Indus Water Treaty of 1960

INDUS WATER TREATY OF 1960 by William H. Thompson [February 2013] The Indus Water Treaty (IWT) of 1960 is an example of a mutually beneficial conflict or, as Kriesberg and Dayton would define it, a constructive conflict. Born of the dissolution of the British Crown Colony of India in 1947, the treaty recognized the mutual needs of India and Pakistan, and the necessity of ensuring continuing access to the waters of the Indus River System for both nations.Although the treaty has survived â€Å"two and a half wars and frequent military mobilizations† as well as a nuclear arms race, current moves by both Pakistan and India regarding dispute mediation threaten to dissolve the treaty. Differences in interpretation, Pakistani mismanagement of its own water resources and the ongoing question of the status of Kashmir each threaten the continued observance of the treaty. Neither nation can afford the loss of this treaty. For each nation this treaty has been a source of ongoing diplomati c relations, requiring annual meetings and open verification of water projects within the covered regions.It has been used to address non-water issues and to placate each other in times of crisis. It has also ensured that water continues to flow between the two, in spite of the strategic advantage that India could gain by stopping that flow. This paper will outline some of the dangers affecting the future of the IWT. It will address the interpretation of treaty clauses by neutral parties and how that has resulted in diplomatic escalation by Pakistan. It will address the very real concern for Pakistan that India has the superior strategic position with regard to control of the Indus System.It will also highlight the inadequate water infrastructure within Pakistan and the affect that this has on the ability of India to complete its own water projects. The paper will describe certain indicators of the health of the treaty. Finally, it will outline two scenarios for the future of the IW T and the likely outcome of each. The goal of addressing these issues is to stress the importance of this treaty over national concerns for control of water and how the mutual control of the Indus system is the best solution for both nations.Before exploring the continued existence of the Water Treaty of 1960, and the potentially far reaching effects of its nullification, it is necessary to provide a brief history of the Indo-Pakistani conflict, especially as it relates to the Kashmiri region and control of the Indus River System. When the British Parliament passed the Indian Independence Act of 1947, its primary concern was achieving a speedy settlement of the partition rather than the stability of the resulting entities.Sir Cyril Radcliffe, the English barrister charged with partitioning the Indian colony into two separate entities, arrived in New Delhi on 8 July 1947 to learn that the date of independence for both newly formed nations of India and Pakistan had already been set fo r 15 August of that same year. The rules for the partition of India and Pakistan, established in negotiations between the British representative Lord Mountbatten, the Indian National Congress representative Jawaharlal Nehru and the Muslim League representative Muhammed Ali Jennah, focused the division along religious lines.In certain provinces with no clear religious majority, most notably those bordering Punjab and Bengal, the citizens of the province were to be given the opportunity to vote over which country to join. Independent princedoms, such as Kashmir, were given the option of joining with either state, but were encouraged to hold a plebiscite if the desires of the people were in doubt. The resulting boundaries would have three far-reaching results.First, the sudden change in citizenship (from nominally British to Pakistani or Indian respectively) resulted in bloodshed and mass-exodus as Muslims moved from India to Pakistan and Hindus moved to India from Pakistan, as well as an almost instantaneous nationalism within both nations. Second, when establishing borders between the states it did so with little regard to natural boundaries, such as rivers, and little thought to allocation of the infrastructure and resources now shared by the two states.What had been created by one central government, such as irrigation systems, canals, and dams, was now controlled by two with no standing agreement over how they should be shared. Finally, in giving the rulers of independent princedoms the right to choose which country to join, the prince was expected to abide by the wishes of his subjects; in the case of Kashmir, the prince made his own choice. Common sense should have dictated that the province becomes the northernmost province of Pakistan: Its people were predominantly Muslim and it controlled the flow of the Indus River into Pakistan.Kashmir as a province of Pakistan was likely the vision of the British, Muslim and Hindu negotiators of the partition. Unfort unately, the status of the various princedoms, including Kashmir, was left to each ruling prince. Although not alone in originating the Indo-Pakistani conflicts, the decision of Hari Singh, the Maharaja of Kashmir, to join India rather than Pakistan has played a vital role in exacerbating them. One oddity of the partition of the former British colony is the Standstill Agreement.This agreement stated that the flow of the Indus between East and West Punjab (India and Pakistan) would remain at the same level from the date of partition until 31 March, 1948 and that Pakistan would pay a set fee for the water that flowed. As Pakistani forces crossed the border of Jammu and Kashmir to protect Muslims and Indian forces were airlifted into Kashmir to defend India’s territorial boundaries, the dams, canals and barrages along Indus tributaries continued to operate and adjust flows to ensure that water reached the fields of Pakistan.And, as these things occurred, Pakistan continued to pa y its water fee to India. However, on 01 April, 1948, with the agreement ending and no new agreement in place, the flow of water stopped. Although India and Pakistan would agree to a resumption of water deliveries, two precedents had been set: Pakistan recognized that it was in an untenable position and India had demonstrated that it would abide by existing agreements but, in the absence of agreement would act in its own best interests.In 1952, the World Bank offered to mediate the dispute over Indus Waters. The resulting treaty, based on the water usage needs of each, water availability in the Indus System and mutual development of the watershed granted India the use of several rivers flowing through Kashmir for power generation, but stipulated that the usage must allow free flow of the waters into Pakistan. Each nation must announce water development plans and allow for the inspection of these projects by engineers from the other nation.It established a Permanent Indus Commission, made up of engineers from each nation, which would meet annually to discuss development issues and treaty implementation and established steps for dispute arbitration. Modern interpretation of the provisions of a treaty established in 1960 have strained the agreement and resulted in an escalation of Pakistan’s arbitration demands. Until 2005 all disputes over water projects had been resolved through the annual meetings of the Permanent Indus Commission. This changed with Indian plans to build the Baglihar Dam, a hydroelectric project, across the Chenab River.Although planning began in 1992, Pakistani engineers first objected to the project in 1999 on the grounds that it blocked the free flow of water within the Indus System in violation of the IWT. India contended that, in spite of the fact that it did not comply with the original treaty, the design of the dam was sound and that it would not only allow for the flow of water but would ensure that water supplies were available throughout the year. Pakistan referred the dispute to the World Bank for neutral arbitration under terms of the IWT.Although the neutral arbiter agreed in principal that the Indian project violated some aspects of the treaty, the violations were determined to be based on â€Å"sound and economic design and satisfactory construction and operation† and the project was allowed to continue. While Pakistan agreed to the decision of the World Bank, its next dispute, over the Kishanganga Hydroelectric Dam, was taken directly to the International Court of Arbitration. Although this level of arbitration is specified in the IWT, it is the first time that any dispute under the treaty has been taken to this level.The fact that Pakistan skipped neutral arbitration in favor of the International Court may be a signal that it mistrusts the neutrality of the World Bank. Although the Court has not yet ruled on the project, a ruling in favor of India may convince Pakistan that the treaty is no longer in its best interests. The escalating arbitration demands of Pakistan reflect some concern over individual water projects, which was reflected in its arbitration request concerning the Baglihar Dam project, and more concern for the strategic implications of the Indian system as a whole.As most agree, no single Indian project could shut down water supplies to Pakistan. However, there is general agreement that India holds the superior position regarding control and usage of the Indus River. And there is agreement that the sheer number of dams along the northern Indus System could indeed have adverse effects on the water available to Pakistan. While Indian water needs are fulfilled by three rivers, the Ganges, the Brahmaputra as well as the Indus,Pakistan is served almost exclusively by the Indus, over which India maintains control. Although India contends that it has never diverted water from Pakistan, the water stoppage of 1948, when East Punjab halted water flow into West Pu njab, is ever present in Pakistani strategic thought. India has the greater GDP, and therefore a greater ability to withstand delays to its water projects, and a larger military, so it cannot be easily intimidated into acceding to Pakistani demands.As Pakistani negotiators have stated, the Indian negotiating strategy is â€Å"one of delay, of foot dragging, of ‘tiring you out’;†¦of â€Å"creating facts†, proceeding with construction plans, even when aware that the plans might well violate the treaty, so that Pakistan, confronted eventually with fait accompli, would have no choice but to cut its losses and accept an unfavorable compromise settlement; and †¦ insisting on a bilateral framework of talks, without intending ever to settle on any but India’s terms. Although Pakistani negotiators may believe that India can drag negotiations on, the reality is that each referral to arbitration has put a great burden on India in time to completion. In the case of the Baglihar Dam, India announced its plans in 1992, began construction in 1999, the project was taken to arbitration in 2005 and the entire project was not completed until 2010. This case is similar to other projects which have taken 10+years from commencement, through negotiation, to completion.Some, especially within Pakistan, have suggested that the treaty is no longer useful, that it is too strategically disadvantageous to Pakistan and that the only solution to the issue is to take control of Kashmir and the northern Indus System. Others have expressed concerns that India’s hydroelectric projects may force Pakistan to abrogate the treaty and spark a war over Kashmir and control of the Indus.Whether concerns over war between the two nuclear nations are meant as a warning or a threat they have come often enough since the dispute over the Baglihar Dam that they must be seen as a real concern. With multiple Indian hydroelectric projects in the planning stage (althou gh the actual number is in dispute), the opportunities for â€Å"hawks† within Pakistan to demand war will continue to place pressure on politicians and the military to accept nothing less than a halt to all projects.The disputes over Indian projects have allowed Pakistan to divert attention away from its own weaknesses with regard to water availability. Although Pakistan often contends that Indian projects on the northern Indus have resulted in a loss of useable water within Pakistan, it is â€Å"a case of wastage and unequal distribution by internal forces† that has resulted in less water availability within Pakistan. This loss in water availability is due to aging transfer systems (pipes, canals), increasing silt levels within dams, corruption and inefficiency and low expenditure on water sector development.Ninety percent of Pakistan’s irrigable water is supplied by the Indus; an aging system of canals, barrages and hydroelectric dams within Pakistan has resu lted in waste within its own water management systems. This is largely a result of heavy sediment composition of the Indus. Water storage systems and canals have filled with sediment over time, resulting in less water availability and susceptibility to flooding, especially during heavy monsoonal rains. The IWT has been used as a means to, if not settle other non-water related disputes, to at least achieve a hearing of them, or to ease the tensions between the nations.Most recently, in 2009, the Pakistan Commissioner of Indus Waters had been asked about developments on the Nimoo-Bazgo Hydro Project and whether his office had inquired about inspecting the development. His response was that â€Å"We would like to go there when the tension between India and Pakistan following the Bombay attacks ease. † In the wake of the Mumbai attacks, the Pakistani official chose to delay his inspection to avoid inciting an already tense situation.India had threatened to pull out of the treaty as a response to cross-border terrorism in 2001-2002, and has used its control of the upper Indus to exert pressure on Pakistan to halt attacks. Although this may be viewed as using its hegemonic power over water flows to exert pressure, the alternative is that war was avoided through the use of the existing treaty. Should either India or Pakistan see the treaty as having outlived its usefulness, the nations have two choices: nullification or renegotiation.Renegotiation would be the most desirable choice for the nations and the region. Indeed, renegotiation of the treaty may be a necessity. Guarantees of water deliveries through the Indus system may be unsustainable if climate change models are correct. Pakistan is currently able to store only 30 days of water, leaving it highly vulnerable to even mild fluctuations in water flow. This vulnerability exists in a period when the Indus is at its highest flow in 500 years due to the melting of the Himalayan glaciers that feed the system. The expectation, although the calculations differ, is that the flow will slow as the glaciers recede, leaving both India and Pakistan struggling for water. Signs that offers to renegotiate are real would have to include two things; 1. Renegotiation would have to be open to public scrutiny and third party mediation and 2. They would have to include cooperative agreements on joint water projects. Renegotiation of the treaty under these conditions would indicate that both parties are committed to the IWT in some form.Nullification may be more difficult to predict. As stated above, the treaty itself has survived at least three and a half conflicts and terrorist incursions. Escalation of hostilities may not be a reliable indicator of nullification. The current escalation of arbitration demands under the current treaty may provide some warning, should Pakistan reject the findings of the current International Court arbitration. Although the current case was brought over the Indian Kishanga nga dam, it is actually a story of two dams.Pakistan is currently building a dam on the same river, the Neelam-Jhelum Dam. Should arbitration be decided in India’s favor, the Kishanganga dam will divert water away from the Neelam-Jhelum, making the dam useless. Should this occur and the two nations are unable to come to some accommodation, Pakistan may determine that the treaty is no longer in its best interest. Without the treaty its guarantees of water flow into Pakistan, the nation may see war as the only alternative. There are two likely scenarios for future developments with regard to the IWT.The first is and most likely scenario is a renegotiation of the treaty. For renegotiation to occur, it would most likely need to be initiated by India, as such an offer would likely be seen by the Pakistani public as bowing to Indian pressure. In addition, were Pakistan to request a renegotiation, India most likely would have the upper hands in discussions. The catalyst for renegoti ation would most likely be the ongoing demands for arbitration from Pakistan and the continuing delays in Indian construction projects.In return for a greater freedom to build on the upper Indus, India would have to offer significant concessions, the most likely being the instigation of joint projects to ensure more efficient irrigation to Pakistani cropland and more effective flood mitigation. Should India successfully convince Pakistan that a new treaty would provide more favorable water availability and would result in less control over the Indus System by India, then the renegotiation could be both a diplomatic and public relations success.The end result would be that both countries would be much better prepared should the flow of the Indus be reduced in the future. The second scenario is less hopeful and also less likely. Should Pakistan determine that the existing treaty is no longer in its best interest and it believes that Indian projects will result in less water availabili ty on the Indus, Pakistan may nullify the treaty. In this case, war would be highly likely to occur as Pakistan attempts to seize control of Kashmir and the upper Indus River.This scenario itself has three likely outcomes. 1. In order to avoid a nuclear war, the international community brokers a cease-fire. India retains control of Kashmir and effectively ends both Pakistan’s claims to the province and any obligations to allow the free flow of water to Pakistan. While Pakistan would still receive some flow, mainly as a result of flood control measures and sediment flushing from Indian dams, it would not be enough water to enable Pakistan to adequately irrigate or to provide fresh water to its people.The aging irrigation infrastructure would continue to deteriorate, compounding an already untenable situation. The threat of nuclear war would hang over the region for the foreseeable future as radical elements within Pakistan are able to seize power and Pakistan becomes a failed, pariah state. 2. As a result of a brokered cease-fire, Kashmir achieves independence. Kashmir brokers its own water treaty with both India and Pakistan: India agrees to maintain the existing hydroelectric dams and water storage in return for continued access to the electricity being generated.Pakistan continues to receive flow from the Indus River, but at lower levels than under the IWT as Kashmir diverts and stores some of the water for its own irrigation. Pakistan’s irrigation and storage systems continue to deteriorate, but at a less noticeable pace than under the first nullification scenario. Radical elements are able to achieve some power within Pakistan, but moderates are able to maintain control and because of the existing water treaty are able to contract assistance from China and the United States to upgrade irrigation and water storage.Although still a nuclear power, Pakistan is unable to maintain parity with India on a military or economic level, effectively dimin ishing the threat of nuclear war. 3. Pakistan achieves strategic surprise and is able to seize control of Kashmir and the upper Indus River prior to the brokered cease-fire. Rather than increasing the flow of water to irrigate, Pakistan maintains the current hydroelectric systems built by India, selling some of the power to India and diverting the rest for its own use.Pakistan fails to address its own interprovincial water sharing issues: In addition to existing squabbles between Punjab and Sindh, it has added Kashmir to the mix with its own demands for irrigation and fresh water. Although Pakistan is able to maintain water flow to support irrigation, it is below the level of the IWT. India and Pakistan continue their adversarial relationship but without the benefits of diplomatic exchange. Radicals within Pakistan are able to exploit the inequitable division of water between the provinces and, in spite of its Muslim majority, Kashmir never becomes a fully integrated part of Pakista n.Because of its need to maintain both a military balance with India and to secure its facilities against domestic terror attacks, it is unprepared for the dropping water flow due to the recession of the Himalayan glaciers feeding the Indus. The region continues to be an international concern as China and the United States jockey for influence. Although the scenarios regarding a nullification of the IWT may be unduly negative, most academic studies agree that the Indus Water Treaty of 1960 is too important to regional relations for either India or Pakistan to seek an alternative.Whether the treaty continues in its present form, which is increasingly unlikely, is renegotiated as part of a larger brokered deal, or is restructured according to some recognition of Indian responsibility to its neighbor, the treaty has survived an ongoing adversarial relationship for 53 years due to both its effectiveness and its utility. With the worldwide potential for resource scarcity, the potential e xists that other nations sharing water resources could model their own disputes on the IWT, but only if Pakistan and India are able to resolve their own ongoing issues.

Ritz-Carlton Hotels

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The most common issue in our world today is the conflict between parents and teenagers. However, it is more than just conflict. What was once a sort of a â€Å"B.F.F.† relationship is now sour and bitter; full of arguments and sometimes fights! The arguing becomes an everyday thing and the parent nor is the child able to prevent it. It becomes natural to both. With that in mind, yes it is conflict, but it’s not forever; just temporary. Ask yourself a question about the situation†¦ Why are the teenage years more difficult with parents? Well, simply because the teenage years are when teens seek identity and parents majority come in-between it. In the present time, 13 years old is when everything goes downhill for the relationship. We start becoming more rebellious, the â€Å"back-talking† starts, conflict problems with other teens, wanting to be independent and unfortunately its good-bye to the video games and hello to the parties! All of this at only age 13. Can you imagine what the next 5 years will be? When a person reaches his or her physical maturity, there is also a change in your mental state as well as physical. We mature much earlier now days. There is no longer a match between our demand for independence, and our ability to actually be independent! Our teenage bodies and minds are screaming â€Å"I am ready to be independent! I want to make my own decisions! I want to be my own boss!† Your parents and society are screaming back at you, â€Å"You are not ready to be independent yet! You have not learned what you need to know yet! You cannot support yourself! You do not yet fully understand the dangers in this world! You are not grown!† . . . . And in the end they are right. We struggle with this unnatural situation. Reference http://www.livestrong.com/article/7895-conflict-between-parents-teenagers-/

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Forbes International Top Ten - Research Paper Example There are many things that Toyota has done in order to outsell its competition. This report will discuss the strategic plan in depth. According to the Maynard (2008) one of the things that Toyota does is to find a way to build their cars better. Some of what they do is to encourage employees to do exercises that will create a more focused employee. This is part of their strategy to use in training centers to make sure that their employees are focused on what they are attempting to do. They have found that through watching how they make cars and improving this at every level, it has created a better opportunity for them to build cars faster (Maynard, 2008). Dawson and Shirouzu (2011) also state that Toyota is downsizing its board of directors in order to make decisions at the higher levels easier. The plan will also include an expansion to China and India before it is completed (Dawson and Shirouzu, 2011). Akio Toyoda (2011) the new head of Toyota has created a vision for his company that provides what they intend to do in the future. It details their strategic plan and provides important information as to how they see themselves and what they are to do. The information they provide is as follows: â€Å"Toyota will lead the way to the future of mobility (par. 7)†. Toyota will creatively stay ahead of its competition. This area includes finding ways to help people in different types of mobility as well as moving them into the future and they will research â€Å"smart grids† (par. 7) in order to understand how to make their company more ecologically friendly. â€Å"Enriching lives around the world (par. 8).† This statement is their commitment to making automobiles that people want to drive. This provides continuous employment for many people and it provides safe vehicles for those who need them. â€Å"With the safest and most responsible ways of moving people (par. 9).† This statement involves Toyota’s commitment to making sure t hat their vehicles are safe and attractive to the eye. They want people to purchase them because they are emotionally moved to do so as well as providing an automobile that is safe and environmentally friendly. â€Å"Through our commitment to quality, constant innovation (par. 10).† According to Toyoda, this is the â€Å"DNA† of what Toyota is and what they are supposed to do. It means that they are always looking for ways to reinvent themselves so that they can provide vehicles that will respond to the needs of people in the future. â€Å"Respect for the planet (par. 11).† This statement is their commitment to taking the environment into consideration when they make automobiles. They are committed to creating a way to manufacture automobiles that â€Å"minimizes waste† (par. 11). These eleven statements are at the heart of Toyota’s strategic plan according to Toyoda (2011) and they are what they want to work with throughout the next several years. In addition, they have a commitment to their employees to make sure that they are reaching goals that they have set. It is important to Toyota that all employees work to a high level of performance and they expect that their stockholders and others will form positive opinions of what they are doing. They are interested in making sure that the people who work for them take into consideration opinions and thoughts of both their customers and their stakeholders (Toyoda, 2011, par. 12). Toyota also emphasizes

The needs of future generations are being met by current policies of Essay - 1

The needs of future generations are being met by current policies of sustainable development. To what extent do you agree with this statement - Essay Example s individuals have become increasingly aware of fragile environment and the need to promote utility of the individual not met them as well is generated at the same time. As a function of seeking to answer the degree and extent to which current policies is development are assisting future generations the following analysis will consider three case study involving pollution, global warming, precise as a function of seeking to answer this broader overarching question. It is the further hope of this particular author that such a level of discussion and analysis will be beneficial with regards to representing the reality in which the current world exists is further utility and sustainability of the plan as well as the individuals that call it home currently those that will one day live upon. Further, even though a global level of understanding relates to this specific topic, a level of analysis will be provided on Singapore; allowing for a more individualized and focused analysis. Firstly, with respect to the issue of recycling, it can be noted that current policies of sustainable development encourage recycling as a means of ensuring that the same level of resources will be available future generations as are currently available. Great progress has been made with respect to encouraging recycling and in some cases incentivizing; actually paying firms and/or individuals to dispose of their waists in a responsible manner so that this process will gain a degree of traction and, reinforce behavior that is exhibited elsewhere throughout society. However, there is a fundamental drawback with respect to the way in which recycling programs are being throughout the developed world. One of the most prominent of these has to do with the fact that recycling programs are not mandatory. Accordingly, those individuals that the recycling is either a hassle or useless towards improving their own lives will not integrated with an continue to dispose of garbage and other waste and an

A review of black lives matter a modern social movement in America Essay Example for Free

A review of black lives matter a modern social movement in America Essay Abstract The prominence of the Black Lives Matter is a reform of social justice in America. There are many layers to the movement including and having to do with feminism, an ideological change, as well as a different psychology. It holds the future of social justice. It has been over a century since the formal end to American slavery, and decades since the Civil Rights Movement in the 1960’s, but social justice and an egalitarian society does not yet exist. But with the knowledge and teachings that can change that attitude, the movement will continue to grow and make the strides that are necessary to change the lives of the people who help make America. Black Lives Matter: Beyond a Movement â€Å"Black Lives Matter† is very well known and everyone has a ready association when hearing it. I have always found it most interesting that this movement has come with so much mockery. Mockery in the sense that for all the people that take it seriously and for the people whose daily lives are affected, there are just as many people if not more who come with the opposition. The opposition make other slogans (i.e. â€Å"All Lives Matter†, â€Å"Blue Lives Matter†, â€Å"White Lives Matter†, etc.) This makes a mockery of the issue (Yancy Butler, 2015). They are saying, your issue is not a real one, get over it. And also saying, maybe you should think about us first, and how you are affecting our lives. It is more than a movement at the end of the day, and this is because the more people do not take the issue seriously, the more Black lives are taken in horrible ways without a valid reason. Even then, a major problem of victim blaming occurs with every one of these cases and the â€Å"Blue Life† that matters are able to get off scot-free and continue their life. So clearly the blue life matters, but what about the Black life that was taken by that blue life (Maclin, 1991)? Why are some people given the right and privilege to decide who gets to live? Of course, everyone is aware of the answers to these hypothetical questions I present. The power of the hierarchy of society and race is deep-rooted inside all of us (Maclin, 1991). And it does not seem to change, it makes strives toward changes, but it does not change like it should. It is constantly the same story day after day, decade after decade, and at this point, century after century. The big question now, asks everyone wh at they are able to do for their country. The minority population and the African-American population create America and they are just as much of the country as any other ethnicity. â€Å"We completely expect those who benefit directly and improperly from white supremacy to try to erase our existence.† (Garza, 2014). I like the usage of the word, improperly, in this article. The author is addressing the movement from the perspective of a Black queer woman. The word fits perfectly into defining so much of society. It is improper, how the current social situation is so prominent today. Roughly 50 years ago was the Civil Rights Movement, that is in the lifetime of many people alive today and yet it does not concern so many that the same issues are here today. The distribution of power and right to life has been given to a select few. Not even given, but taken by a select few, who have and continue to refuse to give it up (Garza, 2014). The right to life, it essentially has been taken from the those who are not white males- the normal (Garza, 2014). And Black lives are given the least regard to their life. Black Lives Matter also serves as an ideology. It even perhaps serves better to be categorized as an ideology. It is an ideology that brought a uniformity to the anger and frustration with the obvious social injustices occurring (Thomas, 2004). Especially, after the tragic killing of Trayvon Martin and the verdict that allowed the killer, George Zimmerman to go back home. After the ruling, the anger and sparked outrage of the Black community, made people worry for Zimmerman. It made the white community worry for their own lives. This truly is irony at its finest (Yancy Butler, 2015). It is more than a misunderstanding of the point. It was disregarded, that this verdict justified the killing of completely innocent Black lives. But rather than worrying about the people who cannot walk down a street, without having to worry about being killed. And their families who have to deal with the death of a loved one and then watch as their killer went free. But then be told that it does not ma tter. And justify it with, ‘all lives matter’ (Yancy Butler, 2015). Through my research, I noticed there are typically two different types of papers written on this topic: either from a perspective looking at the whole or from the perspective of a Black woman. I find this very interesting, there are multiple struggles going on through this entire movement and ideological change. But there is the other issue of Black feminism that also needs addressing. I find it both compelling and significant that Black women both support the greater picture with their fellow man, but also separate themselves because they are different and need something else (Thomas, 2004). Black women are similar to Black men in some ways and similar to white women in some ways, but are different from both of them- they cannot completely identify with either one, in order to find further justice (Thomas, 2004). I have always considered myself a feminist, but unfortunately, not until more recently did I know that there are actually different types of feminism and feminists struggles. There is white feminism and there is also Black feminism and Hispanic feminism (which can be grouped together and separately as well). White women argue for some basic rights such as equal pay and respect to work outside of the home. Whereas minority women fight for even more basic rights than that, such as getting hired for a job (Thomas, 2004). They are not just asking for equal pay in a salary job, but for their job applications to not be thrown away for a minimum wage job because of their name. But just like in all feminism, it is important to give people who believe and support the feminist struggle, to separate themselves from the people who do not (Garza, 2014). This is because until there is true equality, the people who support it are different, and the difference raises attention. But, looking at the b igger picture, white feminism is the issue that is raising attention and not so much of the feminist struggle of minorities (Garza, 2014). The ‘Black Lives Matter’ movement creates a new foundation in psychology. Psychology is in part the study of behavior. But the psychology most people study, leave people out, large groups of people are left out (Thomas, 2004). One of these groups is the Black community and even further is Black women (Thomas, 2004). The issues are established, many people are ignorant of it or they chose to ignore it, but knowledge serves as a powerful tool. Social justice starts from the ground up. The racial institution that America exemplifies all so much prevents Black Americans from accomplishing the ‘American dream’. The Black community is stereotypically living in poverty, living in ‘ghettos’, and cannot get jobs. They are blamed for their own issues, as they deserve what they are getting out of life (Garcà ­a Sharif, 2015). But rather than realizing that it is the racism of America that is keeping any minority and especially the Black community from going anywhere (Garcà ­a Sharif, 2015). Many Black Americans have made great accomplishments, but not only are they ignored and not taught about, but are made to seem as exceptions. Successful Black people are not like the normal Black person, they lucked out or they were given some special situation that allowed them to accompli sh anything. There is a deep-rooted problem even with the Black community, that is given from within. They are told they are hated by society and therefore they should see problems with themselves, too (Garcà ­a Sharif, 2015). No one who is not living outside of the white standard should be proud of themselves, in fact, they need to take it one step farther and be ashamed of themselves. Not only ashamed of their own appearance but be afraid to accomplish anything (Garcà ­a Sharif, 2015). Everyone is held up against the white standard, not only in physical beauty but also with development and academic development and progress. If a Black person is more successful than a white person in academics, then there must be some sort of mistake. There has to be another reason for this, perhaps something is wrong with the Black student. This is just one of the many ways social injustice is justified with society. It becomes a self-fulfilling prophecy with Black children (and even throughout their lives) . They are told they are problems within the classroom and if they accomplish more than a white student, it is not because they are smart. It is probably because they are too hyper or they are not listening to directions. When a person is told they are not good enough, then they will not be good enough (Garcà ­a Sharif, 2015). A person is just as good as other people tell them they are because they take on the role they are told that they have. Black Lives Matter is a â€Å"mode of address† (Yancy Butler, 2015). It is against society telling that an entire group of people that every other life is above yours. So as long as anyone who is not Black is dying, then Black lives and the unjust loss of can be addressed later (or in other words, never) (Yancy Butler, 2015). Black men and women, even boys and girls, are seen and portrayed by society and police as scary, dangerous, aggressive, and violent. Constantly, the testimonies of these murder cases will reveal the cop’s description of the victim as forceful or aggressive (Yancy Butler, 2015). When watching the videos, it is clear this just is not true. But also there is an issue of the cop’s word and racist mindsets, makes people see the video through a lens. It is quite evident that it does not matter how a Black person presents themselves because they are never good enough. The stress, discouragement, outrage, depression, etc., etc. resulting from rac ism and hate acts, is detrimental to the health of the Black community (Garcà ­a Sharif, 2015). The growing importance of social media is resulting in a large part of ‘Black Lives Matter’. Not only education but the usage of social media, I think, will only continue to help the movement toward social justice. What is so amazing about social media is that it is accessible to people all across the country and even the world, within seconds. It does not take more than a few minutes for the entire world to see the video of a horrible killing of another Black life. Or the ruling in yet another unjustified ruling letting goes another cop who killed an unarmed Black man or woman. The videos are hard to watch. And making it even better, once they are on the internet they do not ever really go away. What is so important in a movement, ideology, psychology such as Black lives matter is that the people who have died do not go forgotten. It is crucial that their names are known, giving them a life. Once they are identified and their story is told under ‘young black manâ €™, etc., their story goes with all the other people who are said to not matter. We must be able to see the totally egalitarian society, we must be able to see the possibility of in order for it to ever have a true chance. This movement starts, to say this is done, and we need this- NOW. And we have to listen and respond and see the future as such to move forward successfully. References Garza, A., Tometi, O., Cullors, P. (2014). A herstory of the# BlackLivesMatter movement. Jee-Lyn Garcà ­a, J., Sharif, M. Z. (2015). Black lives matter: a commentary on racism and public health. American journal of public health, 105(8), e27-e30. Maclin, T. (1991). Black and Blue Encounters-Some Preliminary Thoughts About Fourth   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Amendment Seizures: Should Race Matter. Val. UL Rev., 26, 243. Thomas, V. G. (2004). The psychology of Black women: Studying women’s lives in context.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Journal of Black Psychology, 30(3), 286-306. Yancy, G., Butler, J. (2015). What’s wrong with ‘all lives matter’?. New York Times, 12, 156.

Physico-chemical Processes that Occur During Freezing

Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio Physico-chemical Processes that Occur During Freezing Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio