It is important to show that biogeneric drugs are comparable in structure and function to that of the innovator and any differences have to be supported with data showing no influence on these parameters.
India’s rising prominence in the field of biogeneric products (off-patent biopharmaceuticals) also known as Biosimilars (in Europe) or Follow-on-biologics (in US) is evident from the large number of biogeneric products getting marketing approval. A number of biopharmaceutical protein-based products are due to go off-patent in the next few years which will bring more biogeneric brands to the market.
Usually biopharmaceuticals are manufactured through a complex process of fermentation and multiple purification steps; making them more complex and susceptible to alterations as a result of variations in the process. These alterations, even if small, can influence its structure and function significantly. Product quality, safety and efficacy of biopharmaceuticals are highly dependent on the process of production, purification and formulation and subtle differences are often observed between the innovator’s product and biogenerics. Even seemingly small changes in the structure can have significant effect on the drug’s safety and efficacy parameters. Therefore, it is important to show that biogeneric drugs are comparable in structure and function to that of the innovator and any differences have to be supported with data showing no influence on these parameters.
Competition amongst biosimilar players in India
India has, by far, demonstrated the greatest acceptance of biosimilars which is reflected from over 50 biopharmaceutical brands getting marketing approval (Table 1). Biosimilars have established a good reputation among healthcare professionals. Doctors and hospitals are prescribing biogenerics immediately after their launch with interchangeability and substitution among brands being freely practiced.
In recent years the focus within the Indian biopharmaceutical sector has been mainly directed towards the development of biogenerics. This is primarily because it requires much lower R&D spends and time to market, coupled with the capability of Indian biopharmaceutical companies to reverse-engineer the drug development process.
The lower price of biogeneric compared to the innovator’s drug remains one of the key marketing forces. There is a fierce market competition; forcing these companies to drop their prices too fast and too soon, within two to three years of market launch. This price-sensitivity has led to companies not fully observing quality parameters that are essential for biogeneric products. The comparability with the innovator drug is often poor. There are differences in the physico-chemical properties of the protein, use of different host cells for manufacturing, the process used for purification and formulation are different, composition, strength and the level of impurities and container-closure systems. These differences (form the innovator’s product) cannot guarantee that the product will be bioequivalent to the innovator’s product as such differences are known to have serious implications on the drug safety and efficacy.
This article is based on a blind study carried out under careful conditions to look at the differences among various biogeneric brands in comparison to the innovator’s product used as a reference.
Blind comparative study
A comparative blind study was carried out at Intas Biopharmaceuticals Ltd. (IBPL) with the objective of comparing quality parameters such as identity, purity, content and efficacy of various biogeneric brands taken from the Indian market and comparing them with those of the innovators’ drug products. The study was carried out on sixteen commercial brands covering three different biopharmaceuticals, viz. two brands of rHu PEG G-CSF, seven brands of rHu G-CSF and seven brands of rHu Erythropoietin (Table 2) picked from the market. IBPL products were also picked from the market to harmonise any shipment-induced effects and to bring all samples on parity. Precaution was taken at the time of procurement that the age difference between different samples should not be significant. All brands were collected from a single Clearing & Forwarding (C&F) agent to preclude any effects of variation in storage conditions. Samples (including that of IBPL) were shipped using a validated cold-box and a validated cold chain. On receipt at the testing centre, they were verified free of any physical damage. Before analysing them in the laboratory, all samples including the innovator’s product were transferred to sterile microfuge tubes and labelled with a unique code to conceal its true identity. Data was interpreted by comparing all quality parameters with the innovator’s product after decoding. During the analysis, in-house reference standards made for NIBSC or innovator’s product were also used.
The coded samples were blind tested simultaneously by a qualified analyst using validated test methods and instruments.
Comparison among different brands of rHu-PEG-GCSF
This part of the study focusses on the comparison between Neupeg (Manufactured and marketed by IBPL) and Neulastim (innovator’s product marketed by Roche India) with respect to identity, purity and efficacy. The active ingredient of Neupeg and Neulastim is Pegylated Filgrastim [Granulocyte-Colony Stimulating Factor (PEG-GCSF)], which is a 175 amino acid protein conjugated to polyethylene glycol for a total molecular weight of ~39 KDa. The protein is produced by recombinant DNA technology in Escherichia coli. Both the products consist of similar excipients i.e. Sodium acetate, Sorbitol, Polysorbate 20, Sodium hydroxide and WFI except that Neupeg has a drug concentration of 6.0 mg / ml compared to Neulastim, which has 6.0 mg / 0.6 ml. Before analysis Neulastim was diluted to 6.0 mg / ml with the same formulation buffer before assigning codes and testing blind. The quality parameters analysed and the analytical methods used are given in Table 3.
P5/CA/01 and P5/CA/02 were found to be similar with respect to Specific Activity (P5/CA/01 =1.39 x 108 IU / mg and P5/CA/02 = 1.44 x 108 IU / mg); this value represents the average of five independent replicates performed over a period of three days, identity by Peptide map (Figure 1), SDS-PAGE (Figure 2) and Western blot (Figure 3).
Comparison among different brands of rHu-GCSF
The active ingredient of Filgrastim is a 175 amino acid protein (human Granulocyte-Colony Stimulating Factor) with a molecular weight of ~18.8 KDa, which is produced by recombinant DNA technology in Escherichia coli. All brands had the same strength (i.e. 300 mcg / ml) but some had lower fill volumes. Volume was made up to 1 ml with formulation buffer prior to coding of samples and blind testing.
1. Each sample of rHu G-CSF was visually tested. All samples appeared clear and colourless consistent with the specification except sample P1/CA/04 which showed white fibre like particle in one vial out of three vials.
2. Product identity was tested by SDS PAGE (Non Reducing & Reducing) (Figure 6 and 7) and western blot analysis (Figure 8). All samples complied with the specifications i.e. principal band of all brands matched the expected molecular weight of rHu-GCSF (~18.8 kDa) and was matching in position with the principal band of the reference standard. Identity of rHu G-CSF was also verified by size exclusion chromatography (Figure 09A & 09B) and RP-HPLC (Figure 10A & 10B) by comparing retention time of the main peak with that of the reference standard. All samples showed similar retention time on size exclusion chromatography and RP-HPLC.
3. There were significant differences in the protein concentration among brands. Only four brands out of seven were found within ± 10 per cent of label claim. Variation in the protein concentrations among various brands is shown below.
4. During determination of biological activity by cell proliferation assay, significant variation in the specific activity was observed as shown below.
Note: Specific activity was calculated by labelled protein concentration.
5. Relative impurity level of all products with respect to high molecular weight impurities was determined by size exclusion chromatography (Quantitative) results obtained (on percentage impurities scale) are as follows
6. Levels of low molecular weight impurities were determined by semi-quantitative reducing SDS-PAGE (Figures 6 & 7) and results are as follows:
Erythropoietin is a 165 amino acid protein with a molecular weight of ~30.3 KDa, produced by recombinant DNA technology in Chinese Hamster Ovary cells with 40 per cent of the molecular weight contributed by the sugars.
Erythropoietin is heterogeneous glycoprotein hormone, consisting of several different isoforms, primarily due to variation in the type and extent of glycosylation and sialic acid content. The structure of carbohydrate in Erythropoietin plays an important role in determining the circulatory half-life of the drug. The presence of more basic or more acidic isoforms has been shown to reduce specific activity. Additional basic isoforms are also associated with a shorter high life.
Results and Discussion
Given the complex nature of biopharmaceutical products, there can be differences in the quality, safety and efficacy among various brands of biogeneric products. However, these differences can be titrated to a low level by using appropriate analytical tools, applying the concept of Quality by Design, use of orthogonal methods for analysis of in-process material, drug substance and finished product, risk-based approach to product development and characterisation of the process and the product through extended comparability testing using innovator’s product as the reference standard.
In absence of any overarching regulatory guidelines for biogeneric products, Indian biogeneric companies are not following uniform measures to establish comparability with the innovator’s product. With the recent introduction of product-specific monograph for six recombinant proteins (viz. EPO, G-CSF, HBsAg, Interferon-alfa, Factor VIII and Streptokinase) in the Indian Pharmacopoeia, any company marketing these products in India shall be required to meet the quality parameters as laid down in the pharmacopoeia.
Although physico-chemical comparability is not sufficient to establish bioequivalence and identical safety and efficacy profile, it is the least a biogeneric company must adhere to in order to ensure that their products have comparable efficacy and safety as that of the innovator’s product. An interesting observation was that all biogeneric samples of EPO had potency values higher than the innovator’s product, partly attributable to higher amounts of protein content, presumably because overages are permitted in India. Two samples (P9/CA/01 and P9/CA/07) showed greater than 0.5 per cent aggregates. Higher levels of product-related impurities (such as aggregates) are highly undesirable as they are known to be immunogenic. Similarly non-comparative isoform profile (e.g. in the case of EPO) can affect in vivo efficacy and immunogenic response.
Worldwide, there have been over 188 cases of Pure Red Cell Aplasia (PRCA)—a rare adverse event known to occur in patients receiving EPO, many of which were subsequent to the manufacturing changes introduced by Johnson and Johnson for its product EprexTM, (which included a formulation change that was free of Human Serum Albumin (HSA), use of uncoated rubber stoppers and change in the route of administration from intravenous to subcutaneous). This adverse event was attributed to impurities which were leached from the rubber stoppers.
It is now widely known that impurities present in biopharmaceuticals (including protein aggregates) are known to induce immunogenic response to a therapeutic protein. Therefore, it is absolutely important for biogeneric products to have comparable (or lower) levels of impurities as well as a similar impurity profile as compared to the innovator’s product. There are reports citing various factors which confer risk to the patients and have emphasised the need to control them. Yet many biogeneric companies in India seem to be oblivious of these risk-factors or not doing enough to control them. It is, therefore, necessary that Indian regulatory agencies make it mandatory for biogeneric companies to comply with standards laid in Indian Pharmacopoeia for at least six drug substances. It is also necessary for the National Drug Control Laboratory to maintain vigilance on the quality of various marketed products from time to time to ensure that certain standards of quality are maintained for all biogeneric products. It is necessary for companies to maintain comparability of their product to the innovator’s drug at all times.
Biopharmaceuticals are susceptible to degradation and aggregation at elevated temperatures and have to be maintained at +2 to +8oC. Failure to maintain this temperature either during shipment or storage can easily accelerate generation of impurities such as aggregates and oxidised forms. This could well be the reason for such impurities as seen in this study. In a country like India, where in the peak of summer, temperatures can reach above 45oC, a robust cold chain is absolutely required. In Thailand, after the incidence of Eprex-associated PRCA peaked in 2002, there were additional 15 non-Eprex PRCA cases reported with biogenerics, some of which have HAS in the formulation. This has been attributed to poor cold chain maintenance. Similar case of PRCA has been reported with the use of a biogeneric EPO in India.
Extreme caution is exercised by the Indian regulatory agencies before granting marketing authorisation of biogeneric products. Prior to the approval for pre-clinical and clinical studies, it is mandatory for biogeneric companies to provide proof of physico-chemical comparability of the product made from five consecutive batches to the innovator’s drug. It is clear from this current study that in spite of such scrutiny exercised by the regulatory agencies, biogeneric products in India are not comparable to the innovator’s products. It is not known whether the lack of comparability is a result of batch-to-batch variation or a systemic failure to maintain them comparable or a breach in the cold chain affecting their quality. Consistent with our observation, similar comparative study has reported high variability of epoetin products launched in Korea, Argentina, China and India. Although this does not necessarily mean that these products are clinically inferior, maintenance of appropriate internationally accepted quality standards can help to safeguard the interest of biogeneric industry on the long run.
Intas Biopharmaceuticals Ltd. was the only Indian company whose all three biogeneric products were found to be comparable to the innovator brands. All three products (Neupeg, Neukine and Erykine) of Intas Biopharmaceuticals were found to be biosimilar to the innovators’ products (Neulastim, Neupogen, Eprex) with respect to all quality parameters used in the study.
We monitored storage temperatures of various warehouses across India of Clearing and Forwarding agents (who distribute the products in local geographic regions). As a revelation, it was found that many of them did not meet the specified storage conditions. As a corrective and preventive measure, temperatures at all clearing and Forwarding warehouses are now being monitored on a continuous basis. Such measures play a vital role in ensuring patient safety.
Significant difference in the level of purity has been observed among various brands of biogenerics of G-CSF and EPO. The degree of purity for some brands was below the Pharm. Eur. and Indian Pharmacopoeia standards. It is time that the Indian regulators should raise the expectation bar. This can be achieved once it will become mandatory for biosimilar companies to comply with the quality standards as given in the product-specific monographs in the Indian Pharmacopoeia. However, the Reference Standards required for comparison are not available till date. Introduction of National Reference Standards should be done on priority.
In the long run, it will be in the interest of Indian biogeneric companies to observe biogeneric guidelines as per the standards laid down by EMEA, if Indian companies have to emerge as global suppliers of biogeneric products. This is because many regulators are now focussing on EMEA guidelines as a benchmark. It is time for the Indian biogeneric players to go beyond Pharmacopoeia quality standards and also ensure stricter monitoring of temperatures during post-manufacturing storage and distribution.
We wish to thank Vijay Anand, Alkesh Ajameri and Snehal Roy for their valuable contributions to this investigation. We sincerely acknowledge the contribution of Dhananjay Patankar towards helpful discussion and valuable suggestions offered during the manuscript preparation.
Rustom Mody completed his PhD from Department of Microbiology, MS University of Baroda, India. He was a Research Associate for six years at the University of Nebraska Medical Center at Omaha, USA, where he worked in diverse research areas such as immunochemistry, molecular immunology and cancer biology. Currently, He is heading R&D and Quality divisions of Intas Biopharmaceuticals Ltd., with a focus on developing products and intellectual property that could give the company a strategic advantage and global reach.
Vishakha Goradia is working as Senior Executive in Quality Control Department of Intas Biopharmaceuticals Ltd. She is postgraduate in Microbiology from Bhavnagar University and having six years of professional experience in biotech Industry. She joined IBPL in 2003 and is associated with analysis of cell culture based products.
Deepak Gupta is currently heading analytical Quality Control department of India’s only EU-GMP approved biopharmaceutical plant at Intas Biopharmaceuticals Ltd. Being associated with the Indian biotechnology industry for the past eight years, he has been responsible for quality control of several bio-therapeutics products. He joined Intas in 2007 and prior to this he worked for Panacea Biotec Ltd and Biological E. Ltd. He received his MSc in microbiology from Gurukul University.