Chemistry & Biology

Innovations

Pichia Power: India’s Biotech Industry Puts

Unconventional Yeast to Work

Chandra Shekhar

DOI 10.1016/j.chembiol.2008.03.002

As a platform for making recombinant pro-

teins, E. coli is hard to beat. This universal

gut bacterium grows quickly and eagerly,

and—with simple genetic tinkering—will

make virtually any foreign protein. How-

ever, E. coli, a prokaryote, can’t handle

posttranslational modifications; the pro-

teins it makes are often misfolded and in-

soluble, requiring expensive processing

steps to make them usable. Mammalian

cells, in contrast, fold and modify proteins

with ease but are much harder to culture.

One organism that potentially combines

the advantages of bacterial and mamma-

lian expression systems is Pichia pastoris,

a harmless species of yeast that feeds on

methanol. Being a single-celled organism,

it is easy to grow and manipulate, while as

a eukaryote, it can perform complex ma-

nipulations on proteins. These attributes

havemadeitamainstayoftherecombinant

protein industry in India. ‘‘Pichia gives high

yields of well-expressed proteins,’’ says

Harish Iyer, head of research and develop-

ment at Biocon, the first company to mar-

ket recombinant human insulin made using

Pichia. ‘‘It is a wonderful workhorse for us.’’

Pichia’s role in biotechnology dates

back to 1969 when researchers first

learned about the organism’s remarkable

ability to feed on methanol. Since at that

time methanol could be made cheaply

from waste natural gas, oil company re-

searchers were intrigued by the idea of

using this yeast to make protein-rich ani-

mal feed. They developed the technology

to grow the organism to cell densities as

high as 130 g/l. However, the oil crisis of

the 1970s, coupled with a fall in price of

soybeans—a competing source of animal

feed protein—killed this effort.

Researchers then turned their attention

to using this prolific yeast as an expres-

sion system for foreign genes. In the early

1980s, molecular biologists at the Salk In-

stitute Biotechnology/Industrial Associ-

ate, Inc., (SIBIA), in La Jolla, CA, isolated

the principal gene for the yeast’s alcohol

oxidase promoter, which is highly effec-

tive at controlling the expression of for-

eign genes. The team went on to develop

vectors, strains, and tools for genetic ma-

nipulation of the organism. The combina-

tion of a strong promoter and very high

cell densities ‘‘resulted in strikingly high

levels of foreign proteins,’’ recalls James

Cregg, who led the SIBIA team. One of

the team’s first achievements was cloning

the surface antigen of the hepatitis B virus

(HBsAg), a process that was later re-

peated by several Indian companies with

great commercial success. In contrast,

when HBsAg is expressed in E.coli, ‘‘you

just get ‘rocks’ or inclusion bodies of mis-

folded protein that are apparently not pro-

tective against the virus,’’ says Cregg.

Since then, researchers worldwide

have used Pichia to make recombinant

versions of several dozen proteins—bac-

terial, fungal, viral, plant, and human—

with expression levels comparable to

E.coli and higher than tissue cultures.

‘‘Pichia doesn’t have toxins like E.coli

does, or viruses like tissue cultures do,’’

says Cregg. ‘‘It is an excellent expression

system, perhaps one of the best.’’

Despite its advantages, Pichia has not

been widely used in the United States

and Europe to make human therapeutics.

(One of the very few Pichia-derived inject-

able biologics approved for use in the U.S.

is a recombinant human serum albumin

made by Japan-based Mitsubishi Pharma

Corporation.) This is partly because gly-

cosylation patterns in yeast cells tend to

differ from their mammalian counterparts;

studies suggest that glycoproteins made

using fungal systems might trigger ad-

verse immune responses when injected

into mammals. More importantly, many

elements of the Pichia expression system

are protected by patents by Tucson, AZ,

based Research Corporation Technolo-

gies. ‘‘Because of patent concerns, only

a handful of U.S. companies have worked

with Pichia,’’ says Cregg. In contrast, sev-

eral Indian biotechnology companies

have embraced this yeast as a platform

for making recombinant products, en-

couraged by intellectual property regula-

tions that until recently were less stringent

than those in the U.S. and Europe.

In 1997, Hyderabad-based Shantha

Biotech announced India’s first Pichia-

derived product, a hepatitis B vaccine

based on a recombinant form of the

HBsAg antigen. Earlier work by Cregg

and other researchers provided guide-

lines for doing this, but transitioning the

technology from the lab to the factory

required a major effort. ‘‘We initially strug-

gled to get good levels of expression,’’

recalls Revathi Chaganti, then the sole

molecular biologist at the company. The

protein tended to associate with the

membrane, and it was tricky to extract it

without disturbing its structure. ‘‘Even

now we recover only about 30% of it,’’

she says. Most of the published work

dealt with shake-flask cultures; scaling

the system to industrial-size fermentors

was a challenge. ‘‘We started with a 10 L

fermentor and increased it by stages to

750 L,’’ says Chaganti. From such humble

beginnings, the company now makes

more than 100 million units of the vaccine

each year. It sells the product in more

than 50 countries and provides 40% of

UNICEF’s supply. Prior to Shantha’s ef-

fort, the multinational GlaxoSmithKline

was the sole provider of this vaccine in

India, charging more than $10 per dose;

Shanta’s entry into the market caused

its price to plunge, and it now sells at

15 cents a dose.

One organism that potentially combines the advantages of bacte-

rial and mammalian expression systems is Pichia pastoris,

a harmless species of yeast that feeds on methanol.

Chemistry & Biology 15, March 2008 ª2008 Elsevier Ltd All rights reserved 201

During the past few years, the market

for the hepatitis B vaccine in India has

grown fiercely competitive, as several

other companies have entered the fray.

One of the contenders is Indian Immuno-

logicals, a veterinary biologics manufac-

turer for whom this vaccine was the first

foray into recombinant DNA technology

as well as the Pichia expression system.

‘‘We had 25 years of experience with

mammalian cells,’’ says V. Srinivasan, di-

rector of research and development at the

Hyderabad-based company. ‘‘Compared

to that, Pichia was a much easier system

to work with.’’ Downstream processing,

however, required more effort; it was par-

ticularly challenging to attain purity levels

of 97% and above, according to Sriniva-

san. ‘‘It is not the expression system alone

that matters–you have to couple it with

powerful downstream processing,’’ agrees

Krishna Ella, chairman and managing

director of another hepatitis B vaccine

manufacturer,

Bharat

Biotech,

who

switched to Pichia after initially working

with a Saccharomyces platform. Yet an-

other platform, Hansenula polymorpha, a

methylotrophic yeast similar to P. pastoris,

is used by Mumbai-based Wockhardt for

making its version of the vaccine.

In 2000, Biocon, India’s leading bio-

technology company, unleashed Pichia

on insulin, another hot itemin the Indian bi-

ologics market. ‘‘It was a singularly chal-

lenging project,’’ recalls Iyer, who led the

company’s effort. ‘‘Here we were, a band

of 10–20 people, competing with multina-

tionals like Eli Lilly and Novo Nordisk, with

their hundreds of researchers.’’ As with

HBsAg, expressing the protein was the

easy part. ‘‘There wasn’t much difficulty

in transforming the microbe or getting

high yield,’’ says Iyer. The challenge

came from the whole series of activities

required to commercialize the product:

extraction, purification, clinical studies,

regulatory approval, etc. It took the better

part of four years to complete these

stages. ‘‘And then one day we had our first

formulation, a vial with a label that had

Biocon’s name on it,’’ says Iyer. ‘‘It was

a tremendously rewarding moment.’’ The

product, which was released in 2004, is

now registered in around 40 countries

and sells millions of units each year.

Indian manufacturers cite several rea-

sons for choosing Pichia over other plat-

forms. For instance, Chaganti observes

that Pichia-derived proteins, in contrast

to those made using E.coli, have intact di-

sulfide bonds and don’t need expensive

refolding stages. ‘‘All you need to do is

to break open the association with the

membrane, put it on a column, and purify

it,’’ she says. However, Chaganti and

others also noted some shortcomings

with Pichia. She points out that some

proteins give poor yields unless their

gene is optimized to eliminate rare co-

dons. Further, during secretion, glycopro-

teins such as granulocyte-macrophage

colony stimulating factor (GM-CSF) get

over-glycosylated and may get N-termi-

nal amino acids cleaved off. ‘‘Because of

this we could not use Pichia for producing

GM-CSF, though the expression levels

were very good,’’ says Chaganti. Pichia’s

ability to feed on cheap methanol is a ma-

jor selling point; however, even here there

is room for improvement, says Iyer. He

points out that much of the methanol con-

sumed by the organism is burnt to pro-

duce heat instead of the ‘‘useful work’’

of making protein. ‘‘How to shunt the pro-

cess away from heat into work is a major

unsolved problem with Pichia,’’ says

Iyer. ‘‘We need to do some fundamental

work here.’’

Until recently, Indian companies were

more interested in reproducing and

adapting results from the literature than

in performing fundamental research.

However, with recent legislation that

brings Indian patent laws in compliance

with World Trade Organization (WTO)

and Trade-Related Aspects of Intellectual

Property Rights (TRIPS) requirements, the

situation is changing. Shantha Biotech,

for instance, is working for the develop-

ment of a novel Pichia-based expression

system in collaboration with the Centre

for Cellular and Molecular Biology,

a research institute in Hyderabad. Other

companies and research institutes are en-

gaged in similar efforts. ‘‘Our initial effort

was one of indigenization rather than in-

novation,’’ says biochemist P.N. Rangar-

ajan of the Indian Institute of Science in

Bangalore, who developed the Pichia

strain used by Indian Immunologicals for

their hepatitis B vaccine. ‘‘But now we

are doing more basic research and are

hoping to come up with innovative things

in Pichia.’’ He points out that despite

Pichia’s wide use in biotechnology,

many aspects of the organism—such as

how it attains such high cell densities or

how methanol turns on the expression of

its alcohol oxidase promoter—remain

a mystery. To investigate these issues fur-

ther, Rangarajan has launched a new re-

search program at the Institute. ‘‘We are

getting very exciting results,’’ he says.

Chandra Shekhar is a science writer based in

Princeton, NJ.

Chemistry & Biology

Innovations

202 Chemistry & Biology 15, March 2008 ª2008 Elsevier Ltd All rights reserved