Protein Purification Process Engineering

Roger Harrison, Roger Harrison

• Publisher: Routledge
• Year: 2019
• ISBN:
  9781351421003
• Edition: 1
• Topic: Medicina
• Subtopic: Bioquímica en medicina

Offers coverage of the development of protein purification processes for large-scale commercial operations, and addresses process development, scale-up, applications and mathematical descriptions. Technologies currently used at the commercial scale are covered in depth.

1

Organization and Strategy

Roger G. Harrison

University of Oklahoma, Norman, Oklahoma

I. Introduction

During the development of a process to produce a protein, the initial emphasis of the work naturally is on the biological process. This focus on the biological process is often prolonged, because the time to develop this process can be lengthy. As a consequence, there can be a delay in shifting attention to the purification of the protein. However, it is important to realize that key organizational steps should be taken before experimental work on the purification process even begins, and that once the protein purification process development starts, the strategy to use in this development is crucial to the success of the project.

Several factors must be considered that relate to the organization of the work: The facilities and equipment must be appropriate for the job to be performed. The impact and applicability of the Current Good Manufacturing Practices (CGMP) regulations of the Food and Drug Administration (FDA) must be evaluated. The desirability of using a project team approach should be considered.

The complexity of most protein purification processes gives added importance to strategy considerations in the development of these processes. Purification processes for proteins nearly always involve more than one step and frequently involve a multitude of steps. Therefore decisions must be made about which individual unit operations to use and the order in which to use them. This effort is called process synthesis. The economics of the process should be evaluated at various times in the synthesis of the process in order to insure that the process is economically viable.

In addition to the strategy for the overall process synthesis, the strategy to apply in developing each individual process step is important. Four of these strategy considerations stand out, based on the author’s practical experience.

In this chapter, elaboration of these organization and strategy considerations is given.

II. Facilities and Equipment Considerations

Even before the actual process development work on purification begins, the issue of whether present facilities are adequate for the task needs to be addressed. Two situations need to be considered: laboratory scale work and pilot plant scale work.

For process development work at the laboratory scale, a good starting point is a typical protein chemistry laboratory. This would include a spectrophotometer with a UV lamp; a refrigerated centrifuge with centrifuging ability, expressed as relative g force times capacity in liters, on the order of 10,000–15,000; a wide variety of sizes of chromatography columns (glass or plastic) with adjustable plungers; a fraction collector with a UV monitor; a peristaltic chromatography pump; and a homogenizer for disrupting cells (1). Analytical equipment should include a system for analytical gel electrophoresis. In some cases, it may be highly desirable to have an analytical high-performance liquid chromatograph (HPLC) on hand for analyzing samples soon after they are taken.

Numerous operations for the purification of proteins need to be done at near 0°C to minimize proteolytic degradation and bacterial growth. The two options that arise for the lab scale are doing these operations in a refrigerated room and doing them in a chromatography refrigerator. The author has used only the latter option for lab process development work and found this to perfectly satisfactory. Chromatography refrigerators with glass doors, electrical outlets, and access portholes can be obtained with up to at least 75 cu. ft. of capacity.

The pilot plant is in essence a large-scale laboratory. Because of its larger scale, pilot plant equipment often must be constructed differently from laboratory equipment. Some equipment such as columns can be made of glass as in the lab. Other equipment such as vessels must be constructed of stainless steel or a plastic that has good chemical resistance, such as polypropylene. It is advantageous for each vessel to have its own pH probe for local and/or remote reading of pH. This is commonly done with an Ingold-type pH probe, which is made of glass and is capable of being sterilized. For applications involving food or pharmaceuticals, vessels should be of the “sanitary” design, which means that there are no threads on product contact surfaces and that surfaces must be smooth (150 grit or better finish). The sanitary equipment design standards usually employed are the “3A Sanitary Standards” that are published by the Journal of Food Protection, Ames, Iowa.

Pumps are needed in the pilot plant for a variety of operations. All should have sanitary designs and, in some instances, should be able to be sterilized. For low-pressure and very low flow rate applications such as feeding a chromatography column, peristaltic tubing pumps are commonly used. Three of the most widely used sanitary pumps are centrifugal pumps, positive displacement rotary pumps, and flexible impeller pumps; factors in the selection of these pumps have been discussed in detail by Horwitz (2). A less frequently used pump is the diaphragm pump, which has the advantage of being able to be sterilized. This can be obtained with a double diaphragm so that the pump’s hydraulic fluid will not contaminate the product when a diaphragm rupture occurs.

As to the means of keeping process liquids in the pilot plant refrigerated, one common practice is to use jacketed equipment with circulation of a suitable coolant (methanol-water, for example) through the jackets. However, pilot plants have been built with all or part of the equipment in cold rooms. Before using the cold room approach, the processes to be used should be analyzed to determine if the heat transfer will be adequate to maintain temperatures at near 0°C. Pilot plant processes that have appreciable heat generation will generally need to be done in jacketed equipment because the heat transfer coefficient for air in free convection is only a small fraction of the heat transfer coefficient for liquids in forced convection (3). Also, some operations such as precipitation with organic solvents must often be done below 0°C, which means that operation in a jacketed tank will be mandatory.

A number of utilities are needed in the laboratory and pilot plant. The ones required have been listed by Barrer (4). To provide flexibility in using equipment in the pilot plant, it is a good idea for utilities to be distributed to a number of locations, sometimes called utility stations, in the pilot plant. Each utility station contains appropriate outlets for many or all of the pilot plant utilities. If the equipment is modular and on casters, then a great amount of flexibility can be achieved in configuring pilot plant processes. The author both designed and used a pilot plant with utility stations and mobile equipment and found this concept to work out extremely well.

If solvents will be used in the pilot plant, then all the equipment should be designed to explosion-proof specifications. The National Electrical Code classification for electrical equipment and instrumentation is Class I, Group D for the solvents that would potentially be present in a pilot plant to purify proteins (5). If it is possible that solvents may be used at some point during the life of the pilot plant, serious consideration should be given to obtaining explosion-proof equipment initially because of the cost and inconvenience of converting non-explosion-proof equipment later.

III. Good Manufacturing Practices

The Current Good Manufacturing Practices (CGMP) regulations issued by the FDA can have a large impact on how the purification process work is organized, depending on the end use of the product under development. In general, the CGMP regulations cover the design, validation, and operation of a pharmaceutical manufacturing facility. CGMP regulations have been issued in the United States Code of Federal Regulations, Title 21, which is published yearly. The most widely applicable CGMP regulations are those in Part 211 of Title 21 that govern the manufacture of drug products for administration to humans or animals (6). There are supplementary CGMPs for the manufacture of medicated animal feeds (part 225), medicated premixes (part 226), blood and blood components (part 606), and medical devices (part 820).

The FDA has issued helpful guidelines on how to apply and interpret the CGMP regulations. In 1991 a guide for inspection of bulk pharmaceutical chemical manufacturing was issued (7). Bulk pharmaceutical chemicals (BPCs) are defined as being made by chemical synthesis, recombinant DNA technology, fermentation, enzymatic reactions, recovery from natural materials, or combinations of these processes. On the other hand, finished drug products are usually the result of formulating bulk materials whose quality can be measured against fixed specifications. Thus BPCs are components of drug products. This guideline states that “there are many cases where CGMP’s for dosage form drugs and BPC’s are parallel.” The guidelines goes on to say that “in most other cases it is neither feasible nor required to apply rigid controls during the early processing steps.... At some logical point in the process, usually well before the final step, appropriate CGMP’s should be imposed and maintained throughout the rest of the process.” A useful interpretation of these guidelines has been done recently by Moore (8).

In 1991 the FDA issued a very helpful guideline on practices and procedures for the preparation of investigational new drug products that constitute acceptable means of complying with the CGMPs (9). The FDA recognizes that manufacturing procedures and specifications will change as the trials of a new drug advance. However, when drugs are produced for clinical trials in humans or animals, compliance with the CGMPs is required. According to this guideline, this means that “the drug product must be produced in a qualified facility, using laboratory and other equipment that have been qualified, and the processes must be validated.” In contrast, the CGMP regulations do not apply for the preparation of drugs used for preclinical experimentation (such as toxicity studies on laboratory animals). Furthermore, like drugs approved for marketing, investigational drugs have always been subject to the FDA’s inspectional activities.

Each company that is developing processes for protein drug products must carefully design its process development laboratories and pilot plants to be capable of adhering to the CGMPs when required. It is highly desirable that some labs be designated as CGMP labs and others as non-CGMP labs. This type of designation can be done for pilot plants also. However, some smaller firms have only one pilot plant, and for this situation it is not a good idea to be switching back and forth between CGMP and non-CGMP use. It is better to stick with CGMP operation entirely to avoid confusion and to make sure that CGMP procedures are followed when they are supposed to be. Likewise a lab should not be switched back and forth between CGMP and non-CGMP use.

IV. Project Teams

Bringing a new protein to market is often extremely complex, involving a number of professionals from different disciplines who are involved in tasks that frequently must occur simultaneously. There is almost always pressure to bring the product to market as soon as possible. Competition with other companies is often intense. Furthermore, more time will be left on the product’s patent life if the time required to reach the market is relatively short.

A good way to coordinate the various activities in product development is by a project team. For example, the author was on a project team in Phillips’ Biotechnology Division for a new peptide with a molecular biologist, a microbiologist, an analytical chemist, and a marketing professional. This group met often to articulate goals, plan strategy, and discuss results obtained by group members. The project at Biogen on human gamma interferon for use as a pharmaceutical had a team of a group of individuals representing the laboratory research, regulatory affairs, quality control and quality assurance, clinical research, process development, and marketing functions of the company (10). This group met both as a complete team and also in smaller groups.

Since purification usually cannot begin until the protein has been synthesized, the question may arise as to when the purification professional should become involved in the process development effort. Strong arguments have been made that the scientists and engineers involved in purification scale-up should take an active part in the decision making from the start of the process development (11,12 and 13). One example involving recombinant products is the choice of the expression sy...