Figure 2: Sales of "blockbuster" recombinant drugs by category from 20065438+0 to 2005.
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After five years, the ranking of the top three recombinant drugs in the market has not changed, but their respective shares have declined due to the rapid growth of Enli. Enlil has risen to the fourth place tied with GM-CSF in 2005, and interferon-α has dropped to the sixth place. The indication of recombinant human erythropoietin has expanded from renal failure anemia to anemia caused by cancer or cancer chemotherapy, and there has been a lot of clinical evidence that recombinant human erythropoietin can improve the quality of life of cancer patients [26], and its leading position will be more stable in the next five years. The market share of recombinant insulin has declined, but the pulmonary inhaled insulin, long-acting insulin and basal insulin listed this year will support the market not to decline. Interferon β therapy for MS will be challenged by antibody drugs and small molecule drugs, and its development may be inhibited. GM-CSF can effectively reduce the infection caused by neutropenia caused by tumor chemotherapy in clinical use. Long-acting GM-CSF Neulasta is used once in a chemotherapy course, which is highly accepted by doctors and patients and will further accelerate its market share. Enli has made great progress in recent five years, but it will be strongly challenged by antibody drugs. Interferon-α combined with ribavirin is effective in the treatment of chronic viral hepatitis [10, 12], and there will be more room for growth after it is approved in Japan, a major hepatitis country. Next year, Novartis is expected to become a "blockbuster", leading the recombinant plasma protein to substantially adjust the overall market share pattern. Other kinds of recombinant drugs will not form a big market in the next five years.
Figure 3: Changes in the distribution ratio of recombinant drugs in 2001and 2005 (the left figure shows 2006 54 38+0).
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Second, research and development trends
The rapid development of recombinant drugs is inevitable, but there are several problems that must be solved or optimized for sustainable development, including production vectors and output, genetic engineering transformation and post-translation modification, and medication methods.
1, production carrier and output
Insufficient production capacity has become the bottleneck of the development of recombinant drugs. Take Enli as an example. Since 1998 went on the market for half a year, the sales volume in the United States alone has exceeded the global annual demand forecast [27], and the production scale gap is very large. For another example, the local use of HIV protein microbicide can prevent the spread of HIV, but it has not yet entered clinical research because the yield is not enough [28]. There are many drugs not only used in China, but also difficult to use in developed countries. It is estimated that 80% of hemophiliacs have no drugs available, mainly because of insufficient production capacity. Insufficient production capacity also leads to its high price.
Mammalian cells and Escherichia coli are the main production vectors of recombinant drugs (see Figure 4). Escherichia coli is used to express recombinant drugs that do not need post-translational modification, such as insulin, growth hormone, interferon β and interleukin. Glycoprotein recombinant drugs are expressed in mammalian cells except ATryn, which has just been approved for marketing. Activase is the first recombinant drug expressed in mammalian cells, and Epogen is the first blockbuster drug expressed in mammalian cells. CHO cell is one of the most commonly used production vectors. At present, its glycosylation structure is similar to that of human beings, but the glycosylation products are heterogeneous mixtures. BHK battery is the second most commonly used battery. In addition, NSO, HEK-293 and protein expressed by human retinal cells have also been approved. At present, the output of mammalian cells needs to be improved urgently. In the 1980s, the maximum cell density reached 2X 106/ml, the production cycle was 7 days, and the specific product quantity was 50mg/L. According to the data in 2004, the maximum cell density reached 10X 106/ml, the effective expression time reached 3 weeks, and the expression quantity was close to 5g/L, which was. At present, the largest cell fermentor in the world reaches 20,000 liters. Other problems that need to be solved in mammalian cell production system include serum-free medium, delaying apoptosis and improving glycosylation [30]. Although yeast cells can be glycosylated, they are very different from human glycosylation and belong to high xylitol type. The expressed recombinant drug has a short half-life in vivo and has potential immune response. Therefore, the most likely breakthrough in this field is "humanized" Pichia pastoris [3 1], which can produce homogeneous glycoprotein with the same glycosylation as human, and the yield of target protein can reach 15g/L, which is three times that of mammalian cells, which poses a strong challenge to mammalian cell expression system.
Figure 4: Proportion of recombinant drug production carriers in the market.
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Another breakthrough is the plant expression system (molecular agriculture). Plant glycosylation has low immunogenicity and is not easy to induce allergies, but it may change the functions of some glycoproteins. At present, it has been used to express more than 65,438+00 recombinant drug candidates, of which 65,438+0 have entered phase II clinical [28]. The problems to be solved in this system include further improving the expression yield, transforming the glycosylation structure through "humanization" and evaluating the impact of the production system on the environment. Breakthrough transgenic animal production mode will not become the mainstream at least in the near future. The problem is that the glycosylation of emulsified protein in transgenic higher mammals is still different from that in humans, which may lead to antigen changes. The European Committee for Medical Products for Human Use (CHMP) once objected to the listing of ATryn on the grounds that there were too few clinical cases. In addition, the recombinant human acidic α -glucosidase (trade name Myozyme) of Genzyme Company of the United States was originally produced in transgenic rabbit milk, and finally produced by CHO cells, and was approved by FDA [20]. The egg white of transgenic chicken can also express recombinant drugs at a high level, but no drugs prepared by transgenic chicken have been approved yet, and the main problem is glycosylation. Of course, if the drug is used orally and locally, the antigenicity problem is likely to be ignored.
2. Genetic engineering transformation and post-translation modification of recombinant drugs.
High-purity recombinant protein is the same as or highly similar to human endogenous protein, which can avoid immune reaction. However, about 30% of recombinant drugs are modified by genetic engineering or other means (Figure 5), and it is also pointed out that the gene transformation rate of existing recombinant protein drugs in the market is 38%. The purpose of changing the structure of protein is to optimize its pharmacokinetics, but it cannot weaken its biological function and produce new antigenicity.
Figure 5: Proportion of gene modification or post-translation modification of recombinant drugs on the market.
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Taking recombinant human insulin as an example, there are many products whose sequences have been changed by genetic engineering, mainly the amino acid changes at positions B28, B29 and B30. Lispro, the first recombinant human insulin genetically engineered, is a transversion between B28 and B29, which makes it 300 times less likely to produce dimers and multimers than the wild type [32], and can be released into the body more quickly, playing a rapid role. Deletion mutants are also very common. ReFacto (recombinant coagulation factor VIII, with sales of $250 million in 2005) is the deletion mutant [33], which has a good effect on hemophilia patients who use factor VIII inhibitors in vivo. Recent studies have shown that ankyrin repeats (which appear in red blood cells, etc., and consist of 33 amino acids, with β-folded antiparallel and α-helix) are helpful to strengthen the targeted recognition of recombinant drugs, the directionality and stability of membrane proteins [34]. However, genetic engineering should be very cautious when changing the sequence. Some small changes may lead to great changes in the protein conformation, thus inducing an immune response. Post-translation modification mainly includes lipidation and PEGylation. Lipid refers to the fixed-point connection between the valence of fatty acids and protein, thus increasing the affinity between drugs and serum albumin, prolonging the circulation time in serum and playing a long-term role. PEGylation can be divided into mono-PEGylation and PEGylation. By reducing the plasma clearance rate, degradation and receptor-mediated uptake, the long-term goal can be achieved, and the epitope can be shielded to improve the safety of drugs. Pegylated interferon α (peg system and peg intron) and peg GCSF are both successful examples of pegylation [35]. Fusion protein refers to the construction of a protein from different functional domains of different protein through genetic engineering, hoping to have dual functions or new functions. Although a lot of attempts have been made in this regard, only three of them have been approved in the past 25 years, which shows that this is very difficult. Only 1 foreign protein was successful.
3. Changes in the route of administration.
The vast majority of recombinant drugs are administered by injection or intravenous route, and only two are sprays, such as Pulmozyme, which are liquid sprays. Some diseases, such as diabetes, renal failure and anemia, need long-term medication, and the way of injection or intravenous route is very inconvenient, so people have made a lot of attempts in the way of administration. In 2006, a major breakthrough was finally made, and Exubera, a lung inhaled insulin by Pfizer and Aventis, was approved to be listed in the United States and Europe. As a dry powder, lung inhalation dosage form is more stable than liquid spray, and the dosage is easy to master. Of course, Exubera is too expensive, and the British authorities refuse to use it because each patient has to pay an extra $65,438 +08 a week. In any case, this will change the treatment methods of many diabetic patients, relieve their pain, and also stimulate the research upsurge of alternative injection routes of other drugs. Several domestic scientific research institutions and companies have made gratifying progress in studying transdermal drug delivery and pulmonary inhalation drug delivery methods. However, it should be pointed out that pulmonary insulin inhalation started from 1999 [36] and has only been approved so far, so the difficulty can be imagined. In this regard, the biggest technical difficulties are the accuracy of dosage and the stability of drugs.
Three. Problems and challenges faced by recombinant drugs
Some analysts describe the recombinant drug market as a beautiful butterfly. But how long can butterflies fly? How will it evolve? In other words, has the recombinant drug market entered a mature stage? In recent 10 years, there has not been a new product category that has a great impact on the market, R&; D investment is relatively stable (r & 2002-2004 for three consecutive years; D the total amount is stable at $654.38+0.5 billion [37]), which is a sign that the market is too mature and will limit its sustainable development, which is a challenge from itself. Objectively analyzing the development of other treatment technologies, we believe that the recombinant drug market will not encounter real challenges in the near future, but potential threats do exist.
1, the challenge of other therapeutic drugs or methods to the recombinant drug market
Many indications of recombinant drugs are the loss or loss of a protein function caused by a single gene or a clear simple reason, such as hemophilia and type I diabetes, which is very suitable for gene therapy. Imagine, if the gene of recombinant drug can be regulated and expressed in human body, where will the recombinant drug market go? Will gene therapy have a great impact on the recombinant drug market in the short term? In fact, since 1989, about 1 140 gene therapy products have entered clinical research, and only a few have entered clinical phase III research [20]. No 1 products have been listed in the United States and Europe, and many of them have been terminated due to unexpected deaths in clinical research. At present, only China approved the gene therapy based on human p53 gene in June 2003. Similar products in the United States have already entered the third phase of clinical practice, and the reason why they can't go on the market is that the approval of FDA in the United States is based on the change of 5-year survival rate, while China is based on the reduction of tumor. Gene therapy can only enter the market in large quantities if it makes breakthroughs in key areas such as specificity, stability, controllability and antigenicity of target gene delivery. It can be predicted that gene therapy will hardly pose a strong challenge to recombinant drugs in 5- 10 years. The method of inducing stem cells to produce islet-like cells has not made the expected progress, and the road to clinic may be farther than gene therapy. The mechanism of action of antibody drugs or aptamers is a substance that specifically targets binding and inhibits binding, while the mechanism of action of most recombinant drugs is to supplement protein (function). Therefore, both antibody drugs and aptamers will only pose a serious challenge to recombinant drugs with inhibitory mechanisms, such as Enrique and interferon beta.
2. The influence of generic drug era on the market structure of recombinant drugs.
At present, the rule of drug market is that generic drugs will be listed after the patent protection period of new drugs expires, and their price is about 15% of that of new drugs, which greatly affects drug profits. For example, the patent protection of Zokor, the runner-up in prescription drug sales for five consecutive years, expires this year, and its generic drug will not only affect the price of Zokor, but also reduce the overall price of hypolipidemic drugs. Several recombinant drugs in 1980 have lost or will soon lose patent protection. It is estimated that in the next five years, recombinant drugs with a value of more than $10 billion will lose their patents. Will a batch of generic recombinant drugs subvert the recombinant drug market? In 2006, Europe approved the listing of two generic recombinant drugs for the first time. These two drugs are two different versions of human growth hormone Omnitrope and Valtropin. Does this indicate the arrival of the era of generic drugs and recombinant drugs? In fact, the situation of recombinant drugs is far more complicated than that of small molecular drugs. There are guidelines for generic drugs such as growth hormone, Epo, GM-CSF and insulin in Europe, but PEG protein and coagulation factors with complex structure and processing are not considered. How to develop recombinant generic drugs in the United States is still controversial, and the FDA has not issued relevant guiding principles. The most important consideration is safety. Different from small molecule drugs, even if the same gene is expressed in the same cell and similar processing methods are adopted, it is difficult to ensure that the generic recombinant drug is exactly the same as the original drug. Considering the production cost and complexity of processing, the impact of recombinant generic drugs on the existing market is not obvious. However, the era of generic drugs of recombinant drugs is bound to come, which will have a significant impact on the market structure of recombinant drugs.
3. There are risk factors in clinical safety.
Like other drugs, recombinant drugs also have the risk of causing side effects. First of all, the effect of recombinant drugs is not single, or its degree of action is difficult to control accurately, which may lead to serious side effects. For example, some experts believe that the use of recombinant human erythropoietin to correct anemia in cancer patients may promote tumor growth [26]. Similarly, recombinant human growth hormone can stimulate tumor growth and increase the risk of hyperlipidemia and diabetes. However, the treatment of "stroke" with tPA will lead to bleeding tendency, which is closely related to serum matrix metabolic protease 9(MMP9) [33]. Secondly, antibodies against recombinant drugs appear in patients, which are mainly caused by glycosylation differences and new epitopes (especially T cell epitopes) produced by recombination, and the types and degrees of their clinical manifestations are unpredictable. The most common is that the treatment effect is poor or even ineffective. In severe cases, fatal complications may occur, such as erythroaplastic anemia (RBCA) caused by anti-recombinant human erythropoietin antibody [6], and the reasons are unknown. Clinical safety risk is a direct factor affecting the approval speed of new drugs, which is bound to affect the market development.
Fourth, some ideas.
Although the development of recombinant drugs is facing challenges, it will still develop at a faster speed in the near future, and it may become a watershed in the development of recombinant drugs around 2020. The specific time depends on whether the bottleneck problem can be solved and whether alternative therapy can appear. In any case, what we are facing now may be the last development opportunity. The research of recombinant protein is very common in China, and any large research institution has a platform for gene cloning and mutation. Many pharmaceutical companies also have large-scale cell culture and purification systems, and have the conditions to develop and produce recombinant drugs. However, to seize this opportunity, we must calmly analyze the situation and start our work from a high starting point.
1. Objectively select recombinant drugs as the starting point of research and development.
Recombinant human erythropoietin, insulin, interferon-β, GM-CSF, interferon-α and some recombinant plasma proteins occupy most of the market of recombinant drugs. In the past 10 years, only Enli has broken through the above protein categories. It should be emphasized that the characteristics of recombinant drugs determine that these protein types dominate the market, which is a concentrated expression of clinical efficacy and safety, as well as market potential and scale, which can be seen from the increasing sales ratio of "heavy explosives" in the recombinant drug market. Therefore, in order to gain more market share, it is reasonable to choose protein as the starting point of drug research. Recombinant human protease also has a good development opportunity. Fusion proteins are rare in recombinant drugs with specific targeted binding and inhibition mechanism, which is in line with the development trend of tumors and immune diseases. However, 25 years' experience tells us that the fusion protein is difficult to become a therapeutic protein. Because of antigenicity, the foreign protein must wait for the breakthrough of administration route, otherwise the chance is very small.
2, to genetic engineering or other transformation methods to transform the existing large as a breakthrough.
It is not difficult to find that more than half of "blockbusters" have been transformed, and "blockbusters" have transformed new and old products, for example, from Neupogen to Neulasta; PEG- intron A is rapidly replacing intron A, and Pegasys has quickly curbed the development momentum of PEG- intron A, which reminds us that although the market is relatively mature and saturated, there are still great opportunities for "blockbuster" mutants. Of course, this opportunity comes from our thorough understanding of the pathogenesis, chemical and physiological functions of protein, and we must also have a good technical platform to systematically and accurately evaluate the function and safety of the changed protein. Undoubtedly, changing the administration route of "blockbuster" will stand at the forefront of the recombinant drug market and provide a platform for the future antibody drug market.
3. Make breakthroughs in production methods and efficiency, and participate in international competition.
Only enough production capacity can occupy the market and reduce production costs. However, it takes about five years and an investment of 200-400 million dollars to establish a large-scale mammalian cell production base. Therefore, according to the specific situation of China, we can establish a medium-sized mammalian cell culture base and contract the production of international "blockbusters". At present, contracted processing accounts for 25% of the total production capacity, which is also a big market. At the same time, starting with the glycosylation transformation of yeast and the construction of expression system of plants and transgenic animals, China's unique advantages will be rapidly formed and stand out in the international competition. Even if the recombinant drugs are replaced or partially replaced by other therapies around 2020, the established production platform can still be used for the production of new antibody drugs and recombination vaccines, and the overall benefits are remarkable.