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Abhijeet Welankiwar*, Sushant Tope
Govt. college of Pharmacy Kathora naka
Amravati (Maharashtra) 444604.


The validation is a Fundamental segment that supports to a commitment of company towards quality assurance. It also assures that product meets its predetermined quality specification and quality characteristics. Validation of individual step of manufacturing is called as process validation. This Article concerns with the validation of biotechnological process. It is generally complex than validation of traditional synthetic or naturally occurring small molecules of drugs. Its level of complexity depends upon type of biotechnological products. The validation of biotechnological process has 3 Basic aspects they are Risk factors that are needed to be addressed, analytical tools necessary for validation and validation of unit operations.


As per USFDA Process validation is “establishing documented evidence which provides high degree of assurance that a specific process will consistently produce a product meeting its predetermined Quality specifications and Quality characteristics”.

The process validation is of 4 Types: -
1) Prospective validation
2) Concurrent validation
3) Retrospective validation
4) Revalidation

1) Prospective validation: - In prospective validation the validation protocol is executed before the process is put into the commercial use. During the product development stage the production process should be broken down into individual steps. Each step should be evaluated on the basis of Experience or theoretical considerations to determine the critical parameters that may affect the quality of finished product.

2) Concurrent validation: - It is similar to prospective, except the operating firm will sell the product during the qualification runs, to the public as its market price. This validation involves in process monitoring of critical processing steps and product testing which helps to generate documented evidence to show that production process is in a state of control.

3) Retrospective validation: - In this historic data is taken from the records of the completed production batches are used to provide the documented evidence that the process has been in state of control prior to the request for such evidence.

4) Revalidation: - It is the repetition of validation process or part of it. This is carried out when there is any change or replacement in formulation, equipment plan or site location, batch size and in the case of sequential batches that do not meet product specifications and is also carried out at specific time intervals in case of no changes.

Stages of process validation:
There are 3 Stages of process validation they are
Stage 1:- Process design or pre-qualification: The commercial process is defined during this stage based on the knowledge gained through development and scale up activities.

Stage 2:- Process Qualification: During this stage, the process design is confirmed as being capable of reproducible commercial manufacturing.

Stage 3:- Continued process verification: Ongoing assurance is gained during routine production that the process remains in a state of control.

*Biotechnological process validation:
Validation of biotechnology process is a complex task than validation of traditional synthetic or naturally occurring molecules of Drugs. The level of complexity depends upon type of biotechnological products the biotechnology products ranges from the synthetic oligo-nucleotides, monoclonal anti-bodies, synthetic peptides, and synthetic peptides to plasmids, recombinant DNA derived and transgenic proteins, Gene therapy vectors and some cell based therapies. More complex is the product more difficult the validation is this difficulty is linked to its inability to fully characterize the product and manufacturing process other factors which contribute to its complexity are known and unknown risk associated with sources of these biotechnological products. But there are many validation issues that are identical to that are associated with traditional pharmaceutical products includes facility and equipment qualification, validation of water and aseptic process and computer validation. Before beginning the validation of biotechnological process it is very important to evaluate inherent risk factors associated with product source, raw material, processing operation. Validation of biotechnological process has 3 basic areas they are:

1) Risk factors that are needed to be addressed during validation: Validation starts with the good process design which permits the reduction in  risk factors to an acceptable level  basically there are 2 risk factors which are:

A) Product source and raw materials:-
The sources of biotechnological products often include bacteria, yeast, mammalian cells newer sources includes insect cells, transgenic animals, gene therapy vectors, transgenic plants and other each of these posses well known as well as unknown risks. Bacteria and yeast have no  risk associated with the viruses and TSE(Transmissible spongiform encephalopathy) insect cells have potential risk associated with viruses, TSE, nucleic acids and proteins same is associated with mammalian cells, Transgenic animals, gene therapy viral vectors and cellular and gene therapy combinations. But bacterial sources like E.coli have potential risk associated with production of endotoxins, nucleic acids, and protein associated risks often includes cell proteins, aberrant protein products proteins used in cell culture and those associated with process (nuclease employed to reduce viscosity).

B) Processing risk: -
Variability in cell culture leads to an unexpected expression of an adventitious agent. Proteolytic degradation often leads to change in potency or formation of immunogenic products during recovery and purification operation. Variability in processing materials may lead to change in product quality. Leachables from chromatographic resins, filters and equipments components are toxic/may immunogenic. Build up of contaminants may occur with potential for unexpected release into product stream.

2) Analytical tools necessary for validation: Before the biotechnological process can be validated it is essential to validate the analytical methods that provide data that so that process can be understood as well as controlled. The task of analyzing the most of biotechnology process and product is complex. The most frequently employed analytical methods are:




Peptide mapping.



Mass spectrometry

Purity and impurities, molecular weight, glycosylation.



Purity and impurities, carbohydrate analysis.



Purity, impurities, glycoforms.



Potency, tertiary structure of proteins.


Western blot

Protein impurities.


Carbohydrate analysis

Glycoforms, carbohydrate sequence.



DNA, Viruses, mycoplasm.


Nucleic acid sequencing

Genetic stability.

Bioassays are the most important one because they permit direct and correct estimation of tertiary structure of complex proteins and the activity of even more complex biotechnology product. Another complex assays are the host cell protein assays the host cell proteins tends to vary within the cell culture conditions including scale of operation. The inability to pick up all host cell proteins is another problem faced in development and implementation of these assays.



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3) Validation of unit operations which are employed in biotechnology process: Fermentation and cell culture provides the necessary quantities of starting material. For every unit operation there are some validation issues. Some common features applied to all manufacturing steps. The processing time for each unit operation must be firstly defined and the process must be validated within these established time limits. Even the validation of stability is also essential. But the conditions which maintain the intermediate and product stability are ideal for the growth of micro-organisms. Even if micro-organisms are removed they leave potential toxins behind and other harmful substances which can cause product degradation. Temperature, PH, conductivity, product concentration, presence of impurities, processing time and product concentration are all the factors which affect stability and must be validated for intermediate, final bulk, and final product. Even the bioburden and endotoxin specifications should be established for each unit operation. Holding time specification cleaning validation are the important concerns for each unit operations. During the development and the preparation of validation batches firstly control parameters are optimized for each unit operation and then are tested by determining the outputs under worst conditions.

A)    Cell culture:- Is the first step for typical biotechnological product made form eukaryotes the validation parameters varies with type of cell culture lines used for e.g. in the case of Chinese hamster ovary cell lines which are widely used for the production of recombinant DNA derive proteins. The culturing process cannot be validated without firstly defining raw materials and characterizing the cells for which firstly Master cell bank, Working cell bank are prepared and characterized also these cell banks are tested at the end of production and the unprocessed bulk is also tested. The history of cell line, including previous exposure to animal and human substances, is also documented depending upon the information of source and history of cell a testing program is designed and implemented. In case of CHO cell lines characterization involves firstly the correct identification of cells isoenzyme analysis is commonly used for this even the banding cytogenetics provide the most sensitive identity test. Cells are tested for the presence of both endogenous and adventitious virus. These CHO cells have known to have retroviral particles electron microscopy and PCR has been used to detect retroviral load. The kinetics of cell growth, product formation rate, and total product yield is evaluated. The ability to maintain sterility during sampling of bioreactor is also validated. Here are some operating variables and acceptance criteria.

Operating variables

Acceptance criteria


Product content

Dissolved oxygen

Product quality


Cell viability

Motor speed

Cell density and



Of the entire unit operations the cell culture is the most variable .because cells may susceptible to slight variability in operating parameters and when the scale is changed, ph control, temperature, dissolved oxygen must be maintained. Another factor is changes in ability of bioreactor to stir that may include change in stirring mechanism along with the scale. Furthermore the cells also die at different times which result into variable protein impurities so relevant assays must be used in validation to detect these changes and changes of product quality.

B)    Isolation and recovery: - The recovery of biotechnological product involves the use of filtration, clarification and expanded be adsorption like operations. Even for bacterial cells the centrifugation, cross flow filtration, are used. All these operations must be validated within the predetermined specifications (no viable cells, and a defined particulate level) if the product is located within the inclusion bodies product extraction and removal of extraction solution must be validated. The extent to which the product is refolded must be defined. After the bulk harvest is isolated, Stability must be validated for hold period prior to further processing. Specification for the bulk harvest includes PH, conductivity, bioburden, endotoxin and protein concentration, product concentration.

C)    Downstream purification: - Chromatography and the filtration are the primary downstream purification steps. In general there are 3 to 5 purification steps are performed to achieve the required quality in protein product. The degree of purity depends upon product indication, patient population, and the risks associated with the impurities derived from the source material. Regarding the chromatography and filtration process the validation has following key considerations.

1)      Chromatography resins: - Chromatographic resins offer a great deal of surface area and provide clear understanding of separation mechanism. Cleaning and sanitization, as well as column life time are issues that must be addressed while validation. Purification process must be validated in such manner that next lot of resin will not have sufficient variability to cause batch failure. This is achieved by understanding the separation mechanism, how much impurities removed during purification and control parameters whose acceptance criteria met. Such control parameters include the product concentration, total protein concentration, feed stream volume flow rate, ionic strength, PH.

2)      Filters: - Filters used for clarification, sterilization, virus removal, exchange of buffers, removal of small molecules. Filter compatibility must be tested with process conditions to avoid nonspecific binding of product to the filter. Extractables must be defined and limits must be defined based on final product safety studies. Special considerations must be applied for sterilizing filters and those who are designed for the virus removal.

3)      Column packaging and storage: - while validating the biotechnological process the amount of resin pack in a column, its geometry, flow rates, pressure, and hardware of column and wetted materials of construction must be evaluated. Height equivalent to theoretical plate (HETP) and asymmetry determinations can be use to evaluate the quality of column packing, but may limited to some type of columns like on-off type, affinity type. The HETP/asymmetry factor are not relevant to the separation capabilities but can be indicative of other problems associated with the pack columns like the clogging or gross contamination. Specifications for the bioburden and endotoxins. Gas chromatography is also used to evaluate removal of ethanol, which is commonly used in shipping of chromatography resins. Total organic carbon is useful to assess the amount of leakage of resins and validate removal of leakage products prior to reuse of column.

4)      Process validation: - validation of each chromatographic and filtration process step requires the use of orthogonal analytical methods. For each step it is necessary to define the acceptable ranges for all the control parameters. Also a clear understanding of what each step is achieving is essential. Here are some operating variables and their forward processing criteria in validation.

Operating variables

Forward processing criteria

Total protein load

Product purity

Sample volume

Product Yield


Removal of specific impurities



Flow rates




Minor changes in the operating parameters can have major effects on removal of specific impurities. Changes in scale also require validation. Both chromatography and filtration are simple to scale up also shear is not a problem in chromatography but higher chances of it in filtration.

5)      Resin and filter reuse: - Packed columns are used repeatedly for the most of biotechnology process regulatory agencies have expressed that column performance decreases with continued use. So in industries employed resin life time studies both at the small and large scale. Validation of the ability to produce a consistent product for lifetime of the resin is essential. The small scale models are validated by determining the chromatographic performance (determined by the parameters like purity, yield, and removal of specific impurities) is same for both pilot and full scale. Flow and integrity tests are necessary to ensure the filter remains same after usage and cleaning.

6)      Cleaning and sanitization of columns and filters: - Cleaning validation of any component with large surface area can be problematic with the chromatography and TFF the concerns are carryover of product, degraded product, and impurities. These are not sterile process and there is potential for microbial organisms to be retained on the columns resins today the resins and filters are clean and sanitized by sodium hydroxide the techniques like PCR could be effective to validate these cleaning and sanitization operations of columns and filters.

7)      Leakage and Extractables: - leakage form chromatography resins and filters should be investigated. Most of the leakage occurs while use of harsh solutions used for cleaning and sanitization but leakage can also occur during the storage of resins. Validation of removal of leakage products from the product is necessary. This is particularly true when the affinity chromatography is used. The leakage product is usually a complex of ligand and product, which can be immunogenic. Although its amount is very small it should be validated that subsequent steps will remove any potential leachables to an acceptable level, which is determined by performing the risk assessment based on the product dose and indication as well as patient population.

8)      Validation of viral clearance: - it is a major concern for the products which are derived from the mammalian cell culture and the transgenic animals as well as from the viral vectors used for the gene therapy. Validation of viral concern has major concern because of the increased detection of type and the number of viruses that are used for the viral clearance studies. But this validation is not performed at full scale. Inactivation, filtration and chromatography are commonly used for this viral clearance. The inactivation studies often include low PH, solvent treatment, and heat treatment. Removal by chromatography is often leads to variability in operating parameters. Inactivation and filtration are considered to be as robust. Viral detection assays may be used to evaluate the viral clearance which necessitates the use of statistical evaluation. PCR can also be used regardless the assayed which are used there are many variables that must be controlled during viral clearance evaluation they are virus selection, virus titer, buffer interference, buffer cytotoxicity, scale down, suitable spiking and sampling points, freeze-thaw effect on enveloped viruses.

Validation of biotechnological process is a complex task than validation of more traditional synthetic or naturally occurring small molecule of drugs. Its level of complexity is governed by the type of biotechnological product. It requires good process development. Assessment and removal of the risk factors is necessary for the validation of these biotechnological processes and for continued manufacturing of safe and effective product the good process design helps to remove these risk factors. Also for the validation appropriate analytical tools and techniques are necessary. Every unit operation has their own issues like cell culture has high rate of variability so that must be needed to be addressed while validation. The operating variables must meet their acceptance criteria same is here applied to all unit operations.

[1]U.S. Food and Drug Administration. Guideline on General principles of Process Validation. Rockville, MD; May, 1987.
[2]Nash RA, Wachter AH. Pharmaceutical Process Validation. 3rd Ed. New York: Marcel Dekker, INC;1990. P. 252-274.
[3]ICH Q5C. Quality of Biotechnological products: Stability testing of Biotechnological/Biological products. (1995).
[4] Kathiresan k, Kiran k. Basics of validation- Pharmaceutical Perspective. 1 st ed. Chidambaram: K.K. Publishers; 2005. P. 32-46.
[5] ICH Q5A. Viral safety Evaluation of Biotechnology Products derived from cell lines of Human or Animal origin (1997).
[6] Chapman KG. A History of in the United States, Part I. Pharma tech 1991; 15(10): 82-96.



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