticals

Cost componentsProduct I: % cost Smallvials, 24-h cycle

Product II: % cost Largevials, 4-day cycle

Pure drug substance 73 65Solution manufacture 7 10Containers 3 3QC assays 8 7

Source: M.J. Pikal (unpublished data).

23Essential Product and Process Parameters in Summary

carried over in the solution during downstream processing. They mayinclude pH buffers, surfactants and salts. A common additive, phos-phate-buffered saline solution (PBS) ensures the isotonicity of a recon-stituted parenteral product in water, prior to injection. Although from apharmacokinetic standpoint some of the additives in common use maybe innocuous or even beneficial, their presence in the solution mayincrease technical problems that can be encountered during freeze-drying. For instance, the presence of PBS will inevitably require alonger freeze-drying cycle than would be required for the same solutionin the absence of salts. The omission of phosphates and NaCl from theinitial solution is therefore helpful, since it allows for a shorter dryingcycle. On the other hand, reconstitution must then be performed withPBS solution, rather than with sterile water (‘‘water for injection’’),apparently a distinct disadvantage from a marketing standpoint.

3.3 Process Parameters

As already pointed out, the primary process parameters by means ofwhich drying can be directly controlled are shelf temperature, chamberpressure and time. In principle, the condenser temperature can also beadjusted, but in practice it is usually set to the lowest possible value, soas to maximise the ice sublimation rate. Since it is the temperature of thematerial that determines the drying rate, the control over the process canonly be indirectly maintained, usually by coupled adjustments, eithercontinuous or ramped, of the shelf temperature and/or the chamberpressure throughout the drying cycle.Once the process cycle conditions have been decided upon and the

vials have been loaded, the process is automatically controlled by acomputer. The freeze-drier performance is monitored, and a sample of atypical output sheet is shown in Figure 1. It records the time course ofthe production cycle (48 h in this case) in terms of shelf, condenser andproduct temperatures and chamber pressure. As mentioned earlier, theonly directly adjustable parameters are the shelf and condenser temper-atures, and the chamber pressure. We shall defer a detailed discussion ofthe information contained in the chart to several later chapters.

3.4 Multidisciplinary Nature of Freeze-Drying

As will become clearer in the following chapters, the successful appli-cation of freeze-drying is based on a complex interplay of several

24 Chapter 3

scientific principles, some of which are still very poorly explored andhardly appear in university undergraduate curricula. In the author’sexperience, one rarely finds managers or technologists with an overallbroad view of the various stages of freeze-drying. Thus, the trained

Figure 1 Freeze-drier recorder output of condenser temperature (bottom, blue), shelftemperature (black), product temperature (green) and chamber pressure(red). Ordinate scales (from top to bottom): 1 and 2 ¼ bar, 3 ¼ 1C, 4 ¼ mbar

25Essential Product and Process Parameters in Summary

chemist’s experience differs from that of the physicist, the materialsscientist or the chemical engineer. And yet, an overall appreciation ofhow these disciplines interact is of the utmost importance. In addition,experts in the various disciplines within a company will usually be foundin different divisions of a line management structure, a custom notconducive to the fostering of the necessary collaboration.This book aims to highlight the importance of a multidisciplinary

approach to freeze-drying. Nonetheless it has been found necessary,although not really desirable, to separate physical, chemical, economicand engineering and other aspects into different chapters. The followingparagraph is intended to provide a summary of the important disci-plines, some knowledge of which is essential for the development of asuitably formulated product, coupled with a sensible process cycle. Theterms printed in italics are those that are of importance and requireunderstanding by the practitioner.Pharmacokinetics deals with all aspects of the therapeutic effect of

the chosen drug substance, suitably formulated, on its target. It formsthe very basis of the formulation and processing stages. Of secondhighest importance is the formulation stage. In the case of biopharma-ceuticals based on proteins, some experience of protein biochemistry andtechnology is absolutely necessary. The formulation process aims toprotect the bioactive substance from the severe stresses to which it willbe exposed during any drying process, including those directly due toconcentration increases during freezing. The freezing process itself isgoverned by ice nucleation and crystal growth rates in supersaturatedsolutions. It is accompanied by freeze concentration of the solution, aprocess that may result in both crystalline or amorphous solids, or inmixtures. A correct identification of the physical and chemical identitiesof the product during and after processing requires an understanding ofthe analytical techniques available for obtaining the necessary infor-mation, also for an eventual submission to the relevant regulatorybody.The ice sublimation stage depends on the balance of heat and mass

transfer. Secondary drying depends on the diffusion rate of waterthrough the solid matrix. The long-term stability is governed by thematerials science of crystalline and amorphous solids, and it is measuredwith the aid of an array of physical analytical techniques, such as thermalanalysis, X-ray diffraction, spectroscopy and chromatography. For regis-tration purposes, all process and analytical data must be submittedin the required formats, with product identity, purity, safety, stabilityand shelf life playing major roles in the submission for regulatoryapproval.

26 Chapter 3

3.5 Conclusions

In small teams where good communication between individual staffmembers is fostered by imaginative management, it is possible for thenecessary knowledge and experience to be passed on and becomecommon knowledge, so that high-quality freeze-drying can be per-formed. The complex nature of biopharmaceutical freeze-drying makesit unlikely that this can be achieved in larger institutions, because mostline management structures inhibit the flow of information betweengroups devoted to formulation and those whose objective it is to deviseeconomical drying cycles. This was brought home to the author when hisconsultancy work took him to a German company that undertook thefreeze-drying of human blood products on a large scale, but withvariable results. During a round-table discussion, it became apparentthat those pharmaceutical chemists who were responsible for formula-tion development had never met or spoken to their engineering col-leagues, who were responsible for the freeze-drying process. Indeed, for‘‘security’’ reasons, the latter were not informed of the nature of theproducts that were to be freeze-dried. This was a scenario that ourtechnologists also encountered in a number of other mega-pharmacompanies. Usually, even the mention of ‘‘glass transition’’ or its meas-ured value met with blank stares. The complexities were not appreciated,the freeze-drying operation being regarded as a push-button affair,hardly a recipe for success.

27Essential Product and Process Parameters in Summary