Patenting and Biotechnology

 Patenting and Biotechnology

Intellectual property laws are complicated in the field of biotechnology patents.  A biological or biotechnological patent is given to an invention in the field of biology and it lawfully allows the patent holder to exclude others from making, using, selling, or importing the protected invention for a limited period of time. The scope and reach of biological patents include biological technology and products, genetically modified organisms, genetic material, etc, even though it vary among jurisdictions. The patenting of substances or processes wholly or partially of natural in origin is a subject of heavy debate.

A few examples of patented biotechnological products and procedures that are lifesaving are insulin, anti-cancer drugs, autoimmune drugs, pasteurization, etc. It was in 1862, Louis Pasteur patented pasteurization at the French Patent Office. Today this process is used commercially by companies to improve shelf life of juice, beer, eggs, milk, etc.

Patenting scientific advancements in the field of biotechnology is an extremely complicated process.

Biotechnology patents fall under utility patents which is available for the invention or discovery of a new and useful machine, manufacturing process, composition of matter, or process or for improvements to an existing process that are considered new and useful.

When applying for a patent, the inventor must prove that their invention meets certain eligibility requirements. In the United States, the US Patent & Trademark Office set forth five elements for patentability. To qualify as a patent, an invention must fall under subject-matter eligibility, have utility and novelty, be non-obvious, and not have been previously disclosed.

In Europe, the European Patent Office states the requisites as a patentable invention can be a product, a process, or an apparatus and it “must be new, industrially applicable, and involve an inventive step.”

In Japan, the Japanese Patent Act requires that patented inventions must have commercial potential. The “medical activities” such as methods of surgery, therapy, and the diagnosis of human diseases cannot be patented.

In India, Under the Patent Act 1970, every invention must pass a two-step test in order to be patentable

1.         It must not fall in any of the categories specifically excluded under Section 3 of the Patent Act

2.         It must pass the three-pronged test of novelty, inventive step and industrial applicability.

These are the category of nonpatentable inventions in India

(a)        an invention which is frivolous or which claims anything obviously contrary to well established natural laws

(b)        an invention the primary or intended use or commercial exploitation of which could be contrary public order or morality or which causes serious prejudice to human, animal or plant life or health or to the environment

(c)        the mere discovery of a scientific principle or the formulation of an abstract theory [or discovery of any living thing or non-living substances occurring in nature

(d)       the mere discovery of a new form of a known substance which does not result in the enhancement of the known efficacy of that substance or the mere discovery of any new property or new use for a known substance or of the mere use of a known process, machine or apparatus unless such known process results in a new product or employs at least one new reactant. Explanation. -For the purposes of this clause, salts, esters, ethers, polymorphs, metabolites, pure form, particle size, isomers, mixtures of isomers, complexes, combinations and other derivatives of known substance shall be considered to be the same substance, unless they differ significantly in properties with regard to efficacy

(e)        a substance obtained by a mere admixture resulting only in the aggregation of the properties of the components thereof or a process for producing such substance

(f)        the mere arrangement or re-arrangement or duplication of known devices each functioning independently of one another in a known way

(h)       a method of agriculture or horticulture

(i)        any process for the medicinal, surgical, curative, prophylactic [diagnostic, therapeutic] or other treatment of human beings or any process for a similar treatment of animals to render them free of disease or to increase their economic value or that of their products

(j)       plants and animals in whole or any part thereof other than micro-organisms but including seeds, varieties and species and essentially biological processes for production or propagation of plants and animals

(k)       a mathematical or business method or a computer programe per se or algorithms

(l)      a literary, dramatic, musical or artistic work or any other aesthetic creation whatsoever including cinematographic works and television productions

(m)      a mere scheme or rule or method of performing mental act or method of playing game

(n)      a presentation of information

(o)      topography of integrated circuits

(p) an invention which in effect, is traditional knowledge or which is an aggregation or duplication of known properties of traditionally known component or components

Among these, the following are the excluded biotechnology-related inventions,

Section 3(b) – inventions contrary to public morality

Section 3(c) – discoveries, things isolated from nature, plants and animals

Section 3(d) – new forms or uses of known substance

Section 3(e) – mere admixture

Section 3(i) – methods of treatment and diagnosis

Section 3 (j) - Plants and animals in whole or any part thereof other than microorganisms, but including seeds, varieties and species, and essentially biological processes 

Section 3(h) – agricultural or horticultural methods

Section 3 (k) - Computer programs per se and algorithms, mathematical methods

Section 3(p) – traditional knowledge

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Other than section 3, the following sections of the Patents Act, 1970 are also emphasised in the context of patents in biotechnology and allied fields:

·                     Section 2 (1) (j): Novelty, inventive step & industrial applicability of products or processes,

·                     Section 10 (4): Sufficiency of disclosure and the best method of performing the invention

·                     Section 10 (5): Unity of invention and clarity, succinctness and support of the claims

Section 3(b) – inventions contrary to public morality

Inventions for which the primary or intended use or commercial exploitation is contrary to public order or morality or which cause serious prejudice to human, animal or plant life or health or to the environment are unpatentable. Examples include genetic modification of animals which results in suffering of the modified animal without any substantial medical or other benefit, and inventions causing adverse environmental impact.

Section 3(c) – discoveries, things isolated from nature, plants and animals

Discoveries of living things or non-living substances occurring in nature are not patentable subject matter. Thus, micro-organisms isolated from nature and DNA, RNA or proteins isolated from living organisms are unpatentable.

Although naturally occurring micro-organisms are unpatentable, genetically modified micro-organisms and vaccines are patentable, subject to other requirements. Synergistic compositions of new or known micro-organisms can also be patentable, as can processes for isolating such substances. The act was amended in 2002 and “biochemical, biotechnological and microbiological processes” are potentially patentable chemical processes.

Plants and animals or their parts – including seeds, varieties and species – are unpatentable. Biological processes for the production of plants or animals (plant breeding and tissue culture techniques) are also unpatentable.

Although genetically modified plants or seeds are not patentable in India, processes for the genetic modification of plants are patentable.  A sui generis system for protection for plant varieties is available under the Protection of Plant Varieties and Farmers’ Rights Act 2001.

Section 3(d) – new forms or uses of known substance

A new form of a known substance is unpatentable unless it differs significantly in properties with regard to the known efficacy. This provision essentially prevents the evergreening of patents through trivial modifications or incremental innovations.

A mere change of form of a chemical substance with properties inherent to that form would not qualify as enhancement of efficacy of a known substance.  Physico-chemical properties such as better flowability, processability, thermodynamic stability and lower hygroscopicity have nothing to do with therapeutic efficacy.

The mere discovery of any new property or new use of a known substance is also unpatentable. Therefore, a second therapeutic effect of a known drug is unpatentable. In Monsanto (2013) a claim for a method of producing heat, salt and drought-tolerant transgenic plants using cold shock protein was rejected under Section 3(d), since the cold-tolerant property of cold shock protein was already known in the art.

Section 3(e) – mere admixture

The mere admixture of two or more previously known substances is unpatentable, unless it is shown that the combinative effect of such substances is more than the sum of their individual effects. In other words, such a combination should result in a synergistic effect and the synergism must be properly demonstrated by providing experimental data.

Section 3(i) – methods of treatment and diagnosis

The act does not prevent patenting of pharmaceuticals and medical devices. Thus, medicinal compounds, drugs, formulations, stents, surgical sutures and staplers are patentable. However, Section 3(i) precludes from patentability:

 

any process for the medicinal, surgical, curative, prophylactic, diagnostic, therapeutic or other treatment of human beings; or any treatment of animals which renders them free of disease or increases their economic value (or that of their products).

Section 3(i) unpatentable subject matter examples

·         Medicinal methods - A process of administering medicines orally, through injection, topically or through a dermal patch.

·         Surgical methods - A stitch-free incision for cataract removal.

·         Curative methods - A method of cleaning plaque from teeth.

·         Prophylactic methods - A method of vaccination.

·         Diagnostic methods - Identification of the nature of a medical illness by investigating its history and symptoms and applying tests.

·         Therapeutic methods - Prevention and treatment or cure of diseases.

·         Methods of treatment to render animals free of disease or increase their economic value (or that of their products) - A method of treating sheep for increasing wool yield.

The cosmetic application of substances to the body, methods of operating a medical device and the manufacture of artificial prostheses and taking measurements thereof are potentially patentable. Diagnostic methods practised on a living body are unpatentable; however, diagnostic methods carried out on tissues, cells or biological fluid completely removed from the body can be patentable.

Dosage forms, modes of administering medicine or kits designed for the treatment of a disease that comprise nothing more than a dosage pattern are unpatentable.

Section 3(h) – agricultural or horticultural methods

Methods of agriculture or horticulture are unpatentable. Agriculture and horticulture are processes involving multiple steps, such as preparation of soil, sowing, applying manure and fertilisers, irrigation, protection from pests and weeds, harvesting and storage.

For example, a method of reducing mycotoxin contamination of a plant or harvested plant material that involved treatment of seeds with the chemicals before sowing or during sowing in the field for the plant cultivation process is unpatentable.

Section 3(p) – traditional knowledge

An invention which is essentially traditional knowledge or which is an aggregation or duplication of the known properties of a traditionally known component or components is specifically excluded from patentability. India has developed the Traditional Knowledge Digital Library (TKDL), providing information on India’s traditional knowledge in the country related to ayurveda, unani, siddha and yoga. The database is available in five languages (English, German, French, Japanese and Spanish) and assist the major patent offices around the world to carry out prior art searches.

 

References

https://www.labiotech.eu/in-depth/biotechnology-patents-intellectual-property/#:~:text=Biotechnology%20patents%20fall%20under%20the,composition%20of%20matter%2C%20or%20process.

https://www.wipo.int/patents/en/topics/biotechnology.html

https://www.iam-media.com/patenting-biotechnology-indian-scenario

https://indiankanoon.org/doc/874310/

Virus

 Virus

Viruses are the smallest known infective agents and the simplest form of life known. They do not possess any cellular organization and they do not fall into the category of unicellular microorganisms.

Properties of Viruses

1. Viruses do not have a cellular organization.

2. They contain only one type of nucleic acid, either DNA or RNA but never both.

3. They are obligate intracellular parasites.

4. They lack the enzymes necessary for protein and nucleic acid synthesis and are dependent for replication on the synthetic machinery of host cells.

Morphology of viruses

                                                        thebiologynotes.com

Size: Viruses are much smaller than bacteria. Their small size and ‘filterability’ (ability to pass through filters that can hold back bacteria) led to their recognition as a separate class of infectious agents. They were termed as filterable agents upon discovery.

Viruses vary widely in size from 20 nm to 300 nm. The largest among them is pox virus (300 nm) and is as large as the smallest bacteria (mycoplasma).  The Smallest viruses are the parvovirus (about 20 nm).

They are too small to be seen under the light microscope. Some of the larger viruses, such as poxviruses can be seen under the light microscope when suitably stained.

Shape:

Shape of virus varies with different groups of viruses. Most of the animal viruses are roughly spherical, some are irregular and pleomorphic. Poxviruses are brick-shaped, rabies virus is bullet-shaped, tobacco mosaic virus is rod-shaped. Bacteriophages have a complex morphology.

They consist of nucleic acid core surrounded by a protein coat called capsid. The capsid is composed of a large number of capsomers which are polypeptide molecules arranged symmetrically.  The capsid with the enclosed nucleic acid is known as nucleocapsid.

Functions of Capsid

i. It protects the viral genome from physical destruction and enzymatic inactivation.

ii. It provides binding sites for the virus to attach to specific receptor sites on the host cell.

iii. It facilitates the assembly and packaging of virus after replication.

iv. It serves as a vehicle of transmission from one host to another.

v. It is antigenic and specific for each virus type and is of importance in the host’s defence to virus infection.

vii. It provides the structural symmetry to the virus particle.

Viral architecture is grouped into three based on the arrangement of morphologic subunits:

(1) Icosahedral symmetry (2) Helical symmetry (3) Complex structures.

1. Icosahedral Symmetry

An icosahedral (icosa, meaning 20 in Greek) is a polygon with 12 vertices or corners and 20 facets or sides. Each facet is in the shape of an equilateral triangle. Two types of capsomers constitute the icosahedral capsid. They are the pentagonal capsomers at the vertices (pentons) and the hexagonal capsomers making up the facets (hexons).  Example - Adenovirus

2. Helical Symmetry

The nucleic acid and the capsomers are wound together in the form of a helix or spiral. Examples: Tobacco Mosaic Virus and rabies.

3. Complex Symmetry

They do not have either icosahedral or helical symmetry due to complexity of their capsid structure.  Example is Poxviruses.  Some bacteriophage such as T4 phage have icosahedral head and helical tail and thus have complex symmetry.

Enveloped Virus : Virus may be enveloped or nonenveloped (naked). In enveloped Virus, the envelope or outer covering of virus is derived from the plasma membrane of the host cell during their replication. The envelope is lipoprotein in nature. The lipid is of host cell origin while the protein is virus encoded.

Enveloped viruses are susceptible to the action of lipid solvents such as ether, chloroform and detergents, whereas most viruses existing as naked capsids are more likely to be resistant to them.

In virus particle, the glycoproteins appear as projecting spikes on the outer surface of the envelope.  These are known as peplomers (from peplos, meaning envelope). A virus may have more than one type of peplomers, e.g., the influenza virus carries two kinds of peplomers, the hemagglutinin which is a triangular spike and the neuraminidase which is a mushroom shaped structure. Envelope confer chemical, antigenic and biological properties on viruses.

Functions of Peplomers

i. Many peplomers mediate attachment of the virus to the host-cell receptors to initiate the entrance of the virion into the cell.

ii. Some viral glycoproteins attach to receptors on red blood cells, causing these cells to agglutinate (hemagglutination).

iii. Some possess enzymatic activity like neuraminidase which cleave neuraminic acid from host cell glycoproteins.

iv. Glycoproteins are major antigens

Viral Nucleic Acids

Viruses contain a single kind of nucleic acid—either DNA or RNA—that encodes the genetic information necessary for replication of the virus. The genome may be single-stranded or double-stranded, circular or linear, and segmented or nonsegmented. The type, structure and size of nucleic acid are used for classifying viruses.

sciencedirect.com

Viral replication

The genetic information necessary for viral replication is in the viral nucleic acid but since virus lack biosynthetic enzymes they depend on the host cell for replication. The viral replication / multiplication cycle can be divided into six sequential phases

1.  Adsorption or attachment

2. Penetration

3. Uncoating

4. Biosynthesis/Replication

5. Maturation

6. Release

researchgate.net

1. Adsorption or Attachment

Virus come in contact with host cells by random collision followed by specific adsorption or attachment.  This is mediated by the binding of virion surface structures, known as ligands, to receptors on cell surface. In case of influenza virus, hemagglutinin (a surface glycoprotein) binds specifically to sialic acid residue of glycoprotein receptor sites on the surface of respiratory epithelium. In case of human immunodeficiency virus, surface glycoprotein gp 120 acts as a ligand which binds to the CD4 glycoprotein on the surface of mature T lymphocytes. Rabies virus binds to the acetylcholine receptor found on neural cells.

2. Penetration

After adsorption, the virus particle is taken up inside the cell.

Some viruses accomplish this by receptor mediated endocytosis or viropexis.  Here uptake of the ingested virus particles within endosomes occurs. Most nonenveloped viruses enter the cell by viropexis.

Enveloped viruses fuse their membranes with host cell membranes and deliver the nucleocapsid or genome directly into the host cytoplasm.

3. Uncoating

This is the stripping of virus of its capsid so that the nucleic acid is released into the cell. Uncoating is effected by the action of host cell lysosomal enzymes. Genome of most RNA viruses remain in the cytoplasm, while genome of DNA viruses, except poxviruses, are delivered to the nucleus.

4. Biosynthesis/Replication

This phase includes synthesis of the viral nucleic acid, capsid protein and enzymes necessary in the various stages of viral synthesis, assembly and release. In addition to these, some ‘regulator proteins’ are also synthesized which shut down the normal cellular metabolism and direct the production of viral components.

Most DNA viruses synthesize their nucleic acid in the host cell nucleus. The exception is the poxviruses, which synthesize the components in the host cell cytoplasm.

Most RNA viruses synthesize their components in the cytoplasm, except orthomyxoviruses, some paramyxoviruses and retroviruses which synthesise few of the components in the nucleus.

Viral proteins are synthesised in the cytoplasm.

Steps of Biosynthesis

i. Transcription of messenger RNA (mRNA) from the viral nucleic acid.

ii. Translation of the mRNA into ‘early proteins’. These are enzymes required for the synthesis of virus components and to induce shutdown of host protein and host nucleic acid synthesis.

iii. Replication of viral nucleic acid.

iv. Synthesis of ‘late’ or structural proteins, which are the components of daughter virion capsids.

5. Maturation

Assembly of the various viral components into virions occur during maturation.  This may take place either in the nucleus (herpes and adenoviruses) or cytoplasm (picorna and poxviruses).

In case of enveloped viruses, the envelopes are derived from the host cell nuclear membrane (herpes virus) or host cell plasma membrane (orthomyxoviruses and paramyxoviruses).

6. Release

Viruses are released from cells after lysis of the cell or by exocytosis or by budding from the plasma membrane.

Viruses that exist as nucleocapsids are released by the lysis of the host cell (polioviruses) or they may be extruded by a process known as reverse phagocytosis.

Release of enveloped viruses occurs as budding from the plasma membrane without killing the cell.

Eclipse phase

During viral multiplication the virus cannot be demonstrated inside the host cell from the stage of penetration till the appearance of mature virions. This period is known as the ‘eclipse phase’. The time taken for a single cycle of replication is about 15-30 minutes for bacteriophages and about 15-30 hours for animal viruses.

Abnormal replicative cycles

1. Incomplete Viruses

A proportion of daughter virions that are produced may not be infective due to defective assembly.  Such ‘incomplete viruses’ are seen in large numbers when cells are infected with a high dose of influenza virus.  The virus yield will have a high hemagglutinin titer but low infectivity. This is known as the von Magnus phenomenon.

2. Abortive Infections

Abortive infections fail to produce infectious progeny, either because the cell may be nonpermissive and unable to support the expression of all viral genes or because the infecting virus may be defective due to lack of some functional viral genes.

3. Latent Infection

During a latent infection, there will be the persistence of viral genomes and expression of none or a few viral genes, but the infected cell continue to survive.

4. Defective Viruses

Viruses which are genetically deficient and incapable of producing infectious daughter virions without the helper activity of another virus are known as’ defective viruses’. Progeny virions will be formed only if the cells are simultaneously infected with a helper virus, which is a normal virus.

Example

i. Hepatitis D virus and adeno-associated satellite viruses which replicate only in the presence of their helper viruses—hepatitis B and adenoviruses respectively.

ii. Rous sarcoma virus (RSV) cannot code for the synthesis of the viral envelope in the absence of its helper virus (avian leucosis virus).  Infectious progeny of RSV results only if the helper virus contribute to the synthesis of the envelope.

Baltimore classification of viruses based on replication mechanisms

Viruses have been categorized into six classes by Baltimore (1970) based on their replication mechanisms.

Sl. No	Class	Feature
1	Single stranded DNA viruses	DNA molecule moves into the host cell nucleus and is converted into the duplex form. Transcription is achieved by host enzymes. Example - Parvovirus
2	Double stranded DNA viruses	DNA enters the host cell nucleus and uses the host cell enzymes for transcription Example -  Hepadnaviruses,  Poxviruses
3	Single stranded RNA viruses   Depending on the method of mRNA transcription, these are classified into two categories	Positive strand (plus strand, positive sense): The viral RNA acts as the mRNA. Viral RNA is infectious by itself and is translated directly into viral proteins in the host cell cytoplasm. Example -Picorna, Togaviruses.
4		The negative strand (minus sense) RNA viruses: The RNA is ‘antisense’, with polarity opposite to mRNA. They possess their own RNA polymerases for mRNA transcription Example – Rhabdo virus, Orthomyxo virus, Paramyxovirus
5	Double stranded RNA viruses	The DS RNA is transcribed to mRNA by viral polymerases Example - Reoviruses
6	Retrovirus	SS RNA genome is converted into an RNA: DNA hybrid by the viral reverse transcriptase enzyme. Double stranded DNA is then synthesized from the RNA: DNA hybrid. The double stranded DNA form of the virus (provirus) is integrated into the host cell chromosome. This integration may lead to transformation of the cell and development of neoplasia.

 

Demonstration of bacterial pigmentation

 

Demonstration of bacterial pigmentation

Aim:

To observe pigmentation by various bacteria when grown on nutrient agar.

Principle:

When a single bacterial cell is placed on a solid or liquid medium, it begins to divide and eventually, a colony appears in the place. Microorganisms exhibit visible physical differences in appearance in their colonies such as colonial shape and appearance, pigmentation and smell and their growth pattern when grown on different types of media. These differences are called cultural characteristics or morphology and may be used as an aid in identifying and classifying them. Cultural characteristics or morphology are determined by observing microorganisms cultured in nutrient broth and on nutrient agar plates, after incubation.

Chromogenic bacteria produce intracellular pigments that are responsible for the color of the colonies on the agar surface. Some bacteria produce extracellular soluble pigments that are excreted into the medium and that also produce a colored colony. Most microorganisms are nonchromogenic and will appear cream, white, or gray.  Pigmented bacteria are also known as chromobacteria. Bacterial pigments are water soluble or insoluble; water soluble pigments are diffused in the growth medium. Chemically, bacterial pigments are pyrrole, phenazine, carotenoid, xanthophylls and quinine or quinone derivatives. The pigment molecules are synthesized in cell wall or periplasmic space. Only aerobic and facultatively aerobic bacteria are pigmented because, molecular oxygen is essential for pigmentation. Pigment synthesis is also dependent on light, pH, temperature and media constituents like indicator dyes.  In bacteria, pigment formation is associated with morphological characteristics, cellular activities, pathogenesis, protection and survival.

Pigment                                              Bacteria

Purple                                                  Spirillum rubrum

Violet                                                  Chromobacterium violacein

Yellow                                                Xanthomonas campestris

Orange                                                Sarcina aurentiaca

Red                                                     Serratia marcescens

Black                                                   Prevotela melaninogenica

Golden                                                Staphylococcus aureus

Pink                                                     Micrococcus roseus

Fluorescent blue/green                        Pseudomonas aeruginosa

Fluorescent yellow                              Pseudomonas fluorescens

S. aureus is a facultatively anaerobic, Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope, and has round, usually golden-yellow colonies, often with hemolysis, when grown on blood agar plates. The golden appearance is the etymological root of the bacterium's name; aureus means "golden" in Latin.   Some strains of Staphylococcus aureus are capable of producing staphyloxanthin - a golden coloured carotenoid pigment. This pigment acts as a virulence factor, primarily by being a bacterial antioxidant which helps the microbe evade the reactive oxygen species which the host immune system uses to kill pathogens.

Serratia marcescens is a motile,short rod-shaped, Gram-negative, facultative anaerobe bacterium.  S. marcescens produces a reddish-orange tripyrrole pigment called prodigiosin.  Prodigiosin is made up of three pyrrole rings and is not produced at 37°C, but at temperatures below 30°C.

Pseudomonas aeruginosa is a common gram-negative, rod-shaped bacterium.  P. aeruginosa can secrete a variety of pigments, including pyocyanin (blue-green), pyoverdine (yellow-green andfluorescent), and pyorubin (red-brown). The species name aeruginosa is a Latin word meaning verdigris ("copper rust"), referring to the blue-green color of laboratory cultures of the species. This blue-green pigment is a combination of two metabolites of P. aeruginosa, pyocyanin (blue) and pyoverdine (green), which impart the blue-green characteristic color of cultures.

Materials required:

Routine microbiological facilities

Nutrient agar plates

Overnight cultures of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus

Procedure:

Using an inoculation loop nutrient agar plates were inoculated with Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus.  Two plates were kept for Serratia marcescens culture.

All plates were kept at 37oC for overnight incubation.  One set of Serratia inoculated plate were kept overnight at room temperature.

After incubation, the plates were observed for colony morphology.

Observation:

S. aureus formed medium sized, round, golden yellow pigmented colonies

Pseudomonas aeruginosa colonies were large, translucent, with a diffused greenish blue pigment.

Serratia marcescens formed medium sized, round, bright red pigmented colonies

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