WO2007099462A2 - Recombinant pichia pastoris strains and process for the production of recombinant human interferon alpha - Google Patents

Abstract

The present invention relates to the field of production of recombinant human interferon alpha. More specifically, the invention discloses novel recombinant Pichia pastoris strains that have been genetically engineered for efficiently producing two kinds of active recombinant human interferon alpha: human interferon alpha 2a and human interferon alpha 2b. The invention also pertains to optimized processes for producing and purifying said interferon alpha.

Description

Recombinant Pichia pastoris strains and process for the production of recombinant human interferon alpha

The present invention relates to the field of production of recombinant human interferon alpha. More specifically, the invention discloses novel recombinant Pichia pastoris strains that have been genetically engineered for efficiently producing two kinds of active recombinant human interferon alpha: human interferon alpha 2a and human interferon alpha 2b. The invention also pertains to optimized processes for producing and purifying said interferon alpha.

Interferons (IFNs) are essential components of the innate line of defence of vertebrates against infectious agents and tumour development and progression. Recombinant interferon α has been used for almost two decades, as a therapeutic agent in a variety of diseases ranging from viral infections to different types of cancer.

Recombinant human interferons have been expressed in Escherichia coli (Nagata et aL 1980; Remaut et al., 1983; Mizoguchi et al., 1985; Babu et al., 2000), yeast (Hitzeman et al.. 1981 ; Zsebo et al., 1986). baculovirus infected insect cells (Smith et al., 1983; Voss et al., 1993) and animal cells (Rossmann et al., 1993). The yield of recombinant IFNs using E. coli is by far higher than the one obtained using the other systems. However, IFNs are produced in E. coli as insoluble inclusion bodies that need to be subjected to solubilization and refolding steps to allow generation of functional and active proteins (Babu et al., 2000). Such steps are usually time consuming and need extensive optimisation to reach reasonable production yields. Furthermore the products generated in E. coli and intended for human use need to be endotoxin free. This requirement further complicates the production processes using E. coli and impact negatively on the final cost of the products. Yeast and particularly the methylotrophic yeast Pichia pastoris, have numerous advantages as host system for the production of recombinant proteins from higher eukaryotes. They combine the ease, simplicity and cheapness of bacterial expression systems. Yeast is simple to cultivate on inexpensive growth media and the array of techniques for the making of recombinant clones is now well advanced (Clare and Romanos, 1995; Sreekrishna et al., 1997; Cereghino and Cregg, 2000). Like eukaryotes, yeasts provide an adequate environment for post-translational processing and secretion, resulting in a product that is often identical or more similar to the native protein. Moreover, Pichia pastoris grows at very high cell density on minimal medium. The success of the Pichia system is largely due to the use of the strong, tightly regulated alcohol oxidase (AOX l) promoter to drive heterologous protein expression. In wild-type Pichia, this promoter controls the expression of alcohol oxidase 1 , the first enzyme in methanol metabolism (Cregg et al., 1989b), which can account for up to 30% of the total soluble protein (Couderc and Baratti, 1980). Another key feature of the system is that a high cell density can be achieved using a minimal salt medium which is cost effective (Sreekrishna et al., 1989). The strong promoter, coupled with the high cell density fermentation, has allowed production of recombinant proteins at high concentration. The highest yields reported are 22 g per L of S-hydroxynitrile lyase, an intracellular expressed enzyme (Hasslacher et al., 1997), and 1 1 g per L culture volume of secreted gelatine (Werten et al., 1999).

For these reasons, Sudhir et al. (WO 01/68827) have proposed a process for the production of human alpha interferon from genetically engineered P. pastoris. More recently, Villarete et al. (US 2003/0065148) proposed some improvements to this process, applied to Interferon α-1 (also known as IFNaD). In particular, they introduced, in the expression vector, fused to the 5^" extremity of the IFNaD coding sequence, a sequence coding for a signal peptide sequence enabling the secretion of the recombinant protein. Even more recently, Lohray et al. (WO 2004/039996) described the production of !FNα2b using genetically-engineered P. pastoris strains transformed with an expression vector expressing a secreted lFNα2b. The producing strain and the process described in this document are however not optimal, since a complex medium is needed to obtain high production yields.

When using P. pastoris as a producing strain, different phenotypes of the recombinant clones can be obtained regarding methanol metabolism, depending on how the expression cassette is integrated into Pichia genome by homologous recombination. Mut⁺ clones can metabolise methanol quickly since in these clones the AOXI gene is intact, whereas clones in which AOXl gene has been replaced by the expression cassette due to a double cross-over event, show slow growth on methanol (Mut^s).

The Mut phenotype of the recombinant clone is important because protein expression driven by the AOXl promoter requires the presence of methanol. Special care must be taken to keep the methanol concentration below toxic levels while maintaining the induction (Stratton et al., 1998).

Downstream processing is another key issue that needs to be considered during the design of biotechnological processes. Common purification processes of recombinant proteins employ sequential chromatography techniques such as cation exchange, anion exchange, gel filtration, etc. They are therefore costly and time- consuming.

The present invention aims at overcoming at least some of the limitations of the current processes for producing human alpha interferons. A first aspect of the present invention is a nucleic acid molecule comprising a coding sequence encoding a human alpha interferon, wherein said sequence is optimized for expression in Pichia pastoris. Indeed, it has been shown that organisms display a non-random pattern of synonymous codon usage (Grantham et al., 1980; Nakamura et al., 1999), and that exogenous coding sequences using the codons which are more frequently used by the host itself may be more efficiently translated than sequences not respecting this codon bias. Hence, an "optimized sequence" for expression in Pichia pastoris is an artificial coding sequence in which the codons have been chosen according to the codon bias of Pichia pastoris, as disclosed for example by Sinclair et al. (2002).

In a particular embodiment of the nucleic acid according to the invention, the encoded human alpha interferon is HulFNα2a and the coding sequence is SEQ ID No: 1.

Another nucleic acid according to the invention comprises a coding sequence encoding a human interferon α2b, wherein said coding sequence is SEQ ID No: 3. This sequence has been cloned by an RT-PCR approach, from healthy individual leukocytes exposed to the New Castle Disease virus.

The invention also relates to an expression vector for transforming a Pichia pastoris strain, which comprises, in the 5' to 3^" direction and operably linked :

(i) a Pichia /κw/øra-recognized transcription and translation initiation region, (ii) a sequence coding for a secretion signal enabling protein secretion in

Pichia pastoris,

(iii) a coding sequence encoding a human alpha interferon, such as the sequences defined above (in particular, the sequences SEQ ID No: 1 or SEQ ID No: 3, or an optimized sequence for expression in P. pastoris, encoding a HulFNα2b of SEQ ID No: 4), and

(iv) a Pichia pasloris-rtcogmzzd transcription and translation termination region.

A Pichia pastoris strain which has been transformed by a vector as above-described, and which produces human alpha interferon, is also part of the present invention. In a preferred embodiment of this aspect of the invention, the transformed strain has been obtained by transforming a Mut^s KM71 H Pichia pastoris strain.

Preferred examples of Pichia pastoris strains according to the invention are KM71 H/PlCZαrHulFNα2b, deposited at the Collection Nationale de Cultures de Microorganismes (CNCAf) on February 01 , 2006, under the n°I-3562, and

IRDF/INF/1PT05, deposited at the Collection Nationale de Cultures de Microorganismes

(CNCM) on September 1 1 , 2006, under the n°I-3668.

Another aspect of the present invention is a process for producing a human alpha interferon by culturing a Pichia pastoris strain which expresses, under the control of a promoter regulated by methanol, a secretable form of said human alpha interferon. This process comprises the following steps:

(i) inoculation of a synthetic culture medium in a bioreactor, by a preculture of said Pichia pastoris strain, wherein said synthetic medium contains glycerol as a carbon source, and has a defined salt composition; (ii) batch culture in said bioreactor, at 30⁰C, pH5, under agitation and oxygenation;

(iii) fed-batch culture on glycerol at 3O⁰C, pH5, under agitation and oxygenation:

(iv) induction phase: fed-batch culture on methanol, in a synthetic culture medium supplemented with EDTA at a final concentration of 10 mM.

Steps (i) to (iii) represent a biomass build-up phase, resulting in a great amount of P. pastoris cells, whereas step (iv) is an induction phase, during which said cells produce the interferon.

The process of the invention is preferably carried out with a Pichia pastoris strain according to the invention, as described above.

In the above process, the synthetic culture medium is preferably obtained as follows:

- glycerol (40 g/1), K₂SO₄ ( 18.2 g/1), MgSO₄ (7.28 g/1), KOH (4.13 g/1), CaSO₄.2H₂O (0.93 g/1) and 85% orthophosphoric acid (26,7 ml/1) are dissolved in deionised water and sterilized in the bioreactor;

- 5 ml/1 basal salts of fermentation PTM 1 and 2 ml/1 of 500X biotin are added to the medium after sterilization, and culture medium pH is adjusted to 5 by addition of 25% ammonia (w/v), wherein the PTM l solution contains: CuSO₄.5H₂O (6 g I^"1), NaI (0.08 g I^"1), MnSO₄-H₂O (3 g I^"1), Na₂MoO₄^H₂O (0.2 g I^"1), H₃BO₃ (0.02 g I^"1), CoCl₂ (0.5 g I^"1), ZnCl₂ (20 g I^"1), FeSO₄.7H₂O (65 g I^"1), biotin (0.2 g I ¹) and H₂SO₄ 98% (5 ml I^"1).

When performing this process, the fed-batch solution used in step (iii) preferably contains glycerol (450 g/1), PTM l (8 ml/1) and 500X biotin (5 ml/1), and is added at a rate of 15 ml/l/h.

The fed-batch solution used in the induction phase (step (iv) of the above-described process) preferably contains methanol at 100% (973 ml/1), 500X biotin (5 ml/1) and PTM l (8 ml/1) and the feeding rate is modulated to keep the methanol residual level under 0.6% (v/v). The induction phase lasts for at least three days, preferably 4 or 5 days, during which the following components are added to the bioreactor at regular intervals of 24 hours: casaminoacids at a final concentration of 0.1 % (w/v) and Yeast Nitrogen Base at a final concentration of 0.1 % (w/v).

In a particular way of performing the above process according to the invention, step (iii) is initiated once the optical density at 600 nm of the culture in step (ii) has reached 80, and the fed-batch culture of step (iii) lasts until the dry weight reaches at least 100 g/1, preferably 1 10-120 g/l.

In addition, it can be advantageous, when performing the production process according to the invention, to change the culture medium between the biomass build-up and the induction phases. Jn this case, the cells are harvested, resuspended in fresh medium and returned back to the bioreactor, between steps (iii) and (iv). Hence, the induction phase takes place in a medium which is completely free of complex medium, since the small volume of medium of the preculture is removed before the beginning of the induction phase. Indeed, it can be noted that the preculture used in step (i) is not necessarily done in a synthetic medium. The induction medium is preferably supplemented with EDTA (final concentration of 10 mM).

An example of detailed conditions for performing the production process according to the invention is described in Example 7 below. In this non-limiting example, the bioreactor is a 5-liter reactor, equipped with a process control system of data, and in which the batch culture is carried out with an initial culture volume of 2 litres inoculated with the 200 ml preculture in BMGY medium, in the following conditions: temperature = 30⁰C, stirrer speed = 800 rpm, pH maintained at 5 by addition of 25% ammonia, dissolved oxygen set at 40% of air saturation by stirrer cascading and enrichment of the inlet air with pure oxygen when required. The conditions of stirring, pH, and oxygenation are preferably the same during the induction phase. Other aspects of the present invention are processes for purifying human alpha interferon secreted in the culture medium by recombinant yeasts. A first purification process according to the present invention comprises the following steps:

(i) eliminating the biomass by centrifugation, (ii) filtering the supernatant through a 0.1 μm cartridge in the presence of

Triton X-IOO at 1%,

(iii) desalting the permeate by diafiltration using a 5 kDa cartridge and, for example, lactic acid 50 mM pH4,

(iv) purifying the supernatant by a cation exchange chromatography, for example on a Sepharose SP column, as disclosed in Example 8.1 below.

Alternatively, the desalting step (iii) can be performed by chromatography on a Sephadex G25 column.

A second process according to the invention, for purifying human alpha interferon secreted in the culture medium by recombinant yeasts, comprises the following steps:

(i) dilution of the culture medium with a solution of lactic acid (25 mM). NaCl (0.1 M), pH4, to an optical density at 600 nm equal to 100;

(ii) purification of the diluted medium using an expanded bed chromatography on a cation exchange support, for example a Streamline SP XL device as described in example 8.2 below.

When performing any of the purification processes according to the invention, a further step of purification by gel filtration, using sephacryl S-100, can be added.

Of course, these purification processes are preferably performed on medium obtained at the end of the induction phase (iv) of the production process described above, and/or on culture medium obtained after said human alpha interferon has been produced by a recombinant Pichia pastoris strain according to the present invention.

The invention is further illustrated by the following figures and examples. Legends to the figures

Figure 1 : Lane 1 shows the 498 bp RT-PCR reaction product from HulFN-a2b cDNA using the 5' EcoRl forward primer: FlFNα2b and the 3^" STOP/Notl reverse primer: RIFNα2b. Aliquots of 5μl were electrophoresed on a 0.7% agarose gel, TBE 1 X with the 1 Kb DNA ladder (lane L).

Figure 2: Isolation of pP!CZαA/rHulFNα2b recombinant plasmid containing the cDNA sequence encoding human IFNα2b by "colonies-PCR'^" based method. Clones containing the 498 bp !FNα2b gene (Lane 2, 3. 4, 5, 6, 7, 8, 9 10, 1 1 , 12 and 14) as well as lane T+ (Amplified !FNα2b product), show a 498 bp band. Lane T- corresponds to the mixed PCR solution.

Figure 3: Isolation of pPlCZαA/rHuIFNα2b recombinant plasmid containing the cDNA sequence encoding human IFNα2b by restriction analysis using BgIU restriction enzyme. Lane 1. 2, 3 and 4 represent respectively the clones 2. 4. 9 and 12 containing the !FNα2b gene, while lane 5 corresponds to the non recombinant pPlCZαA plasmid (empty). 20μl Aliquots of i?g/I]/digested products were electrophoresed on a 0.7% agarose gel, TBE 1 X with the DNA markers (Boehringer Manheim. Germany) (lane L).

Figure 4: Fig. 4A: pPlCZαA/rHuIFNα2b recombinant plasmid nucleotide sequence. Sequencing result obtained using the forward Aoxl/F sequencing primer. Fig. 4B: pP!CZαA/rHulFNα2b recombinant plasmid nucleotide sequence. Sequencing Result obtained using the reverse Aoxl/R sequencing primer.

Figure 5: Map of the pPICZαA/rHulFNα2b P.pastoris expression plasmid. The rHu\FNa2b gene was inserted between the 5" EcoRl and the 3^"Notl restriction site. The TGA stop codon was introduced at the 3' end of rHu\¥Ncc2b gene, to produce a native C-terminal rHu\VNa2b protein. Figure 6: Analysis of the codon usage of the Human IFNa 2b encoding cDNA sequence.

Figure 7: Analysis of the codon usage of the synthetic rHuIFNα2a encoding cDNA sequence. Figure 8: Clone design Fig. 8A: Design of the first HuJFNα2a synthetic fragment 1 by PCR-assembly using the synthetic primers: FaI . FbI . Fc I . Fig. 8B: Design of the second HulFNα2a synthetic fragment 2 by PCR-assembly using the synthetic primers: Fa2. Fb2 and Fc2. Fig. 8C: construction Design of pPlCZαA/rSynHulFNα2a recombinant plasmid.

Figure 9: 1% agarose gel Analysis of Purified PCR-Assembly product. Lane 1 represents the 200 bp first synthetic PCR-assembly product using the primers: FaI, FbI and FcI (Figure 8A). Lane 2 shows the 267 bp second synthetic PCR-assembly product using the primers: Fa2, Fb2, Fc2 (Figure 8B). Lane L, represent the 100 bp DNA Ladder from Invitrogen.

Figure 10: 1 % agarose gel Analysis of Bglll digested transformed clones Isolation of the Pl recombinant plasmid clone (pGEX4Tl containing the first HuIFNα2a synthetic fragment) was performed by restriction analysis using BgIlI restriction enzyme as recommended by the manufacturer (Amersham-Biosciences). Lanes Ll and L2 represent respectively the lOObp DNA ladder (Invitrogen) and lKiloBase DNA marker (Amersham- Biosciences). Lane 1 corresponds to the control BgIIl digestion product of the recombinant plasmid pGEX4Tl/ HulFNα2b. Lanes 2 to 1 1 correspond to the putative clones containing the 186bp first synthetic IFNα2a sequence.

Figure 1 1 : Nucleotide sequence of recombinant plasmid "Pl", pGEX4Tl containing the first HulFNα2a synthetic fragment.

Figure 12: Fig. 12A: Isolation of the recombinant plasmid clone pGEX4Tl containing the HuIFNα2a Synthetic fragment 2 inserted into the right sense, was performed by colonies-PCR method using the 5' Ffgsl and 3' RfgsII primer pair. Lane 1 for clone 1 , 4 for clone 4, 5 for clone 5, 6 for clone 6 and 8 for clone 8, show a 450 bp band corresponding to the amplified PCR product of the 186 bp fragment Synthetic 1 + the 264 bp right sense inserted fragment Synthetic 2. Lane L, represents the 100 base-Pair Ladder from (Amersham-Biosciences). Fig. 12B : Isolation of the P2 recombinant plasmid clone was performed by restriction analysis using EcoRl and Xhol restriction enzyme. Lane 2 corresponds to the EcoRl/Xhol digested product from Clone 1 (Figure 12A). The electrophoresed profile shows a 498 bp band corresponding to the 498 pb full length Synthetic Human lFNα2a gene. Lane 1 corresponds to the digested product from Pl cloning vector (pGEX4Tl containing the first HuIFNα2a Synthetic fragment 1+ the native -COOH 48 bp sequence). Lane L represents the l kb DNA Ladder from Promega, while lane M corresponds to the 100 base-Pair Ladder from (Amersham-Biosciences).

Figure 13: Fig. 13A: Isolation of pPlCZαA/ Synthetic Human IFNα2a recombinant plasmid containing the cDNA sequence encoding the Synthetic human !FNα2a by "colonies-PCR" based method using the Forward 5^; Ffgsl and the reverse 3' RIFNα2b primer pair.Clones containing the 498 bp !FNα2a gene (Lane 1 , 2, 3, 4, 5, 6, 7, 8, and 9) show a 498 bp PCR-product band, corresponding to the full length Synthetic Human lFNα2a. Lane T- corresponds to the mixed PCR solution. Lane L represents the 100 base-Pair Ladder from Amersham-Biosciences. Fig. 13B : Isolation of pPlCZαA/ Synthetic Human IFNα2a recombinant plasmid containing the cDNA sequence encoding the Synthetic Human !FNα2a by restriction analysis using EcoRl and Notl restriction enzyme. Lane 1 corresponds to the EcoRl/Notl pPlCZαA cloning vector digested product. Lanes 2, 3, 4 and 5 represent respectively the clones 2, 3. 4 and 5 containing the 498 bp Synthetic Human !FNα2a. Lane L represents the lkb DNA Ladder from Promega.

Figure 14: Fig. 14A: pPlCZαA/ Synthetic Human lFNα2a recombinant plasmid DNA Sequencing Result using the forward Aox l/Fsequencing primer. Fig. 14B : pPlCZαA/ Synthetic Human !FNα2a recombinant plasmid DNA Sequencing Result using the reverse Aoxl/R sequencing primer.

Figurel 5: The Sacl digested product from the recombinant pPlCZαA/ Synthetic Human !FNα2a. Lane 1 shows the electrophoresis profile on 0.7% agarose gel, TBE IX of 2 μl phenol/Chloroform/JAA purification product from the digested Sacl pPlCZαA/ Synthetic Human !FNα2a recombinant plasmid. Lane L represents the l kb DNA Ladder from Promega.

Figurelό: The BstX l digested product of the recombinant pPlCZαA/rHulFNα2b. Lane 1 shows the electrophoresis profile on 0.7% agarose gel, TBE I X of 5 μl phenol /Chloroform/ Al A purification product from the digested BstXl pP!CZαA/rHulFNα2b recombinant plasmid. Lane L represents the I KiloBase DNA marker (Amersham-Biosciences).

Figure 17: Fig. 17A: rHulFNα2b level obtained after 48 h of methanol induction from different selective Zeocin clone^'s growth (A) Coomassie-stained-SDS- PAGE gel (15%). The arrow indicates the position of rHu!FNα2b; while Cl , C2, C3, C4,

C6, Cl, C9. Cl O, C 12. C 13, C14 and Cl 5 stands for the corresponding number of clone.

Cl , C2, C3, and C12 were only selected on 500μg/ml zeocin YPD plates, clones 4, 6, 10,

20 were only selected on l OOOμg/ml zeocin YPD plates, while C7, 13 and 14 were selected on 2000μg/ml zeocin YPD plates. Fig. 17B: lmmunoblot with Anti- human IFNα polyclonal antibody of the highest level producing clone. Aliquots of l Oμl from each recombinant KM71 H culture supernatants were electrophoresed on a denaturized conditions with the RPN756 JVl W markers (lane L for A and B figure).

Figure 18: Evolution of biomass (Fig. 18A), residual methanol and methanol flow rate (Fig. 18B), through out the culture of the recombinant clone of Pichia pastoris in a 5-1 bioreactor producing IFNα2b. on a minimal salt medium.

Figure 19: SDS-PAGE 15% analysis of culture supernatant at different induction time of the recombinant clone of Pichia pastoris methanol fed batch culture.

Figure 20: Cation exchange chromatographic purification of rHulFNα2b on SP-Sepharose column. Coomassie blue stained SDS-PAGE analysis of column fractions. Gel A : Lane 1 shows the crude supernatant. Lane 2 shows the flow through fraction, Lanes 3. 4. 5, 6, 7 correspond to washing fractions. Lane 8 shows the 0.1 M NaCl lactic acid 50 mM I mI elution fraction. Gels B, C and D show elution fractions with 27 ml step gradient from 0.2 to 1 M NaCl/ lactic acid 50 mM pH 4 (lanes 9 to 32).

Figure 21 : Silver (A) and Coomassie blue (B) stained SDS-PAGE analysis through size exclusion purification step. The pooled fractions (from fractions 12 to 25) obtained during cation exchange purification on SP sepharose column. Lanes 2 to 17 correspond to the different fractions collected during size exclusion step on Sephacryl S- 100 column. Lane L shows RPN756 MW marker.

Figure 22: Silver (I) and Coomassie Blue (H) stained SDS-PAGE analysis through Streamline SP-XL purification step. Lanes 1 and A represent the harvest from HCD culture diluted to OD₆oo to 100. Lanes 2 and B represent the flow through fraction. Lane 3 shows the first wash fraction. Lanes 4 and C, 5 and D, and 6 and E represent the eluted fraction with Lactic Acid 25mM I M NaCl. Lane L shows RPN756 MW marker. EXAMPLES

Example 1: Construction of Recombinant pPICZαA/rHulFNα2b expression vector

Human Interferon α2b cDNA was cloned by an RT-PCR approach using mRNA prepared according to standard procedures, isolated from healthy individual leukocytes exposed in vitro to the New Castle Disease virus. The cDNA was amplified using the FlFNα2b Forward primer designed to introduce an EcoRJ site at the 5^" end of the gene (TGGAATTCTGTG ATCTGCCTC A A ACCC A (SEQ ID NO: 5)) and the RlFNα2b reverse primer designed to contain the Notl site and an TGA STOP codon at the 3' end of the gene (ATTCTGCGGCCGCTCATTCCTTACTTCTTAAACTTTC (SEQ ID NO: 6)). The

498 bp purified RT-PCR product (Figure 1) was digested with EcoRl and Notl restriction enzymes and inserted into the (3.6 kb) plasmid pPlCZαA (Invitrogen, Groningen, the Netherlands) to generate the pPICZαA/rHulFNα2b expression vector. The E.coli Top] OF" (recA^'/endA ) transformed clones were selected on low salt Luria- Bertani (LB) medium with 25μg/ml zeocin. Isolation of pPlCZαA/rHulFNα2b recombinant plasmid containing the cDNA sequence encoding human IFNα2b was performed by colonies-PCR method using the 5^" FIFNα2b Forward and 3^" RlFNα2b reverse specific primers {Figure 2), according to procedures described in Current Protocols in Molecular Biology (Ausubel et al.) and by restriction analysis using BgIlI restriction enzyme as recommended by the manufacturer (Amersham-Biosciences)

(Figure 3). Finally, the nucleotide sequence of the positive clones was checked by automated DNA Sequencing Analysis using the "ABl-PRlSM ³⁷⁷ DNA sequencer of Perkin-Elmer Applied Biosystem. The Aoxl/F (Figure 4a) and Aoxl/R (Figure 4b) from the "Easy select Pichia protein expression kit'^" (Invitrogen, Groningen, the Netherlands), were used as the sequencing primers.

1.1 pPICZaA cloning vector features pPlCZαA cloning vector has been designed as Escherichia coli/P pastoris shuttle vector, containing: An origin of replication for plasmid propagation and maintenance in E.coli strain line, the CoIEl origin (pUC-derived): bases 2866- 3539.

For both P. pastoris and E.coli, the selectable marker gene, is the Sh ble gene from Streptoalloteichus hindυstanus: bases 2163-2537 that confers resistance to bleomycin-related drug zeocin (Cregg et al.. 1985; Cregg et al., 1989a; Higgins et al., 1998) With a transcription elongation factor 1 gene promoter (TEF 1 promoter/ GenBank accession number Dl 2478, DOl 130/ bases 1683-2094) from Saccharomyces cerevisiae that drives expression of Sh ble gene in P. pastoris strain, and the E.coli constitutive promoter droved Sh ble gene expression: the EM7

(synthetic prokaryotic promoter): bases 2095-2162.

An 1202 bp fragment expression cassette from AOXl gene composed of the 5' AOXl promoter (bases: 1 -940) that allows methanol-inducible, high level expression in P.pastoris, and a second short sequence of 260 bp required for transcription termination TT : bases 1341 -1601 (Koutz et al., 1989) that permits efficient 3' RNA processing, including polyadenylation: base 1520, for increased mRNA stability.

Between the promoter and the terminator sequence there is the multiple cloning sites (MCS), bases 1208-1274, containing the 10 unique restriction enzyme sites for insertion of foreign sequences.

In the pPlCZαA AOXl gene, the alcohol oxidase open frame (ORF) is preceded by an unusually long 5^? untranslated region (UTR) of 823 bp: bases 1-823.

- The 5' end of AOXl mRNA: base 824 and the 3' end of mRNA: base 1514.

The Saccharomyces cerevisiae α-factor secretion signal (bases 941 -1207), allows the secretion of most proteins from P.pastoris host strain. The initiation ATG (bases 941-943) in the α-factor signal sequence in pPIZCαA corresponds to the native initiation ATG of the AOXl gene. - The priming site of sequencing primers used to check the integrity of the plasmid construction was respectively: the 5' AOXl priming site: bases 855-875 and the 3^r AOXl priming site: bases 1423-1443.

For expression of C-terminal fusion tagged proteins, pPlCZαA contain both C-terminal myc epitop tag (Glu-Gln-Lys-Leu-Ile-Ser-Glu- Glu-Asp-Leu-Asn (SEQ ID NO: 7)): bases 1275-1307, that permits detection of fusion protein by anti-myc Antibody; and the C-terminal polyhistidine tag: bases 1320-1340 that encode 6 histidine that form a metal binding site for affinity purification of fusion protein. - The Sacl (base: 209), Pmel (base: 414), and BstXl (base:

707) are the unique restriction enzymes that we can use to permit linearization of the recombinant vector at the 5^" AOXl locus for efficient integration into P.pastoris genome.

1.2 pPICZaA/rHu\FNa2b recombiant plasmid features The recombinant expression plasmid pPlCZαA/rHuIFNα2b (Figure 5) was constructed using the pPlCZαA cloning vector as described in Example 1.1. The gene coding for recombinant Human Interferon α2b (rHuIFNα2b) was cloned in frame and downstream of the α-factor signal sequence at the 5" in frame EcoRI restriction site and the 3^" Notl restriction site. To express rHulFNα2b without any C-terminal peptide (C-terminal myc epitop tag and C-terminal polyhistidine tag), we introduced at the 3' end of gene a TGA STOP codon: resulting in a native C- terminal HuIFNα2b secreted protein (Figure 5). The TGA stop codon was introduced in the RIFNα2b reverse primer that was used to amplify the human alpha interferon cDNA. This primer was designed so as to introduce the TGA stop codon in frame with cDNA coding sequence.

1.3. Bacterial media composition:

To have active zeocin, salt concentration in the medium must remain low (< 90 mM) and the pH must be equal to 7.5. Low salt LB medium contains 1 % Tryptone, 0.5 % NaCl and 0.5 % Yeast extract at pH 7.5. Zeocin was added to a final concentration of 25 μg/ml.

Example 2: Construction of Recombinant pPICZαA/rHulFNα2a expression cassette with optimized codon usage

The concept that organisms display a non-random pattern of synonymous codon usage was established by Grantham et al. (1980) and has been confirmed by the explosion of sequence data available from recent genome sequencing projects (Nakamura et al., 1999). Moreover, all organisms investigated to date show some general bias towards a subset of the 61 possible sense codons (Nakamura et al., 1999; Li et al., 1991). While several hypotheses have been reported to explain the origin of this bias, a model involving selection for translational efficiency has been well supported for prokaryotes, unicellular eukaryotes, and to some extent, for insects (Bagnoli et al., 1995; Bulmer, 1987; Powell and Moriyama, 1997). Significant experimental work has been done to examine the effect of varying codon bias in heterologous expression systems, with the assumption that disparate patterns of codon bias between the cloned gene and the expression host will have significant impact on the level of recombinant protein produced. The optimization of coding regions towards the codon bias of the host cell has led to an average 10 to 50 fold increase in heterologous protein production in several host cells expression (Andre et al., 1998; Apeler et al., 1997; Brocca et al., 1998; Nagata et al., 1999; Ushijima et al., 1998; Zolotukhin et al., 1996).

The inventors have decided to study whether codon optimization of the Human Interferon α 2a cDNA towards the bias of P. pastoris could have a positive impact on expression levels. Hence, they have synthesized an artificial gene, according to the codon usage bias of P. pastoris as reported by Sinclair et al. (2002), as decribed below.

2.1 Design of a Pichia pastoris codon-optimized human interferon a2a cDNA and construction of the expression cassette

Leukocytes were isolated from a healthy individual and exposed in vitro to the New Castle Disease virus. RNA was prepared from these infected cells and used to copy the human lFNα2b mRNA by RT/PCR. The IFNα2b cDNA was cloned into the PICZaA plasmid and its nucleotide sequence determined and analysed by using the nearest neighbor analysis "graphical codon usage analyzer" software available through the WEB at http://www.geneart.degcua.schoedl.de/ or from ExPASy Proteomics tools at www.Swissprot.org (Figure 6).

The inventors have decided to change both the R (AGA) at position 23 to L (AAA) and the rarest codons according to the codon usage bias of P. pastoris as reported by Sinclair et al. (2002). Once the codons to be changed were chosen, the novel Human IFNα2a cDNA sequence was analysed "graphical codon usage analyzer" software as described above. The result shown in Figure 7, confirms that the rHulFNα2a synthetic sequence contain only the codon usage bias of Pichia pastoris organism.

The codon analysis of the synthetic Human !FNα2a sequence (Figure 7), was used to design the first HulFNα2a synthetic fragment ] obtained by PCR-assembly method using the synthetic primers: FaI (SEQ ID NO: 8), FbI (SEQ ID NO: 9) and FcI (SEQ ID NO: 10) and the two external primers: the 5' forward Ffgsl (TGGAATTCTGTGATTTGCCTCA (SEQ ID NO: H)) and the 3' reverse Rfgsl primer (GAAGATCTGCTGGATCATCTC (SEQ ID NO: 12)). While, primers: Fa2 (SEQ ID NO: 13), Fb2 (SEQ ID NO: 14), Fc2 (SEQ ID NO: 15) were used to generate the second synthetic Human IFNα2a cDNA fragment 2, using the two external primers: the 5' forward Ffgsll (TGAAGATCTTCAATTTGTTCTCCAC (SEQ ID NO: 16)) and the 3' reverse Rfgsll primer (TGAAGATCTCATGATTTCTGCTCTGA (SEQ ID NO: 17)). Details steps of the cloning strategy are depicted in Figure 8A, B and C.

Gene synthesis and fragment assembly were based on the PCR method described in Figure 8A and 8B, using Taq DNA polymerase from Amersham-Biosciences.

The purified 200 bp DNA fragment corresponding to the first HulFNα2a synthetic fragment PCR-assembly product (Figure 9, lane 1) was cloned into the recombinant plasmid pGX4Tl/rHulFNα2b previously restricted at the 5' EcoR\ and the 3' BgIU restriction sites, generating a plasmid containing only the 48 bp corresponding to the 16 amino acids HuIFNα2a COOH terminus. According to the codon bias usage in P. pastoris, the 16-C-terminal amino acids of the HuIFNα2a does not contain any rare codon.

Isolation of the Pl (Figure 8A) recombinant plasmid clone (pGEX4Tl containing the first HulFNα2a synthetic fragment) was performed by restriction analysis using BgIW restriction enzyme (Figure 10) as recommended by the manufacturer (Amersham Biosciences). Finally, the sequences of the positive clones were checked by DNA Sequence Analysis using the "ABl-PRI SM ³⁷⁷ DNA sequencer of Perkin-Elmer Applied Biosystem. The 5"pGEX primer was used as the sequencing primer (Figure 11). The second 267bp HulFNα2a synthetic fragment PCR-assembly product (Figure 9, lane 2) was introduced into the BgIlJ digested Pl recombinant plasmid.

Isolation of the P2 (Figure 8B) recombinant plasmid clone pGEX4Tl containing the HulFNα2a Synthetic fragment 2 inserted into the right sense, was performed by colonies-PCR method using the 5' Ffgsl Forward (SEQ ID NO: 10) and the 3' Rfgsll reverse specific primers (SEQ ID NO: 16) (Figure 12A) and by restriction analysis using EcoRl+ Xhol restriction enzymes as recommended by the manufacturer (Amersham-Biosciences)) (Figure 12B).

The full length synthetic HulFNα2a was digested with EcoR\ and No/1 restriction enzymes and inserted into the pPlCZαA plasmid to generate the pP]CZαA/rSynHuIFΝα2a expression vector (Figure 8C).

The E.coli Top 1OF' (recA /endA ) transformed clones were selected on low salt LB medium with 25 μg/ml zeocin. Isolation of pPlCZαA/ Synthetic HulFNα2a recombinant plasmid containing the cDNA sequence encoding synthetic human lFNα2a was performed by colonies-PCR method using the 5' Ffgsl Forward (SEQ ID NO: 1 1) and the 3^" RlFNα2b reverse specific primers (SEQ ID NO: 6) (Figure 13A), according to procedures described in Current Protocols in Molecular Biology (Ausubel et al.) and by restriction analysis using EcoK\ and No/1 restriction enzymes as recommended by the manufacturer (Amersham-Biosciences) (Figure 13B). Finally, the nucleotide sequence of the positive clones was checked by automated DΝA Sequencing Analysis using the "ABl- PRlSM ³⁷⁷ DΝA sequencer of Perkin-Elmer Applied Biosystem. The Aoxl/F (Figure 14A) and Aoxl/R (Figure 14B) from the "Easy select Pichia protein expression kit" (Invitrogen, Groningen, the Netherlands), were used as the sequencing primers.

2.2 pPICZaA/rSynHu\YHa2a Integration via electroporation of Mu? KM71H Pichia pastoris host strain

The recombinant pP!CZαA/rSynHulFNα2a P.postoris expression vector was propagated in E.coli strain Topi OF' (recA /endΛ ) in presence of 25 μg/ml zeocin.

Plasmid DNA was isolated using Qiagen plasmid miniprep kit from transformed E.coli clones. It was digested according to manufacturer's instruction, by Sac] restriction enzyme that does not cut within rSynHulFNα2a cloned gene (Amersham-Biosciences). The digested DNA was purified by phenol/chloroform/lAA purification method following standard protocols (Figure 15). Ten μg of linearized recombinant plasmid DNA was used to transform KM71H (aoxl ::ARG4) P.pastoris strain via electroporation method (Bio-Rad GenePulser, 1500 charging voltage (V), 25 μF capacitance, and 200 resistance), as described in the EasySelect Pichia protein expression manual (Invitrogen Catalogue No. K 1740-01). After 3 days of incubation at 30⁰C on YPDS solid medium containing zeocin at 100 μg/ml, 20 KM71 H Mut⁵ transformant clones were retained for further study. Gene insertion occurred at the aoxl::ARG4 (KM71 H) loci from a single crossover event between the loci of the two AOXl regions on the pPlZαA vector: the AOXl promoter or the AOXl transcription terminator (TT). This results in insertion of one or more copies of the recombinant plasmid (pPICZαA/rSynHulFNα2a) upstream or downstream of the aoxl::ARG4 gene.

Example 3. Transformation of Pichia pastoris Mut^s KM71H (aoxl::ARG4) host strain 3.1 Pichia.pastoris Muf KM71H phenotype

Strains with AOX mutations are sometimes better producers of foreign proteins than wild type strains (Tschopp et al., 1996; Chiruvoulu et al., 1997). Additionally, these strains do not require the large amounts of methanol routinely used for large-scale fermentation of Mut⁺ strains. Pichia pastoris KM71H (His4 aoxl ::ARG4) is a strain in which the codons 16 through 227 of AOXl have been replaced by the S. cerevisiae wild type ARG4, creating a disturbed AOX l gene (Cregg and Madden, 1987a). Since the strain must rely on the weaker AOX2 for methanol metabolism, AOX 1 -disturbed clone shows a slower growth phenotype (Mut^s) on minimal methanol medium as compared with MuI⁺ strain (Sreekrishna et al., 1997). However, since the AOXl locus was not completely deleted, these Mut^s strains retain the ability to induce expression at high levels from the AOXl promoter (Chiruvoulu et al., 1997).

3.2 pPlCZaA/rHu\FNa2b Integration via electroporation of Muf KM71H Pichia pastoris host strain

The recombinant pPlCZαA/rHulFNα2b P.pastoris expression vector was propagated in E.coli strain Topi OF' (recA /endA ) in presence of 25 μg/ml zeocin. Plasmid DNA was isolated using Qiagen plasmid miniprep kit from transformed E.coli clones. It was digested by BstX\ restriction enzyme that does not cut within the rHuIFNα2b cloned gene (Amersham-Biosciences). The digested DNA was purified by phenol/chloroform/AlA purification method following standard protocols (Figure 16). Ten μg of linearized recombinant plasmid DNA were used to transform KM71 H (aoxl ::ARG4) P.pastoris strain via electroporation method (Bio-Rad GenePulser, 1500 charging voltage (V), 25 μF capacitance, and 200 ohms resistance), as describe in the EasySelecl™ Pichia protein expression manual (Invitrogen, Catalogue N°. K 1740-01). After 3 days of incubation at 30⁰C on YPDS solid medium containing zeocin at l OOμg/ml, 20 KM71 H Mut^s transformant clones were retained for further study. Gene insertion occurred at the aoxl::ARG4 (KM71H) loci from a single crossover event between the loci of the two AOXl regions on the pPICZαA vector: the AOXl promoter or the AOXl transcription terminator (TT). This results in insertion of one or more copies of the recombinant plasmid (pP!CZαA/rHulFNα2b) upstream or downstream of the aoxl::ARG4 gene.

Example 4: Isolation of Multi-Copy expression clones Numerous examples reporting the effect of expression cassette copy number on protein production in P.pastoris were reported (Sreekrishna et al.. 1997; Clare et al., 1991a; Sreekrishna et al., 1989; Romanos et al., 1991 ; Clare et al._; 1991 b; Sreekrichna 1993). However, a strain that contains a single copy of the expression cassette can be sufficient for optimal production (Cregg et al.. 1985; Cregg et al., 1987b, Sreekrishna et al., 1990; Sreekrishna et al., 1993). Additionally, in some cases, an increase in copy number has a negative effect on the production level (Thill et al., 1990). Thus, the effect of gene copy number is unpredictable. A solution to this problem is to evaluate the production level of different clones as a function of gene dosage, by employing clones selection based on increased level of resistance to an antibiotic marker (Higgins et al., 1998; Clare et al 1991 a; Scorer et al., 1994). Thus, to isolate the multi-copy expression clones, a quick and directed way based on the selective clones' growth on increasing concentration of zeocin (Higgins et al.. 1998) was carried out. Furthermore, strains transformed with expression cassettes containing the zeocin can be selected directly by resistance to the drug marker by plating the putative recombinants clones on YPDS plates containing different concentrations of zeocin: 500, 1000 and 2000 μg/ml. Additionally, populations of transformants can be screened for multicopy expression cassettes strains, simply by plating on increased concentration of zeocin in the selection plates. In the present case, the inventors found a correlation with the highest zeocin resistance clones and the highest expression level clones (Figure 17).

Three clones : 7, 13 and 14 were able to growth on 2000 μg/ml zeocin plate. The highest level rHuIFNα2b producing P.pastoris clones (clones 7, 13, and 14) were identified by a comparative analysis with 12 different selective zeocin clones' growth (clones : 1 , 2, 3, 4, 6, 7, 9, 10, 12. 13, 14. and 20). Visual comparison of band intensity of SDS-PAGE Coomassie Brillant Blue stained (Figure 17A) and western blot (Figure 17B) of the culture supernatants, was carried out. One of the three highest producing rHulFNα2b clones (Clone 7) was chosen for high cell density culture. Example 5: Synthetic primers, cDNA and Amino acid sequences of the two engineered genes of Pichia pastoris clones producing human IFNα Synthetic rHulFNα2a (AAA/23) cDNA sequence

5'TGTGATTTGCCTCAAACCCACTCCTTGGGTTCCAGAAGAACCTTGATGTTG TTGGCACAGATGAGAAAAATCTCTTTGTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCC ACAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCTTGCATGAGATGATC CAGCAGATCTTCAATTTGTTCTCCACAAAGGACTCTTCTGCTGCTTGGGATGAGACCTTGTTGG ACAAATTCTACACTGAATTGTACCAGCAGTTGAATGACTTGGAAGCCTGTGTTATCCAGGGTGT TGGTGTTACAGAGACTCCATTGATGAAGGAGGACTCCATTTTGGCTGTTAGAAAATACTTCCAA AGAATCACTTTGTATTTGAAAGAGAAGAAATACTCCCCTTGTGCCTGGGAGGTTGTCAGAGCAG AAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAATGA 3' (SEQ lD NO: J)

Synthetic rHulFNα2a (K/23) Amino Acid sequence NH2-

CDLPQTHSLGSRRTLMLLAQMRK]SLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEIVIIQOIFNLFS TKDSSAAWDETLLDKFYTELYQQLNDLEACV]QGVGVTETPLMKEDSILAVRKYFQRITLYLKEKK YSPCA WEVVRAEIMRSFSLSTNLQESLRSKE- COOH (SEQ ID NO: 2) rHulFNα2b (AGA/23) cDNA inframe Sequence

5'TGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCT CCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTT CCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGA TCCAGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCT AGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGG GGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATACTT CCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGA GCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAAT GA 3' (SEQ ID NO: 3) HUMAN IFN ALPHA 2b (R/23) AMINO ACID SEQUENCE

NH2-

CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIOQIFNLFS TKDSSAAWDETLLDKFYTELYOQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQR]TLYLKEKK YSPCAWEVVRAEIMRSFSLSTNLQESLRSKE- COOH (SEQ ID NO: 4)

FaI 5'-

TGGAATTCTGTGATTTGCCTCAAACCCACTCCTTGGGTTCCAGAAGAACCTTGATGTTGTTGGC ACAGATG AGA AA A ATCTCTTTGTTCTCC-3- (SEQ ID NO: 8) FbI

5 -AAACTCCTCCTGTGGAAATCCAAAGTCATGTCTGTCCTTCAAGCAGGAGAACAAAGAGAT-S ' (SEQ ID NO: 9)

FcI 5'- CCACAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCTTGCATGAGATGA TCCAGCAGATCTTC-3 (SEQ ID NO: 10)

Ffgsl 5 -TGGAATTCTGTGATTTGCCTCA-3' (SEQ ID NO: 11)

Rfgsl 5 -GAAGATCTGCTGGATCATCTC-S- (SEQ ID NO: 12)

Fa2 5'-

CAGATCTTCAATTTGTTCTCCTCCACAAAGGACTCTTCTGCTGCTTGGGATGAGACCTTGTTGGA CAAATTCTACACTGAATTGTACCAGCAGTTGAATGAC-S- (SEQ ID NO: 13) Fb2

5'-

GTATTTTCTAACAGCCAAAATGGAGTCCTCCTTCATCAATGGAGTCTCTGTAACACCAACACCCT GGATAACACAGGCTTCCAAGTCATTCAACTGCTG-3^" (SEQ ID NO: 14)

Fc2 y_-

GCTGTTAGAAAATACTTCCAAAGAATCACTTTGTATTTGAAAGAGAAGAAATACTCCCCTTGTG CCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCT-S' (SEQ lD NO: 15)

FfgsII

5'-TGAAGATCTTCAATTTGTTCTCCAC-S' (SEQ ID NO: 16) RfgsII

5'-TGAAGATCTCATGATTTCTGCTCTGA-S- (SEQ ID NO: 17)

Example 6: Shake-flask production of secreted rHu]FNα2b

The Expression of the native C-terminal HulFNα2b secreted protein is controlled by the methanol-regulated promoter. The Saccharomyces cerevisiae α-factor secretion signal drives the rHulFNα2b expression to the extracellular domain. The α-factor secretion signal is a prepro peptide consisting of a 19-amino acids signal (pre) sequence followed by a 70- amino acids peptide (pro) sequence containing three consensus N-linked glycosylation sites and a 7- amino acids sequence Kex2 endopeptidase processing site (Kugan and Herskwitz 1982). The processing of the α-factor secretion signal involves three steps. The firs step consists in the removal of the pre signal by the signal peptidase in the endoplasmic reticulum. The second step, includes the Kex2 endopeptidase cleavage between Arg-Lys of the pro leader sequence, followed by a third step in which Stel 3 protease cleaves after the 2 Glu-Ala repeats residues pro-sequence (Brake et al., 1984).

A single colony of pP!CZαA/rHulFNα2b Mut^s KM71H Pichia Pastoris transformants was used to inoculate 50ml of BMGY in a 500ml flask and incubated at 30⁰C and 250 rpm for 24h. The cells were pelleted by centrifugation at 3000 g for 10 min, resuspended in 5ml BMMY medium containing 0.5 % methanol and incubated at 30°C and 250 rpm. During induction, methanol was added every 24h to a final concentration of 1 %. After 2 days (48 h) of induction, the cells were harvested by centrifugation at 10000 g for 20 min at 4°C; the supernatant was collected and stored at -20⁰C for protein expression analysis.

6.1. Media composition for the Shake-flask production Yeast media composition: Yeast extract-peptone-dextrose medium

(YPD) contained 2% peptone, 1 % yeast extract and 2% dextrose, whereas YPDS was supplemented with ] M sorbitol. Zeocin was added to a final concentration of l OOμg/ml. The buffered minimal glycerol-complex-medium (BMGY) was prepared with 2% peptone, 1% yeast extract, 1 % glycerol, 1.34% yeast nitrogen base (YNB) with ammonium sulphate but without amino acids, and 4x10 ⁵ biotin in 100 mM potassium phosphate buffer at pH 6.0. The buffered minimal methanoJ-complex-medium (BMMY) has the same composition as BMGY, except that 1% methanol is used instead of glycerol.

6.2. Secreted rHu\¥Ha2b protein expression analysis

To analyse the extracellular expression of rHuIFNα2b recombinant protein in MuI KMl]H Pichia pastoris host strain, the supernatants were analysed using SDS-PAGE. Electrophoresis was performed using 15% SDS-polyacrylamide gel stained with Coomassie Brillant Blue as described by Laemilli.

The recombinant fusion protein is specifically detected by Western blot-

ECL Assays (Amersham Biosciences) performed according to the manufacturer's instructions using 1 :400 dilution of Anti- human IFNα polyclonal antibody (Endogen), followed by the Anti Goat/sheep IgG peroxidase conjugated monoclonal antibody (Sigma) used as the second antibody. Example 7: High cell density culture

The recombinant clone of P. pastoris isolated from 100 μg zeocin/ml- YPD plate, was grown in 10 ml BMGY medium in a 125 ml baffled flask for 12h at 30⁰C and 250 rpm. This culture was used to inoculate 200 ml of BMGY medium in a 2-liter baffled flask for 12 h at 30⁰C and 250 rpm.

High cell density cultures were carried out in a 5-liter bioreactor, equipped with a process control system of data.

The batch culture was carried out with an initial culture volume of 2 litres inoculated with the 200 ml preculture, the culture was carried out at the following conditions: temperature = 30⁰C, stirrer speed = 800 rpm, pH maintained at 5 by addition of 25% ammonia, dissolved oxygen was set at 40% of air saturation by stirrer cascading and enrichment of the inlet air with pure oxygen when required.

The fed batch culture on glycerol was started once the optical density at 600 nm (OD) reached 80. After a 24 hours of a fed batch culture on glycerol at a rate equal to 15 ml/l/h, the cell dry weight reached at the end of this phase, was equal to 1 10 -120 g/1.

The cells were then harvested from the fermentor centrifuged at 3500 rpm (200Og), 4°C and 15 min; the cells were then resuspended in fresh medium and returned back under aseptic conditions, to the bioreactor containing 2.8 liters of synthetic medium that was supplemented with EDTA at 10 mM (final concentration), which avoids the proteolysis of the protein of interest. A fed batch culture on methanol was then started, the methanol feeding rate was modulated to keep its residual level under 0.6% (v/v). Residual methanol was measured by gas chromatography. The induction phase lasts for 5 days. The yield achieved was equal to 500 -600 mg/1. The composition of the media used for bioreactor culture were as follows:

Batch medium: glycerol (40 g/1), K₂SO₄ ( 18.2 g/1), MgSO₄ (7.28 g/1),

KOH (4.13 g/1), CaSO₄.2H₂O (0.93 g/1) and 85% orthophosphoric acid (26,7 ml/1) dissolved in deionised water and sterilized in the bioreactor. 5 ml/1 basal salts of fermentation PTMl and 2 ml/1 of 500X biotin were added to the medium after sterilization, culture medium pH was adjusted to 5 by addition of 25% ammonia (w/v).

The PTM l solution contains: CuSO₄.5H₂O (6 g I^"1), NaI (0.08 g I^"1), MnSO₄-H₂O (3 g I^"1), Na₂MoO₄^H₂Q (0.2 g I^{" 1}), H₃BO₃ (0.02 g I^"1), CoCl₂ (0.5 g T¹), ZnCl₂ (20 g r¹), FeSO₄.7H₂O (65 g T¹), biotin (0.2 g 1^"') and H₂SO₄ 98% (5 ml I ¹). Fed-batch medium contains glycerol (450 g/I), PTMl (8 ml/I) and 500X biotin (5 ml/1). Methanol fed-batch solution contains methanol at 100% (973 ml/1), 500X biotin (5 ml/1) and PTM l (8 ml/1). During the induction phase, the following components were added to the bioreactor at a regular interval of 24 hours: casaminoacids at 0.1% (w/v) (final concentration), Yeast Nitrogen Base at 0.1 % (w/v) (final concentration).

Figure 18 illustrates the evolution of biomass, residual methanol and methanol flow rate, throughout the culture of the recombinant clone of Pichia pastoris in a 5 1 bioreactor , on a minimal salt medium.

Figure 19 shows SDS-PAGE 15% analysis of culture supernatant at different induction times of the recombinant clone of Pichia pastoris methanol fed batch culture.

Example 8: Downstream processing steps 8.1. Method 1:

At the end of the culture, the medium was centrifuged at 3500 rpm (2000 g). 4°C for 15 minutes. The supernatant was supplemented with Triton X-100 at 1 % (final concentration), then further clarified by a microfiltration cartridge (a polysulfone membrane, cut-off equal to 0.1 μm) (Millipore) at a flow rate of 1.5-1.8 1/min.

The IFTM alpha containing permeate, was harvested and then desalted using diafiltration or chromatography method. Diafiltration was carried out using a 5 KDa cartridge (Amersham

Biosciences) and lactic acid 50 mM pH4. The retentate was concentrated 10 fold with the same cartridge. All the steps were carried out at room temperature.

Desalting was performed on a column packed with sephadex G25

(Amersham Biosciences). The column was equilibrated with 5 column volumes of lactic acid 50 mM pH4 0.15 M NaCl at a flow rate of 3 cm/min. The supernatant was loaded into the column at a flow rate of 4 cm/min, elution is carried out using 2 column volumes of lactic acid 50 mM pH4 at a flow rate of 3 cm/min.

The concentrated retentate or the desalted supernatant containing IFN alpha, was loaded on Sepharose SP column (Amersham Biosciences, 50 mg protein/ml column material). The column was first washed with 5 column volumes, then activated with 5 column volumes with lactic acid 50 mM pH4, I M NaCl. The concentrate is loaded into the column, a washing step with the buffer lactic acid 50 mM pH4, is carried out until the OD₂₈O becomes equal to zero. Elution of the bound IFN alpha is carried out using 9 column volumes of a stepwise gradient of 0.1- 1 M NaCl, at a flow rate 2.5 cm/min. IFN alpha is eluted from an NaCl concentration equal to 0.4 M to 0.8 M. Fractions containing pure IFN alpha were pooled.

The emergence of proteins in the fractions is monitored by measuring the absorbance at 280 nm. Fractions containing protein were analysed by SDS-PAGE and Western blot.

Interferon α can be further purified on a Sephacryl-SlOO column using PBS pH 7.3 at a flow rate of 15 cm/h.

Figure 20 illustrates SDS-PAGE 15% analysis of the different fractions collected during the purification of the desalted harvest containing IFNα on Sepharose SP column.

Figure 21 illustrates SDS-PAGE 15% analysis of the different fractions collected during the size exclusion purification

8.2. Method 2

At the end of the culture, the broth was diluted 4-5 fold with lactic acid 25 mM 0.1 M NaCl pH4 to an OD₆₀O equal to 100 (0.3 % (w/v) dry cell weight). The diluted broth is loaded into an expanded bed cation exchange adsorption on STREAMLINE SP XL (Amersham Biosciences).

The flow velocity during expansion/equilibration, adsorption and wash was 300 cm/h, which caused the bed to expand to 3 times during expansion/equilibration and feed application. The buffer used during expansion/equilibration and wash was lactic acid 25 mM pH4. Desorption of the IFN alpha from the adsorbent was performed with downward flow in sedimented mode using lactic acid 25mM pH4 I M NaCl. The flow velocity during the desorption was 100 cm/h.

Figure 22 shows Coomassie-blue and silver stained SDS-PAGE 15% gels of the different fractions obtained through the purification of rHulFNα2b produced at HCD culture on streamline SP XL column.

Example 9: Analysis of IFN alpha preparations

IFN alpha samples were analysed on 15% SDS polyacrylamide gels under standard reducing conditions. Samples were reduced with mercaptoethanol before electrophoresis. Protein bands were visualized with Coomassie blue staining.

IFN alpha samples were also analysed by SDS electrophoresis followed by silver staining. The IFN alpha content of various samples obtained during HCD culture and purification was determined by ELlSA using monoclonal antibodies and lFNα2b standard produced in-house.

Example 10: Biological activity of rHuIFNα2b The biological activity of the rHuIFNα2b preparation was determined by antiviral and Gene Report assays (Meager et al., 2001 ; Meager 2002).

The antiviral assay is based on the ability of rHulFNα2b to inhibit the cytopatic effect caused by encephalomyocarditis virus (EMCV) on "glioblastoma cell line" 2D9. Furthermore cell lines (HEK 293P) stably transfected with IFN-inducible promoter sequence (ISRE) linked to SEAP gene (secreted alkaline phosphatase) were used to perform the Gene Report assay. The amount of SEAP in supernatant is proportional to the amount of IFNα in the sample. The SEAP produced is measured using the p-NPP (para- nitrophenyl phosphase) which is a chromogenic substrate for most phosphatases.

The purified was calibrated against the IFNα WHO international standard (code: 95/566). The IFNα has a specific activity varying from ~ 1.57 to 2.85 10⁸ 10⁸lU/mg.

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Claims

1. A nucleic acid molecule comprising a coding sequence encoding a human alpha interferon, characterized in that said coding sequence is optimized for expression in Pichiapastoris.

2. The nucleic acid according to claim ] , wherein said human alpha interferon is HulFNα2a and said coding sequence is SEQ ID No: 1 .

3. An expression vector for transforming a Pichia pastoris strain, characterized in that it comprises, in the 5' to 3^" direction and operably linked :

(i) a Pichia /?αstorø-recognized transcription and translation initiation region,

(ii) a sequence coding for a secretion signal enabling protein secretion in Pichia pastoris,

(iii) a coding sequence as defined in claim 1 or claim 2. and

(iv) a Pichia pastoris-xεcogmzzd transcription and translation termination region.

4. A Pichia pastoris strain producing human alpha interferon, characterized in that it has been transformed by a vector according to claim 3.

5. The Pichia pastoris strain according to claim 4, characterized in that it has been obtained by transforming a Mut^s KM71 H Pichia pastoris strain.

6. The Pichia pastoris strain according to claim 4 or claim 5, characterized in that it is 1RDF/FNF/1PT05. deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on September 1 1 , 2006, under the n° 1-3668.

7. A nucleic acid comprising a coding sequence encoding a human interferon α2b, wherein said coding sequence is SEQ ID No: 3.

8. An expression vector for transforming a Pichia pastoris strain, characterized in that it comprises, in the 5^* to 3^" direction and operably linked,

(i) a Pichia pastoris-recognized transcription and translation initiation region,

(ii) a sequence coding for a secretion signal enabling protein secretion in Pichia pastoris,

(iii) a coding sequence as defined in claim 8. and

(iv) a Pichia pastoris-recogni^'zed transcription and translation termination region.

9. A Pichia pastoris strain producing human alpha interferon, characterized in that it has been transformed by a vector according to claim 8.

10. The Pichia pastoris strain according to claim 9. characterized in that it has been obtained by transforming a Mut^s KM71 H Pichia pastoris strain.

1 1 . The Pichia pastoris strain according to claim 9 or claim 10, characterized in that it is KM71 H/P]CZαrHulFNα2b, deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) on February 01. 2006, under the n° 1-3562.

12. A process for producing human alpha interferon by culturing a Pichia pastoris strain which expresses and secretes said human alpha interferon, wherein the expression of said interferon is under the control of a promoter regulated by methanol, characterized in that it comprises the following steps:

(i) inoculation of a synthetic culture medium in a bioreactor, by a preculture of said Pichia pastoris strain, wherein said synthetic medium contains glycerol as a carbon source, and has a defined salt composition;

(ii) batch culture in said bioreactor, at 30⁰C, pH5. under agitation and oxygenation;

(iii) fed-batch culture on glycerol at 30⁰C, pH5, under agitation and oxygenation; (iv) fed-batch culture on methanol, in a synthetic culture medium supplemented with EDTA at a final concentration of 10 mM.

13. The process of claim 12. wherein the Pichia pastoris strain is according to any of claims 4-6 and 9-1 1.

14. The process of claim 12 or claim 13, wherein the synthetic culture medium is obtained as follows:

- glycerol (40 g/1), K₂SO₄ ( 18.2 g/1), MgSO₄ (7.28 g/1), KOH (4.13 g/1), CaSO₄.2H₂O (0.93 g/1) and 85% orthophosphoric acid (26,7 ml/1) are dissolved in deionised water and sterilized in the bioreactor;

- 5 ml/1 basal salts of fermentation PTM l and 2 ml/1 of 500X biotin are added to the medium after sterilization, and culture medium pH is adjusted to 5 by addition of 25% ammonia (w/v), wherein the PTM l solution contains: CuSO₄.5H₂O (6 g I^"1), NaI (0.08 g I ¹), MnSO₄-H₂O (3 g I^"1), Na₂MoO₄.2H₂O (0.2 g I^"1), H₃BO₃ (0.02 g I^"1), CoCl₂ (0.5 g I^"1), ZnCl₂ (20 g r¹), FeSO₄JH₂O (65 g I^"1), biotin (0.2 g I^"1) and H₂SO₄ 98% (5 ml I^"1).

15. The process according to any of claims 12 to 14, wherein the fed- batch solution used in step (iii) contains glycerol (450 g/1), PTM l (8 ml/1) and 500X biotin (5 ml/1) and is added at a rate of 15 ml/l/h.

16. The process according to any of claims 12 to 15, wherein the fed- batch solution used in step (iv) contains methanol at 100% (973 ml/1), 500X biotin (5 ml/1) and PTM l (8 ml/1) and the feeding rate is modulated to keep the methanol residual level under 0.6% (v/v).

17. The process according to any of claims 12 to 16, wherein the induction phase lasts for at least three days, preferably 4 or 5 days, during which the following components are added to the bioreactor at regular intervals of 24 hours: casaminoacids at a final concentration of 0.1 % (w/v) and Yeast Nitrogen Base at a final concentration of 0.1 % (w/v).

18. The process according to any of claims 12 to 17, wherein step (iii) is initiated once the optical density at 600 nm of the culture of step (ii) has reached 80, and wherein the fed-batch culture of step (iii) lasts until the dry weight reaches at least 100 g/1, preferably 1 10-120 g/1.

19. The process according to any of claims 12 to 18, wherein between steps (iii) and (iv), the cells are harvested, resuspended in fresh medium and returned back to the bioreactor.

20. The process according to any of claims 12 to 19, wherein the preculture used in step (i) is in BMGY medium.

21. A process for purifying human alpha interferon secreted in the culture medium by recombinant yeasts, characterized in that it comprises the following steps: (i) eliminating the biomass by centrifugation,

(ii) filtering the supernatant through a 0.1 μm cartridge in the presence of Triton X-100 at 1%,

(iii) desalting the permeate by diafiltration using a 5 kDa cartridge, and

(iv) purifying the supernatant by a cation exchange chromatography.

22. The process of claim 21 , wherein the desalting step (iii) is performed by chromatography on a Sephadex G25 column.

23. A process for purifying human alpha interferon secreted in the culture medium by recombinant yeasts, characterized in that it comprises the following steps: (i) dilution of the culture medium with a solution of lactic acid (25 mM), NaCl (0.1 M), pH4, to an optical density at 600 nm equal to 100; and

(ii) purification of the diluted medium using an expanded bed chromatography on a cation exchange support.

24. The process according to any of claims 21 to 23, comprising an additional step of purification by gel filtration using sephacryl S-100.

25. The process according to any of claims 21 to 24, wherein said human alpha interferon has been produced by a recombinant Pichia pastoris strain according to any of claims 4-6 and 9-1 1.

26. The process according to any of claims 21 to 25, wherein said human alpha interferon has been produced by a process according to any of claims 12 to 20.