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Commercial biologics produced by yeast

What are current the commercial biologics produced by yeast?

Saccharomyces cerevisiae, Escherichia coli, and mammalian cells are the most widely used host systems for biopharmaceutical protein production, accounting for 15%, 31%, and 43% of biopharmaceutical products developed, respectively.

The main strength of E. coli is its capacity for fast and hardy growth in bioreactors using simple media, although producing eukaryotic proteins in E. coli often results in inclusion body formation and/or low specific yields.

Mammalian cells are ideal for incorporating typical eukaryotic post translation modifications, such as glycosylation,however, the culture of mammalian cells is relatively slow, requires complex media, and is vulnerable to viral contaminations. As unicellular eukaryotic microbial host cells, yeast offers unique advantages in biopharmaceutical protein production.

The use of yeasts enables an ideal combination of hardy growth on simple media in large-scale bioreactors with the capacity of the desired post-translational modifications and feasibility in genetic manipulations.

Dozens of pharmaceutical products produced in S. cerevisiae including vaccines and blood factors have been marketed since the first industrial production of recombinant human insulin in S. cerevisiae in 1987, several of which are blockbusters

Here we summary a list of commercial biologics produced by yeast.

System	Protein	Brand Name	Therapeutic area
S. cerevisiae	Hepatitis (or plus other infectious disease) vaccines (I)	Comvax	H. influenzae type B and hepatitis B infection in infants
		Recombivax	Hepatitis B
		Euvax
		Engerix-B
		Fendrix
		Ambirix	Hepatitis A and B
		Twinrix
		Pediarix8	Various conditions inducing hepatitis B in children
		Tritanrix-HB	Diphtheria, tetanus, pertussis, and hepatitis B
		Infanrix Hep B
		Infanrix-Penta	Diphtheria, tetanus, pertussis, polio, and hepatitis B
		Infanrix-Hexa	Diphtheria, tetanus, pertussis, hepatitis B, polio, and H. influenzae type B
		Hexavac
		Procomvax	H. influenzae type B and hepatitis B
		Primavax	Diphtheria, tetanus, and hepatitis B
		HBVaxPro	Hepatitis B in children and adolescents
	Lepirudin (S)	Refludan	Heparin-induced thrombocytopenia type I
	Desirudin (S)	Revasc	Venous thrombosis
	Insulin (S)	Actrapid, Velosulin, Monotard, Insulatard, Protaphane, Mixtard, Actraphane, Ultratard	Diabetes mellitus
	Insulin aspart (S)	Novolog, Novolog FlexPen, Novolog Penfill, NovoRapid, NovoRapid Penfill, Novomix 30, Novolog mix 70/30
	Insulin detemir (S)	Levemir, Levemir FlexPen
	GLP-1 (S)	Victoza	Type 2 diabetes
	Glucagon (S)	GlucaGen	Hypoglycemia
	GM-CSF (S)	Leukine	Cancer, bone marrow transplant
	HGH (S)	Valtropin	Dwarfism, pituitary turner syndrome
	PDGF (I)	Regranex	Lower extremity diabetic neuropathic ulcers
		GEM 125	Periodontal defects
	HPV vaccine (I)	Gardasil	Cervical cancer caused by human papillomavirus (HPV)
	Rasburicase (I)	Fasturtec, Elitex	Hyperuricemia
P. pastoris	Ecallantide (I)	Kalbitor	Hereditary angioedema
	Insulin (S)	Insugen	Type 2 diabetes
	Human serum albumin (S)	Medway	Blood volume expansion
	Hepatitis vaccine (I)	Shanvac	Hepatitis B
	IFN-α 2b (S)	Shanferon	Hepatitis C, cancer
	Ocriplasmin (I)	Jetrea	Vitreomacular adhesion (VMA)
	Anti-IL-6R Ab (I)	Nanobody ALX-0061	Rheumatoid arthritis
	Anti-RSV Ab (S)	Nanobody ALX00171	Respiratory syncytial virus (RSV) infection
	HB-EGF (I)		Treatment of interstitial cystitis/bladder pain syndrome (IC/BPS)
	Collagen (I)		Medical research reagents/dermal filler
H. polymorpha	HBV vaccine (I)	Hepavax-Gene	Hepatitis B
Y. lipolytica	Pancrelipase (S)	Creon, Ultresa, Viokase	Exocrine pancreatic insufficiency

Ref: Yeast synthetic biology for the production of recombinant therapeutic proteins

Genetic and other difference among various yeast system

What are the main differences among various yeast system?

Saccharomyces cerevisiae is one of the best-characterized eukaryotes and most widely used host systems for biopharmaceutical production since the early days of genetic engineering and recombinant protein production.

Recently, nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis have been developed as alternative hosts for the production of recombinant proteins.

Here we summary the main differences among various yeast system.

Yeast species	Attributes
Saccharomyces cerevisiae	Favorable public acceptance GRAS status The most well studied of simple eukaryotes Amenable to both classical genetics and modern recombinant DNA techniques Versatile vector systems (episomal, integrative, copy-number regulated) are available (Invitrogen) A wide range of mutant strains Well-established fermentation and downstream processing Hypermannosylation with immunogenic terminal _-1,3-linked mannose residues Genome sequencing: Reference strain S288C; 12 157 Kb (6273 ORFs); Accession number PRJNA128
Pichia pastoris	GRAS status Tightly regulated, methanol-inducible AOX promoters A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes Can grow rapidly on inexpensive media at high cell densities (up to 150 g DCW L_1) Integrated vectors developed that help genetic stability of the recombinant elements, even in continuous and large-scale fermentation processes Well-established commercial vector systems and host strains (Invitrogen) A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal _-1,3-linked mannose residues Genome sequencing: Reference strain GS115; 9216 Kb (5040 ORFs); Accession number PRJNA39439, PRJEA37871
Hansenula polymorpha	GRAS status polymorpha Stringently regulated strong promoters (MOX, FMDH, etc.) A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes Stable, multicopy integration of foreign DNA into chromosomal locations Thermotolerant (growth up to 45 _C), resistant to heavy metals and oxidative stress Can assimilate nitrates A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal _-1,3-linked mannose residues Genome sequencing: Reference strain DL1; 9056 Kb (5325 ORFs); Accession number PRJNA60503
Schizosaccharomyces pombe	An oleaginous yeast, based on its ability to accumulate large amounts of lipids GRAS status Can grow in hydrophobic environments, that is able to metabolize triglycerides, fatty acids, n-alkanes, and n-paraffins as carbon sources for the bioremediation of environments contaminated with oil spills Can secrete a variety of proteins via cotranslational translocation and efficient secretion signal recognition similar to higher eukaryotes Availability of a commercial expression kit (YEASTERN BIOTECH CO., LTD.) Salt tolerance A lesser extent of hypermannosylation compared to S. cerevisiae; a lack of the immunogenic terminal _-1,3-mannose linkages Genome sequencing: Reference strain CLIB122; 20 503 Kb (7042 ORFs); Accession number PRJNA12414
Kluyveromyces lactis	A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes Lactose-fermenting present in milk and whey, and the strong, lactose inducible LAC4 promoter Very high cell density (> 100 g DCW L_1) Able to use both integrative and episomal expression vectors An available easy-to-use reagent kit for K. lactis protein expression (New England Biolabs) Terminal N-acetylglucosamine and no mannose phosphate Genome sequencing: Reference strain NRRL Y-1140; 10 689 Kb (5502 ORFs); Accession number PRJNA12377

Ref: Yeast synthetic biology for the production of recombinant therapeutic proteins

Summary of recombinant biological molecules used Pichia pastoris for production

The P. pastoris has also been established as a versatile cell factory for the production of thousands of biomolecules both on a laboratory and industrial scale.

Product	Used strain	Used vector	Usage
Lycopene and __carotene	X_33	pGAPZA	Feed supplements
Plectasin	X_33	pPICZ_A	Antibacterial peptide
Bovine lactoferrin	KM71H	pJ902	Transferrin and antibacterial protein
Bovine IFN__	GS115	pPIC9K	Prevention and therapy of viral diseases
Apidaecin	SMD1168	pPIC9K	Antibacterial peptide
hPAB__	GS115	pPIC9K	Antibacterial peptide
Tachyplesin I	GS115	pGAPZ_B	Antibacterial peptide
Snakin_1	GS11	pPIC9	Antimicrobial peptide
PAF102	X_33	pGAPZA	Antifungal peptide
Pisum sativumÊdefensin 1	GS115	pPIC9K	Antifungal peptide
Class I chitinase	KM71H	pPICZ_A	Antifungal peptide
Ch_penaeidin	KM71H	pPIC9K	Antimicrobial peptide
Hispidalin	GS115	pPICZ_A	Antimicrobial peptide
Fowlicidin_2	X_33	pPICZ_A	Antimicrobial peptide
Parasin I	X_33	pPICZ_A	Antimicrobial peptide
CecropinA_thanatin	X_33	pPICZ_A	Antimicrobial peptide
Type I collagen			Connective tissue
Human serum albumin	GS115	pPIC9K	Maintaining osmolarity and carrier in blood
Legumain	X_33	pPICZ_A	Lysosomal protease
Goat chymosin	X_33	pPICZ_A	Hydrolysis of __casein
Carrot antifreeze protein	GS115	pPIC9K	Inhibition of gluten deterioration
Proinsulin	SuperMan5	pPICZ_A	Treatment of diabetes mellitus
hIFN__	X_33, GS115, KM71H, CBS7435	pPICZ_, pPIC9, pPpT4aS	Critical cytokine for innate and adaptive immunity
IL_1_	GS115, SMD1168, X_33	pPICZ_A	Proinflammatory cytokine
IL_3	X_33	pPICZ_A	Multipotent hematopoietic cytokine
IL_11	GS115	pPINK_HC	Thrombopoietic growth factor
IL_15	X_33	pPICZ_A	Differentiation and proliferation of T, B, and NK cells
Cyanate hydratase	GS115	pPICZ_A	Detoxification of cyanate and cyanide
Human antiplatelet scFv antibody	X_33	pPICZ_A	Treatment of atherosclerosis
__Amylase	X_33	pPICZ_A	Starch saccharification
Human epidermal growth factor	GS115	pPIC9K	Generation of new epithelial and endothelial cells
Bromelain	KM71H	pPICZ_A	Oedematous swellings
Keratinocyte growth factor	X_33	pPICZ_A	Epithelialization_phase of wound healing
DM64	X_33	pPICZ_A	Anti_myotoxic
Trypsin	GS115	pPIC9K	Hydrolysis of proteins in the digestive system
Human sialyltransferase	KM71H	pPICZ_B	Pharmacological uses
Transglutaminase	GS115	pPIC9K	Restructured meat products
Streptokinase	X_33	pPICZ_A	Thrombolytic medication
Staphylokinase	GS115, KM71H	pPICZ_A	Thrombolytic medication
TFPR1	X_33	pPICZ_A	Adjuvant

Table:Summary Of Recombinant Biological Molecules Used Pichia Pastoris For Production

Ref: Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

Common Recombinant subunit vaccine expressed in Pichia pastoris

Subunit vaccines often suffer from poor immunogenicity and require certain helper molecules known as adjuvants to induce or enhance an appropriate immune response to the antigen. T‐cell activation is crucial in inducing protective immune responses. Antigens mannosylated by P. pastoris have shown to have enhanced antigen presentation and T‐cell activation properties compared with their nonglycosylated counterparts. Therefore, glycoproteins derived from P. pastoris have the potential to function as adjuvants.

Construct name	Used strain	Used vector	Targeted disease
PIMP‐V1 and PIMP‐V2	KM71	pPICZαA	Malaria
P1‐3CD	PichiaPink	pPink‐HC	Hand–foot–mouth disease
DENV‐3 E	KM71	pPICZ‐A	Dengue
CHIKV‐C‐E3‐E2‐6K‐E1	GS115	pPIC9K	Chikungunya
Gp350	GS115	pPICZαA	EBV infection
RBD219‐N1	X‐33	pPICZαA	SARS
VP2–VP5–Fc	GS115	pPIC9K	Infectious bursal
F protein	GS115	pPICZαA	Newcastle
OmpA	GS115	pPIC9K	P. mirabilis infection
BoNT Hc	X‐33	pPICZ‐A	Botulism
Tc52	GS115	pPICZαA	Chagas
Apa	GS115	pPIC9K	Tuberculosis
HBHA	GS115	pPIC9K	Tuberculosis
CFP10‐Fcγ2a	GS115	pPICZαA	Tuberculosis
ESAT6‐CFP10‐Fcγ2a	GS115	pPICZαA	Tuberculosis
ESAT6‐Fcγ2a	GS115	pPICZαA	Tuberculosis
CFP10‐HspX‐Fcγ2a	GS115	pPICZαA	Tuberculosis
ESAT6‐HspX‐Fcγ2a	GS115	pPICZαA	Tuberculosis
Glycoprotein D	GS115	pPIC9K	HSV‐2 infection
OmpA‐Fc	GS115	pPIC9K	Bordetellosis

Table: Common Recombinant Subunit Vaccine Expressed In Pichia Pastoris

Ref: Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

What are the common vector/plasmids used in the Pichia pastoris Protein expression system?

The expression of any recombinant gene in P. pastoris has three phase: (a) Cloning of a new gene into a suitable expression vector, (b) insertion of the cloned vector into P. pastoris host genome; and (c) trial of the potential different strains for the expression of the recombinant integrated gene.

What are the major components in the expression vector of P. pastoris?

The expression vectors in the P. pastoris expression system are one of the major components of this system. These vectors are composed of three sequences:

Promoter sequence (most often AOX1) in 5′ region
Transcriptional termination sequence in 3′ region which is essential in the processing and polyadenylation of messenger RNAs
Single or multiple cloning sites, necessary for the insertion of the gene of interest.

Why expression vector of Pichia pastoris need to be linearized?

The episomal vectors can replicate either autonomously in the cytoplasm or as part of a chromosome. But the vectors used in P. pastoris have no stable episomal status; therefore, they should first be linearized with enzymes and then be integrated into the P. pastoris chromosome.

Common plasmids used in the P. pastoris expression system to produce extracellular and intracellular proteins are listed in Tables 1 and 2, respectively.

Vector name	Marker gene	Used strain	Recombinant protein
pPIC3.5K	His4, Kan, Amp	KM71	Maltooligosyltrehalose synthase
		SMD1168	Camellia sinensisÊheat shock protein
		GS115	Pleurotus ostreatusÊlaccases
		GS115	Rhizopus oryzaeÊLipase
		GS115	HSA/GH fusion protein
pPICZ	Shble	X_33	Aquaporin
		KM71	Membrane protein
		KM71	Dengue virus envelope glycoprotein
pHIL_D2	His4, Amp	GS115	Prostaglandin H synthase_2
		GS115	CatA1 and SODC
		KM71	RhodococcusÊnitrile hydratase

Table 1. Common Pichia pastoris expression vectors for the production of intracellular proteins

Vector name	Marker gene	Used strain	Recombinant protein
pPIC9K	His4, Kan, Amp	GS115	Xylanase
		GS115	Porcine circovirus type 2
		GS115	Endo_1,3(4)_b_d_glucanase
		GS115	Staphylokinase
pPICZ_	Shble	SMD1168	Human chitinase
		GS115	Human topoisomerase I
		GS115	Human interferon gamma
		X_33	C_reactive protein
		SuperMan5	Insulin
		X_33	Human RNase4
pHIL_S1	His4, Amp	GS115	Rabies virus glycoprotein
		GS115	Rhizopus oryzaeÊLipase
		KM71	Camel lactoferricin
pGAPZ_	Shble	GS115	Acyl homoserine lactonase
		SMD1168	Variable lymphocyte receptor B
		X_33	Human gastric lipase
pJL_SX	FLD1, Amp	MS105	Formaldehyde dehydrogenase
pBLHIS_SX	His4, Amp	JC100	Leukocyte protease inhibitor

Table 2. Common Pichia pastoris expression vectors for the production of secretory proteins

Ref: Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

Representative SARS-CoV And MERS-CoV RBD-Specific Neutralizing Antibodies

Virus neutralizing antibodies induced by vaccines or infected virus play crucial roles in controlling viral infection. Currently developed SARS-CoV- and MERS-CoV-specific nAbs include monoclonal antibodies (mAbs), their functional antigen-binding fragment (Fab), the single-chain variable region fragment (scFv), or single-domain antibodies [nanobodies (Nbs)]. They target S1-RBD, S1-NTD, or the S2 region, blocking the binding of RBDs to their respective receptors and interfering with S2-mediated membrane fusion or entry into the host cell, thus inhibiting viral infections.

Representative SARS-CoV and MERS-CoV RBD-specific nAbs are summarized below.

Ab name	Source	Neutralizing activity	Neutralizing mechanism	Protective efficacy	Refs^b
S230.15 m396 mAbs	Human	Neutralize human (strains GD03, Urbani, Tor2) and palm civet (strains SZ3, SZ16) SARS-CoV infection	Recognize epitopes (residues 408, 442, 443, 460, 475) on SARS-CoV S1 protein, interfering with RBD–ACE2 receptor interaction	Protect mice against challenge of SARS-CoV (strains Urbani, rGD03, or rSZ16)	[2]
S109.8 S227.14 S230.15 mAbs	Human	Neutralize human (Urbani, GZ02, CUHK-W1), palm civet (HC/SZ/61/03), and raccoon dog (A031G) SARS-CoV infectious clones containing S variants	Inhibit the binding of SARS-CoV RBD–ACE2 receptor	Protect mice against challenge of SARS-CoV infectious clones (Urbani, GZ02, HC/SZ/61/03) or mouse-adapted strain (MA15)	[3]
80R scFv, mAb	Human	Neutralize live SARS-CoV (strain Urbani) infection	Recognize epitopes on SARS-CoV S1 (residues 261–672), blocking RBD–ACE2 binding and inhibiting syncytium formation	NA	[4]
CR3022 CR3014 scFv, mAb	Human	Neutralize live SARS-CoV (strain HKU-39849) infection; CR3022 could neutralize CR3014 escape variants	Recognize epitopes on SARS-CoV RBD (residues 318–510); CR3022 binds SARS-CoV-2 RBD with high affinity	CR3014 protects ferrets against SARS-CoV (strain HKU-39849) infection	[6]
33G4 35B5 30F9 mAbs	Mouse	Neutralize human (strains GD03, Tor2) and palm civet (SZ3) pseudotyped SARS-CoV infection	Recognize epitopes on SARS-CoV RBD, blocking RBD–ACE2 receptor binding	NA	[5]
MERS-27 m336 MERS-GD27 MCA1 mAbs, Fabs	Human	Neutralize divergent strains of pseudotyped and live (strain EMC2012) MERS-CoV infection	Recognize a number of key epitopes on MERS-CoV RBD protein, blocking RBD–DPP4 receptor binding	Prophylactically and therapeutically prevent and treat MERS-CoV (strain EMC2012) challenge in hDPP4-Tg mice, rabbits, or common marmosets	[7,8]
4C2 h hMS-1 mAbs	Humanized	Neutralize divergent strains of pseudotyped and live (strain EMC2012) MERS-CoV infection	Recognize epitopes (residues 510, 511, 553) on MERS-CoV RBD protein, blocking RBD–DPP4 receptor binding	Prevent MERS-CoV (strain EMC2012) challenge in Ad5/hDPP4-transduced or hDPP4-Tg mice	[7]
Mersmab1 4C2 D12 mAbs	Mouse	Neutralize pseudotyped and live (strain EMC2012) MERS-CoV infection	Recognize a number of key epitopes on MERS-CoV RBD protein, blocking RBD–DPP4 receptor binding	NA	[7]
HCAb-83 Nb	Dromedary camel	Neutralizes live MERS-CoV (strain EMC2012) infection	Recognizes epitope (residue 539) on MERS-CoV RBD protein	Prophylactically prevents MERS-CoV (strain EMC2012) challenge in hDPP4-Tg mice	[8]
NbMS10-Fc Nb	Llama	Neutralizes multiple strains of pseudotyped and live (strain EMC2012) MERS-CoV infection	Recognizes epitope (residue 539) on MERS-CoV RBD protein	Prophylactically and therapeutically prevents and treats MERS-CoV (strain EMC2012) challenge in hDPP4-Tg mice	[8]

Ref:

[1]Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses
[2]Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies
[3]Structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge
[4]Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association
[5]Cross-neutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein
[6]Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody
[3]MERS-CoV spike protein: a key target for antivirals
[8]Advances in MERS-CoV vaccines and therapeutics based on the receptor-binding domain

Nanobody Engineering and strategies targeting the immune stroma of tumors.

In the last decade, cancer immunotherapies have produced impressive therapeutic results. However, the potency of immunotherapy is tightly linked to immune cell infiltration within the tumor and varies from patient to patient. Thus, it is becoming increasingly important to monitor and modulate the tumor immune infiltrate for an efficient diagnosis and therapy. Various bispecific approaches are being developed to favor immune cell infiltration through specific tumor targeting. The discovery of antibodies devoid of light chains in camelids has spurred the development of single domain antibodies (also called VHH or nanobody), allowing for an increased diversity of multispecific and/or multivalent formats of relatively small sizes endowed with high tissue penetration. The small size of nanobodies is also an asset leading to high contrasts for non-invasive imaging. The approval of the first therapeutic nanobody directed against the von Willebrand factor for the treatment of acquired thrombotic thrombocypenic purpura (Caplacizumab, Ablynx), is expected to bolster the rise of these innovative molecules.

Figure 1. Nanobody-based formats in development for tumor immunotherapy and imaging.
(A) Camelids specificity domains derived of conventional IgG1 or HcAbs (IgG2 and IgG3).
The nanobody crystal structure shown is pdb entry 6GZP. (B) Formats of nanobody engineered
molecules discussed in this review. Nb: nanobody; ARD: antigen recognition domain; TAA: tumor
associated antigen.

Figure 2. Nanobody-based strategies targeting the immune stroma of tumors. Nanobody-derived
immunomodulatory molecules are under investigation to increase anti-tumor immunity (orange
arrows) and prevent tumor-driven immune suppression (blue arrows). TAA: Tumor associated antigen;
IC: Immune checkpoint; ARD: Antibody recruiting domain.

Ref: Nanobody Engineering: Toward Next Generation Immunotherapies and Immunoimaging of Cancer. Antibodies (Basel). 2019 Jan 21;8(1):13. doi: 10.3390/antib8010013.

what are the unique features of nanobody and the applications

What are the unique features of nanobodies

Nanobody show some striking advantages versus conventional Abs that are noteworthy since they combine desirable features of mAbs with some of the beneficial properties of small molecule drugs.

They can bind a broad range of epitopes showing affinities in the nanomolar or even picomolar range. This implies that the affinities of Nbs are comparable or superior to those of conventional mAbs, reaching picomolar affinity constant range.

Possibility of formatting or multimerization. Given the small size and their monomeric nature of Nbs, they can be genetically engineered to obtain modular blocks that can be fused to form a new construct which will enable multispecificity and versatility.

They are highly stable and soluble. Nbs present optimal biophysical and biochemical properties including solubility, thermal tolerance, and proteolytic resistance. The folding of CDR3 loop and the hydrophilic content of the framework-2 region gives them high solubility in aqueous solutions and lack of aggregation.

Deep and fast tissue penetration and rapid blood clearance. Since passive intercellular diffusion rate within a tissue depends on the molecular size and is approximately inversely proportional to it, a 15-kDa monovalent Nb should improve the penetrability when compared with the performance of conventional mAbs (150 kDa).

Recognition of hidden epitopes. Crystallographic studies of conventional Abs have revealed that in most cases the Ag-binding surface is flat or concave.

Immunogenicity. Camelid VHH domains present high degree of homology with human VH domains, which means that to date they have not shown any unexpected immunogenicity reactions, which can be a potential concern for clinical applications

Production costs. mAbs are large multimeric proteins that usually undergo post-translational modifications. Hence, their production requires sophisticated machinery only found in eukaryotic systems.

Applications of nanobodies as diagnostic and therapeutic tools

Detection of proteins and microorganisms
Detection of small molecules
Imaging applications
Therapeutic tools

Nanobody production scheme using a phage display library. The genetic information can be obtained by active immunization using an
immunogen or using non-immunized animals by collection of blood which contains the lymphocytes. Increasing the diversity using semisynthetic libraries and randomly introducing amino acids in the CDR region is possible in order to improve the binding capabilities.
After the insertion of the plasmid in a bacterophage, the phagemid library is ready for this selection. Phage ELISA is the usual way to perform the biopanning. After the best candidate is selected, the production of the nanobody can be expressed in bacteria, yeast, or mammalian cells through its corresponding expression vector

Ref: Nanobody: outstanding features for diagnostic and therapeutic application. Anal Bioanal Chem. 2019 Mar;411(9):1703-1713. doi: 10.1007 s00216-019-01633-4. Epub 2019 Feb 8.

Single B Cell Cloning and Production of Rabbit Monoclonal Antibodies

Monoclonal antibodies are among the most significant biological tools used in medicine and biology that
have revolutionized the field of diagnostics, therapeutics, and targeted drug delivery systems for many
diseases. Among them, rabbit monoclonal antibodies have attracted significant attention for having high
affinity and specificity. During the past few decades, different techniques have been developed to produce
monoclonal antibodies. Single B cell cloning technology offers many advantages compared to other
methods and has been used to generate monoclonal antibodies from different species including rabbits.
This review briefly describes some of these methods, with main focus on single B cell cloning and
production of rabbit monoclonal antibodies.

Comparison between monoclonal and polyclonal antibodies

Diagram summarizing the generation of antigen-specific rabbit monoclonal antibodies by single B cell
cloning. PBMCs peripheral blood mononuclear cells, FACS fluorescence-activated cell sorting, RT-PCR reverse
transcription polymerase chain reaction, VH variable domain of heavy chain, VL variable domain of light chain,
LS leader sequence, HCD heavy-chain constant domain, LCD light-chain constant domain, HC heavy chain, LC
light chain, ELISA enzyme-linked immunosorbent assay

Ref: Single B Cell Cloning and Production of Rabbit Monoclonal Antibodies. Book: Genotype Phenotype Coupling pp 423-441

Genomic Basics

Human Genome

When someone refers to the genome, they often mean all of the information contained within an individual’s DNA. In fact, human genome actually refers to all of human DNA plus the proteins required to read and maintain it, as well as the many particles that help store and give shape to your DNA. Think of the entire genome as a library, and DNA as a genome encyclopedia.

DNA as a Genome Encyclopedia

The information in human genome encyclopedia is organized into about 21,000 gene chapters which are located within 23 chromosome volumes. Each individual has two nearly identical copies of each chromosome volume, one copy from each parent.

All of the entries in human genome encyclopedia are written in a special language—the DNA code. The DNA alphabet has only four letters, A, C, T, and G, representing the four different chemical bases. In total, each person has more than three billion DNA letters in each set of their 23 chromosomes.

The DNA Code

Although it may appear to be one long string of DNA letters, by studying the DNA code, researchers have learned that it also contains a complicated system of punctuation. Special codes signal when the DNA letters are part of a gene. Three letter combinations of DNA refer to specific amino acids. Within a gene, the amino acids provide instructions that can be read by special substances in the body to determine what type of protein needs to be made.

The order of all these DNA letters is called your genome sequence. When someone refers to your genome sequence, they mean the unique combination of DNA sequences from chromosome 1 through your sex chromosomes (XX or XY). Although we have two copies of each chromosome set, we only report one complete sequence.

This is because most of the DNA sequence is the same between the two chromosome sets. Whenever there are differences, this is noted in the person’s one, combined genome sequence. One person’s genome sequence is very similar to another’s. In fact, more than 99% of the human genome sequence is common to all people. This makes sense because we are all the same species (humans), and our bodies tend to have similar features (for example, two arms, two eyes, ten toes) and work in similar manners.

Commercial biologics produced by yeast

Genetic and other difference among various yeast system

Summary of recombinant biological molecules used Pichia pastoris for production

Common Recombinant subunit vaccine expressed in Pichia pastoris

What are the common vector/plasmids used in the Pichia pastoris Protein expression system?

Representative SARS-CoV And MERS-CoV RBD-Specific Neutralizing Antibodies

Nanobody Engineering and strategies targeting the immune stroma of tumors.

what are the unique features of nanobody and the applications

Single B Cell Cloning and Production of Rabbit Monoclonal Antibodies

Genomic Basics

Human Genome

DNA as a Genome Encyclopedia

The DNA Code

Mission

Location

Contact