Comprehensive Review on Manufacturing Process and Characterization of Biosimilar Drugs

According to the World Health Organization, the prevalence of deadly neoplasms, endocrine, blood and immune disorders would detect an extraordinary risk in coming decades followed by increased demand of affordable treatments. Keeping this incident in mind, the development of similar drugs is increasing globally. The sanction of such drugs depends on the conformity with international regulations, which starts with physical presentation of similarities that lie in their physiochemical and functional properties in contrast to the relating product. Consecutive clinical studies are done to physically present similar pharmacological behaviour and to curtail the distrust in relation to their safety points and effectiveness. Here we are to present a physical and biological testing of biosimilars and its reference product, by making use of different procedures and approaches, to judge the physicochemical and biological peculiarities with every possible impact it can cause on its pharmacokinetics, pharmacodynamics, and immunogenicity.


Introduction
Biological or biopharmaceuticals are drugs that are manufactured from living cells with the help of various biological processes and assume certain biological products and substances like hormones (Dranitsaris et al., 2011). Biosimilar drugs are simply the mimics of the original biological drugs that have been authorized, that's why they are similar but not exacts (Schellekens and Casadevall, 2004). The effective substance of Biosimilars medicine is very analogous to one of the biological reference medicine and is used in same quantity and treats the same disease (Roger, 2006). Biosimilars are entity based , regulatory based and market based (Duerden, 2007).
Biologics are way to complex as compared to other fields. The complex 3D structure of biologic products set on by production process. However, even minor changes in these processes can cause great changes in macromolecular structure and capability of biologic drugs. Even minor variations in these processes can cause tremendous affect. For example, impurity levels or level of material gathering and post-translational alterations that can lead to serious safety applications such as loss of effectiveness, abominable immunogenic responses (immunogenicity) and other adverse events. The European Medicines Agency as well as the FDA has recognized that bio similar products don't act identical to the original medicine version. They act differently (Schellekens, 2009), and have also published huge collection of regulatory archives (Cai et al., 2013).
Scientific advancements in recombinant genetic engineering techniques and refinements in manufacturing processes has made its path to success at a very great pace in the field where biological proteins are used in disease treatment (Cai et al., 2011;Tsiftsoglou et al., 2013;Lee et al., 2012;Nowatzke et al., 2011). Biological medicines play pivotal role in treatment for harmful and fatal diseases, such as cancer, hepatitis, autoimmune disorders, neurodegenerative diseases, and orphan diseases. But the treatment cost using biological medicine is costly as compared to a "classical" chemical or synthetic medicine (Chu and Pugatch, 2009). Biosimilars medicines are being launched in the market where the copyrights or patents of certain original biological drugs have terminated. This is a best way to release cheap alternatives in the market and bloom the competition. Like this these medicines are easily available and accessible for the patient easiness. There will be an economic benefit to health sector because of these alternatives. (Camacho et al., 2014).

Manufacturing Procedure of Bio Similar
 A biosimilar is medicine that is very similar to a sanctioned original product with respect to their structural and functional features and possess a very low degree of differences as compared to the original biological drug in terms of safety, purity, and capability.  Small molecule generics are produced through chemical synthesis using a manufacturing process that is done under controlled environment and one can generally predict the results coming (Oldfield, 2011), the production of biosimilar is complicated and the curative effects of the biologic molecule rely greatly on each individual step of the development process (Kuhlmann and Covic, 2006;Lee et al., 2012).  Biosimilars are developed from living cells through genetic engineering and their production relies on developed cell-expression systems and well-controlled manufacturing processes (Fig. 1).  Many of these steps require full development by the new manufacturer because the knowledge gained through the production of reference product is not publically available (Lee et al., 2012).  Production rooted from living cells results in a dissimilar mixture of parent drug, abridged chunks and structural isoforms, (Lee et al., 2012) and the products have an in-rooted sensitivity to posttranslational variations of the parent drug or fragments. (Lee et al., 2012;Tsiftsoglou et al., 2013;Oldfield, 2011;Kuhlmann and Covic, 2006;Cai et al., 2013;Wiecek and Mikhail, 2006).  The initial protein sequence of a biosimilar is exact to the reference product; there can be a little modification in 3-dimensional structure as a result of changes in the manufacturing process that will occur between products,

Analysis of Biosimilars
For the registration of Biosimilars drugs, "bio similarity" needs to be authentic between the physiochemical and biological frameworks of bio similar and originator clusters. In order to test the safety results and effectiveness of Biosimilars as compared with reference many medical, preclinical and, clinical studies must be done as per regulatory guidelines developed by EMA and the FDA. The development and validation of bio analytical methods is very essential (Phinney, 2014). Q5E ,and Q6B (Baber, 1994) provide well-explained guidelines on physiochemical and structural features that could be considered well fitting in the appraisal of comparability as listed below: This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT physiochemical parameters extremely sensitive to concentration and sample matrix. Isolation or purification of an active constituent from the reference formulation is carried out by extraction method; therefore, there could be possibility to a change in physiochemical or structural properties of active constituent and ultimately offers influence on comparability of product and process related impurities should also include in the evaluation of Biosimilar (Cai et al., 2013).

Pharmacokinetic (PK) Assays
The consensus within the industry that produce PK assays for biosimilar sequences is that one assay, when used, should be able to measure both biosimilar and originator components in biological matrices (Cai et al., 2011;Cai et al., 2013). These assays are obligated to quantify the biosimilar and reference products such that they have similar precision and accuracy in order to qualify for nonclinical and clinical studies. In order to fulfil these requirements, we should utilize a single analytical standard to develop a single PK assay that measures the biosimilar and reference products in serum matrix. (Colbert et al., 2014).

Immunogenicity Assay
Most programs that deal with the production of therapeutic protein utilize this assay that consists of three step assays; screening, confirmatory and bioassay that decide neutralizing antibodies (Swanson, 2007). Screening assay is highly sensitive, has moderate selectivity and high throughput (Koren et al., 2008). The confirmatory assay, on the other hand, is also highly sensitive but eliminates falsepositive results due to greater degree of selectivity. After passing through these two assays, the samples are then examined with a bioassay to separate out the samples that can neutralize the biological effect of the drug. It means that the biosimilar drug is equal, or less, immunogenic than the originator. The biggest advantage of using the 'one-assay' approach is that it does not have the 'between assay' variability introduced by the 'two-assay' approach (Cai et al., 2012). ELISA and electrochemi-luminescent assay (ECL) used to assess immunogenicity in comparison trials versus its references medicinal products.
Basic ELISA ELISA (enzyme-linked immunosorbent assay) or enzyme immunoassay (EIA), is a plate-based assay technique which detects and measures substances such as hormones, proteins, peptides and antibodies.
Basic ELISA is distinguished from other antibody-based assays in terms of its separation technique and measurement results. Specific and non-specific interactions are detached via serial binding to a solid surface which is usually a polystyrene multi wellplate. Its measurement results can also be attained (Fig. 3).
First of all, the antigen in a sample is immobilized to the wall of the wells of a microtiter plate by either using a "capture antibody" or direct adsorption to the plate's surface. The capture antibody, mainly used in a specific ELISA type called "sandwich ELISA", has to be specific to the target antigen. After this step, a detection antibody is added to form an antigen-antibody complex by binding to the adsorbed antigen. The detection antibody either provides a binding site for a labelled secondary antibody or is either directly conjugated to an enzyme, such as

Electrochemical-luminescence (ECL)
Due to high demands on purity, sensitive analytical techniques are essential in the biopharmaceutical industry. Electrochemical-luminescence (ECL), more sensitive and can analyse multiple impurities simultaneously and hence can possibly replace ELISA. The three constituents that are required for the detection reaction are; a molecule which has the ability to accept and donate electrons, an electrode and a reporter molecule which has the luminescent capability. The electron carrier in the system is tripropylamine (TPA) and the reporter molecule is a ruthenium (II) trisbipyridine-(4-methyl-sulfone) NHS ester, known as a SulfoTAG TM according to MSD. TPA donates an electron to the electrode while in vicinity to the electrode. Then TPA turns into a radical by losinga proton (H+) to the aqueous phase. Side by side, the SulfoTAG loses an electron to the electrode and then electron is transferredto it from the radical, which excites the SulfoTAG. A photon, with wavelength ~ 620 nm is emitted when it returns to the original energy state. (Yang et al., 1994, Lu et al., 2006.

Ion Exchange Chromatography
Ion chromatography (or ion-exchange chromatography) is a separation technique that uses the affinity to ion exchanger to separate ions and polar molecules. It is applicable on almost any kind of charged molecule like large proteins, amino acids and small nucleotides. There are two types of ion chromatography; anion-exchange and cation-exchange.
Huge amount of studies during the last decade describe charge profiles of proteins and ion exchange chromatography (IEX) is the standard procedure to characterize the protein charge variants (Lau et al., 2010). Molecules are separated based on their respective charged groups in this technique. Analyte molecules are retained on the column following coulombic (ionic) interactions. Molecules interact electrostatically with opposite charges on the stationary phase matrix which consists of an immobile matrix of charged ionisable functional groups or ligands (Fig. 4). This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT

RP-HPLC Peptide Mapping Analysis
It is the chemical or enzymatic handling of a protein to produce peptide fragments and then separate and identify these fragments in a productive way. For bio-therapeutic proteins and peptides, peptide mapping is detailed examination for the identification of minor and even isobaric differences in protein primary structure which include errors in the transcription of complementary DNA, point mutations. Peptide mapping by RP-HPLC is often used for quality control of therapeutic proteins. Since tryptic digestion generates a complex mixture of peptides (typically 25-100, depending on the size of the protein). Separation optimization is often carried out intuitively by altering both column length and gradient slope. A protein is selectively sliced by chemical digestion or enzymes (proteases) into smaller fragments in peptide mapping. The peptide fragments are typically analysed by gradient reversed-phase high performance liquid chromatography (HPLC) using an acetonitrile (ACN)/water gradient with added trifluoroacetic acid (TFA) after the digestion of protein. Critical traits of effective peptide mapping analyses are retention time precision and peak area precision.

Amino Acid Analysis
Amino acid was analysed by performing reaction with the hydrolysis peptide bonds through 6 M HCL, pre column derivatization followed by the o-phthalaldehyde and 9fluornyl methylchloroformate (FMOC-Cl) separated fluorescence exposure by RP-HPLC. Firstly samples were de-salted by PD-10 column (GE Healthcare). The internal standard mixture sample is hydrolysed by 6M HCL at 110C for 24 hour under concentrated pressure. Now dry hydrolysates under concentrated pressure, and transferred to HPLC vials by reconstituted in 50ml of 0.1M HCL. The HPLC ampoules were then full on the illustration plate of an Agilent 1200 HPLC system for automated derivatization of the unconventional amino acids. All accepted proline primary amino acid contained amino acid as derivatized OPA in presence of 3-mercaptopropionic acid. Amino acid was derivatizationnorcaline by using internal standard. By the conduction of RP-HPLC amino acid is analysed with the fluorescence detection. Using the standard mixture amino acid retention times and fluorescence response calibrated for the calculation of sample of the amino acid concentration, we used the units of pmol/ml a curve plot show for the response ratio vs from the respective calibration. The amount ratio and the related molar ratio to leucine was calculated (Jung et al., 2014).

Differential Scanning Calorimetry (DSC)
Differential Scanning Calorimetry (DSC) is a technique used to depict the stability of a protein or other biomolecule directly in its natural form. When heated at a constant rate, the heat change associated with the molecule's thermal denaturation is measured. Macromolecules and macromolecular assemblies (>5000 Daltons), such as proteins and nucleic acids can form well-defined structures that experience thermally-induced conformational changes. During these rearrangements, the heat is absorbed due to redistribution of non-covalent bonds. This heat is measured by differential scanning calorimeters. A biomolecule in solution is in equilibrium between its native (folded) and denatured (unfolded) conformations. Molecule becomes more stable with the higher thermal transition midpoint (Tm). DSC is used to measure the enthalpy (∆H) of unfolding resulted from heat-induced denaturation.

Size-Exclusion Chromatography (SEC-HPLC)
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic methodwhich involves separation of molecules in solution based on their size and molecular weight. Its speed and reproducibility gives it an edge over others for routine and validated analyses (Brange et al., 1992;Yu et al., 2008).The column used is filled with a porous material. Due to permeation deep into pores, smaller dissolved molecules flow more slowly through the column whereas large dissolved molecules flow quickly through the column because they do not enter the pores. As a result, larger molecules elute from the column earlier than smaller molecules and hence, the molecules are separated by size. This is the separation principle of size exclusion chromatography.

Agarose gel electrophoresis (AGE)
Agarose is a natural linear polymer. It is mined from seaweed and when heated in a buffer and allowed to cool, forms a gel matrix by hydrogen-bonding. For most applications, only a single-component agarose is needed without any polymerization catalysts. Therefore, agarose gels are simple and fast to prepare.

Agarose gel concentration
Size of fragments to be resolved determines the percentage of agarose used. The concentration of agarose is referred to as a percentage of agarose to volume of buffer (w/v), and agarose gels are normally in the range of 0.2% to 3% (Smith, 1993).The faster the migration of the DNA fragments, the lower the concentration of agarose.

Electrophoretic buffer systems
Buffers are an integral part of any electrophoresis technique because the effective maintenance of pH within the matrix determines the effective separation of nucleic acids. Moreover, the electrophoresis buffer's composition and ionic strength (salt content) affects the electrophoretic mobility of DNA (Somma and Querci, 2006). This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT bubbles, cool it for 20 mins and take off the combs and uncover the cello tape.

Loading Buffer
This buffer is added to the DNA fragment that has to be electrophoresed. To rise the density of the DNA solutions glycerol or sucrose is added, a part from that the samples goes into the tank i.e. running buffer and not descend into the gel patch. Xylene cyanol or bromophenol blue are the dyes accommodated in the gel loading buffer to enable the examination of the sample during loading of the gel and process of electrophoresis.

Voltage/current applied
Higher the voltage or current more rapidly the DNA drift, but too much high voltage or too much low voltage has a disadvantage. In the former, thin lines of different colors on the band appears particularly for the DNA whose size fall between 12≥15kb. In addition to that the temperature as well as the current of the buffer extends. This will ultimately deliquesce the gel. For standard size gel it is advisable not to exceed 5-8 V/cm and 75 mA or 100 mA for mini gels. In later case the movability of small (≤1kb) DNA is decreased and band expansion occur due to separation and dispersal.
Visualizing the DNA The DNA can be localized inside the agarose gel directly by using colorant with small concentration of interpolate strikingly bright Ethidium Bromide dye under ultraviolet light.

Procedure
The DNA sample is combined with the loading dye and filled into the well with caution by using capillary tube or pipetman. Now as the sample loaded in respective wells the negative terminal i.e. cathode is connected at the top end of the gel and that the positive terminal i.e. anode is connected towards the bottom of respective gel. The utmost volume loaded on to well-formed from 1.5 mm thickness tooth of the comb is 30 µl by switching on the D.C. power pack the process of electrophoresis starts and gel starts running at 5v/cm. as soon as the bromophenol blue dye reached 1 cm above the bottom end the power supply is disconnected and both the gel along with platform is stained in plastic tray containing 0.5µg/ml ethidium bromide in germ free distilled water. After 30-45 minutes the platform and gel is washed out with distilled water and the gel is gently inserted into the UV trans-illuminator by keeping the platform in oblique direction once the UV light is switched on DNA bands are seen and photographed at f 5.6 for almost 10 seconds using orange filter.

Surface Plasmon Resonance
The first biosensor developed almost a decade ago based on surface Plasmon resonance (SPR), the need of this technique has increased undeviatingly (Jönsson et al., 1991).
To study the molecular interactions surface Plasmon resonance (SPR) binding analysis methodology is used (Schuck, 1997;Ramakrishnan et al., 2006). To reveal the interactions of two different molecules (one is mobile and one is fixed on thin gold film) SPR which is an optical techniques is used. As described in the work, an amine coupling reaction is used to immobilize affinity purified fusion polypeptides on a sensor chip that is inserted into the flow chamber of a Biacore 3000 instrument. As the second polypeptide is added the flow through analyse to chamber cause adhesion to the immobilized polypeptide ligand, producing a slight change in refractive index at gold surface (Kretschmann and Raether, 1968), which can be measured with accuracy. Through the ratio of rate constants binding affinities can be achieved tends to give uncomplicated characterization of protein-protein interaction. Without the use of radioactive or fluorescent labeling of polypeptides SPR can detect mass concentration (Stenberg et al., 1991) before measurement, presenting a huge favor in reducing time and complication of the studies (Fig. 5).
In the fields of biochemistry, biology and medical sciences surface Plasmon resonance (SPR) become a significant optical bio sensing technology reason being real time, label free and noninvasive nature (Karlsson, 2004). SPR device is based on the Kretschmann configuration (Kretschmann, 1972) and composed of the following components: light source, prism, gold film, and detector. A testing cell adheres to the gold film and may be filled with different solutions to be studied. When a light beam spread in the prism and experience the interaction of the gold film and the solution, TIR takes place and the fading wave forms as long as the incident angle is greater than the critical angle. Normally, the intensity of the reflected light does not change with the incident angle under the condition of TIR. However, at a specific angle larger than the critical angle, the fading wave exhilarates the delocalized electrons or Plasmon's of the gold film, causing SPR; the intensity of the reflected light becomes smaller intensely at this point. SPR angle is defined as the incident angle at which the minimum reflectivity is observed (Tang et al., 2010).

Protein Quantification by Mass Spectrometry
The very first stage to characterize the proteins structurally is the determination of molecular weight. There are two types of light Ionization Techniques, MALDI (Matrix-Assisted Laser Desorption Ionization and ES/ESI (Electrospray Ionization). As compared to ESI-MS, the MALDI-MS technique analyses intact proteins or peptides which contain greater amount of impurities (e.g., salts). MALDI causes fragmentation of the protein during ionization that can be used to get more information about the fragments. This technique requires sample to be placed in matrix that absorbs appropriate wavelength light. Matrix generates heat and forms ions of analyte and what is around it. The mass measurement precision and accuracy can be Y. Mehmood (2018) Int. J. Appl. Sci. Biotechnol. Vol 6(2): 53-62 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT further improved by using all the observed multiply charged ions (typically better than 0.01% for masses up to 100 kDa) (Smith et al., 1990). Two different approaches are applied to determine the sequence of a protein (Fig. 6): bottom-up (Henzel et al., 1993) and top-down (Ge et al., 2002).

Amino Acid Analysis
Hydrolysis of peptide bonds with 6 MHCl was performed which was trailed by pre-column derivatization using ophthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC-Cl), separation by RP-HPLC, and fluorescence detection in order to carry out this analysis. Columns were first used to de-salt the samples and then aliquoted into hydrolysis tubes containing internal standards. Hydrolysis of the sample/internal standard mixtures was then carried out under reduced pressure using 6 MHCl for 24 h at 110C. The hydrolysates were dried under reduced pressure, re-formed in 50ml of 0.1 MHCl and transferred to HPLC vials. For deriving liberated amino acids automatically, the HPLC vials were then laden onto the sample tray of HPLC system. Derivatization of amino acids containing a primary amino group (all except proline) was achieved with o-phthaldialdehyde (OPA) in the presence of 3-mercaptopropionic acid. Proline was derivatized by reacting it with FMOC-Cl. Norvaline was utilized as the internal standard used for amino acid derivatizations except for proline derivatization in which sarcosine was employed. RP-HPLC with fluorescence detection was utilized to analyse the amino acids. A standard mixture of amino acids helped in calibrating retention times and fluorescence responses were calibrated. Using plots of the response ratio vs. the amount ratio shown from respective calibration curves, we calculated the amino acid concentrations of samples in units of pmol/ml. Then we calculated the molar ratio with respect to leucine.

Sterility Testing
To carry out this testing, aseptic conditions were frequently observed by suitable selection of the working area and appropriated controls as stated in on GMP documents (24,1995).
Test article under sterile testing should be monitored for the traces of elements that will affect the microorganisms' development within the growth media under analysis. This testing is usually known as the Bacteriostasis/Fungistasis (B/F) test or sterility test validation, qualification or verification [10]. Given sample type is passed through the method suitability test only once if no additional changes occurred to the product or any formulation or manufacturing process did not take place. However, it could be executed frequently to confirm no significance in the product or process affecting the sterility assay results (24,1995).

Safety Pharmacology
Drugs or biomaterials can affect the functioning of vital organ systems, which includes the cardiovascular and central nervous systems. Evaluation of these effects before human exposure is necessary either studying them separately or as additions to toxicity studies. Vitro tests to determine the biological and pharmacological activity at the cellular level are often followed by safety tests in animal systems. Generally, there is no need of standard safety pharmacology studies (as commonly conducted for pharmaceuticals) for biotechnology-derived products. Expected and unexpected pharmacological effects of the test materials are outlines in these studies based specifically on factors related to required clinical activity.

Duration
Adjustable -dependent on the test system

Test System/Animal System
In Vivo: selected pharmacologically relevant species; if there is no previous information or there is difficulty in deciding the species relevance, more than one species is specified. In Vitro: relevant animal species are used to extract cell lines; may forecast the optimal species most suitable for in vivo studies. Dose Administration Variable, dependent on the likely effective dose range and test system.

Parameters
In Vivo: Safety studies may be mandatory for the following systems and specific target organs: 1. Respiratory (lungs and bronchi) 2. Gastrointestinal and hepatic (esophagus, stomach, intestines, liver) 3. Renal (kidney) 4. Blood 5. Cardiovascular (heart and blood vessels) 6. Nervous/neurobehavioral (CNS and behavior) 7. Endocrine (thyroid and other endocrine glands) In Vitro: 1. Biological activity at cellular stage can be easily evaluated on following criterion 2. Receptor occupancy 3. Receptor affinity 4. Binding and transport kinetics 5. Production and or secretion of specific proteins in response to test material (for example, antibody production in response to test antigen) 6. Other pharmacological effects on cellular function.

Inactivation Test
Every individual purified bulk material is to be tested on mice for effective inactivation of the virus before preservatives and other substances are added and induced. The test should be performed with undiluted purified bulk material injected into the cerebrum into at least 20 mice, but each should be weighing between 15 and 20 g. These mice shall be kept under observation for about 14 days. Any symptoms caused by the virus shall be observed by Immuno-florescence evaluation procedures. At the end of the observation period, no cytopathetic effects should be observed.

Considerations for phase III clinical comparability trials
The purpose of Phase III clinical comparability trial is to eliminate defects in relation to the reference product following completion of physicochemical, biologic, and preclinical investigations, as well as PK, PD and immunogenicity investigations in humans (Food and Administration, 2012). It also depends on the available history of safety measures with the reference product may lead to extensive phase III clinical investigation of the biosimilar (Food and Administration, 2012).
In order to carry on biosimilar developments, phase III clinical equivalency trials should physically show that whether there is an increased or decreased activity in relation to reference product. (Food and Administration, 2012). The goal is to demonstrate that any level of variation in efficiency or safety between the biosimilar and reference product is less as compared to the standard "clinical equivalency" margin (Dranitsaris et al., 2013). Such an equivalence margin is the foundational crux of the trial and is based on the historical difference noted between the treatment effect or the reference product versus placebo (Kay and Smolen, 2013). Trial designs for biosimilar development are not unlike designs for any other biologic product with similar considerations of patient population, sample size, endpoints, and study duration.

Conclusion
Extensive structural and functional comparison of biosimilars and reference products is the foundation of biosimilar development. The comparison exercise must be scientifically tailored using state-of-the-art analytical tools and sensitive tests to detect small product-related differences between the biosimilar and the reference product. Structural differences determine the amount of biological, non-clinical and clinical studies that are needed for development of biosimilar proteins. The primary aminoacid sequence must be the same for the biosimilar and the reference product in Europe and in the USA. As review edhere, a number of biosimilar techniques are mentioned. As highlighted here, the correct technique selection results in saving significant time and money.